1: J Hered. 2005 Nov 2; [Epub ahead of print] Exclusion of Candidate Genes for Canine SRY-Negative XX Sex Reversal. Kothapalli K, Kirkness E, Pujar S, Van Wormer R, Meyers-Wallen VN. the J. A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates. PMID: 16267164 [PubMed - as supplied by publisher] --------------------------------------------------------------- 2: Zoolog Sci. 2005 Sep;22(9):1045-50. Molecular cloning and expression in gonad of Rana rugosa WT1 and Fgf9. Yamamura Y, Aoyama S, Oshima Y, Kato T, Osawa N, Nakamura M. Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda University, Tokyo, Japan. Sry (sex-determining region on the Y chromosome) is required for testicular differentiation in mammals. In addition to Sry, other genes such as WT1, Fgf9, Dax1, Dmrt1 and Sox9 are widely accepted to be involved in the sex determination in vertebrates. However, the roles of these genes during sex determination still remain unclear in amphibians. This study was undertaken to examine the expression of WT1 and Fgf9 in the developing gonad of amphibians. We first isolated the WT1 cDNA from the frog Rana rugosa. Like WT1 in mice, R. rugosa WT1 showed 2 isoforms; i.e., one had an additional 3 amino acids, KTS, included between the third and fourth zinc fingers. However, 17 amino acids in exon 5 of mammalian WT1 could not be found in R. rugosa WT1, which is also the case in turtle and chicken. The mRNA of both isoforms (+KTS, -KTS) was detected in the lung, kidney and testis, but not in the ovary and muscle of adult frogs. The 2 isoforms were expressed first in the embryos at stage 23. Thereafter, the expressions remained constant in the gonad attached to mesonephros of both sexes during sex determination. We next isolated the R. rugosa Fgf9 cDNA encoding 208 amino acids. The amino acid sequence of Fgf9 had similarity greater than 92% with chicken, mouse and human Fgf9s, suggesting that Fgf9 is highly conserved among vertebrate classes. Fgf9 was expressed in the ovary of an adult frog strongly, but in the lung weakly. In contrast, the Fgf9 mRNA was hardly detected in the kidney, testis and muscle. Moreover, Fgf9 did not show a sexually dimorphic expression pattern during sex determination in R. rugosa. The results, taken together, suggest that both WT1 and Fgf9 are expressed in the indifferent gonad prior to sex determination without any difference in the expression between males and females. Thus, it seems unlikely that they are a key factor to initiate the divergence leading to testicular or ovarian differentiation in R. rugosa. PMID: 16219985 [PubMed - in process] --------------------------------------------------------------- 3: Gen Comp Endocrinol. 2005 Oct 6; [Epub ahead of print] Nucleotide sequence and embryonic expression of quail and duck Sox9 genes. Takada S, Ota J, Kansaku N, Yamashita H, Izumi T, Ishikawa M, Wada T, Kaneda R, Choi YL, Koinuma K, Fujiwara SI, Aoki H, Kisanuki H, Yamashita Y, Mano H. Division of Functional Genomics, Jichi Medical School, 3311-1 Yakushiji, Kawachigun, Tochigi 329-0498, Japan. Sox9 is a member of the Sry-type HMG-box (Sox) gene family. It encodes a transcription factor and is thought to be important for sexual differentiation in chicken. In the present study we have isolated Sox9 cDNAs from quail and duck, and examined the expression patterns of the corresponding genes in early embryonic gonads by whole-mount in situ hybridization. We developed a polymerase chain reaction-based protocol to identify the sex of quail and duck embryos before its morphological manifestation. Sox9 expression was first detected on days 5 and 7 in the gonads of male quail and duck embryos, respectively, and was not apparent in female gonads at these stages. These expression patterns are similar to that of chicken Sox9. Our results thus suggest that the expression of quail and duck Sox9 is associated with testis differentiation. PMID: 16216246 [PubMed - as supplied by publisher] --------------------------------------------------------------- 4: Biol Reprod. 2005 Oct 5; [Epub ahead of print] Homozygous Inactivation of Sox9 Causes Complete XY Sex Reversal in Mice. Barrionuevo F, Bagheri-Fam S, Klattig J, Kist R, Taketo MM, Englert C, Scherer G. In the presence of the Y-chromosomal gene Sry, the bipotential mouse gonads develop as testes rather than as ovaries. The autosomal gene Sox9, a likely and possibly direct Sry target, can induce testis development in the absence of Sry. Sox9 is thus sufficient but not necessarily essential for testis induction. Mutational inactivation of one allele of SOX9/Sox9 causes sex reversal in humans but not in mice. As Sox9(-/-) embryos die around day 11.5 (E11.5) of mouse embryonic development at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can only be analyzed ex vivo in organ culture. We have therefore conditionally inactivated both Sox9 alleles in the gonadal anlagen using the Cre / loxP recombination system, whereby Cre recombinase is under control of the cytokeratin 19 promoter. Analysis of resulting Sox9(-/-) XY gonads up to E15.5 reveals immediate, complete sex reversal, as shown by expression of the early ovary-specific markers Wnt4 and Foxl2 and by lack of testis cord and Leydig cell formation. Sry expression in mutant XY gonads indicates that down-regulation of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our results provide in vivo proof that, in contrast to the situation in humans, complete XY sex reversal in mice requires inactivation of both Sox9 alleles, and that Sox9 is essential for testogenesis in mice. PMID: 16207837 [PubMed - as supplied by publisher] --------------------------------------------------------------- 5: Dev Biol. 2005 Nov 1;287(1):111-124. Epub 2005 Sep 26. Sertoli cell differentiation is induced both cell-autonomously and through prostaglandin signaling during mammalian sex determination. Wilhelm D, Martinson F, Bradford S, Wilson MJ, Combes AN, Beverdam A, Bowles J, Mizusaki H, Koopman P. Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. We have raised an antibody specifically recognizing endogenous mouse SRY protein and used it to investigate the molecular and cellular mode of action of SRY in testis determination. We find that expression of SRY protein closely mirrors the expression of Sry mRNA in mouse genital ridges and is detectable for 6 to 8 h after the mRNA ceases to be detectable. The subset of somatic cells that expresses SRY begins to express SOX9 almost immediately. Since these SOX9-positive cells go on to develop as Sertoli cells, it appears that SRY expression marks the pre-Sertoli cell lineage and leads to up-regulation of Sox9 expression cell-autonomously. However, a small proportion of SOX9-positive cells did not appear to express SRY, possibly reflecting the additional involvement of paracrine signaling in activating Sox9 transcription in these cells. We confirmed by ex vivo cell mixing experiments that SRY is able to engage receptor-mediated signaling to up-regulate Sox9 expression. Finally, we showed by employing specific inhibitors that the causative signaling molecule is prostaglandin D(2) (PGD(2)) and that PGD(2) can induce Sox9 transcription in cultured XX gonads. Our data indicate a mechanism whereby Sry uses both a cell-autonomous mechanism and a PGD(2)-mediated signaling mechanism to stimulate expression of Sox9 and induce the differentiation of Sertoli cells in vivo. PMID: 16185683 [PubMed - as supplied by publisher] --------------------------------------------------------------- 6: Bioessays. 2005 Oct;27(10):1011-25. Regulated nucleocytoplasmic transport in spermatogenesis: a driver of cellular differentiation? Hogarth C, Itman C, Jans DA, Loveland KL. Monash Institute of Medical Research, Monash University, Melbourne, Australia. This review explores the hypothesis that regulation of nucleocytoplasmic shuttling is a means of driving differentiation, using spermatogenesis as a model. The transition from undifferentiated spermatogonial stem cell to terminally differentiated spermatozoon is, at its most basic, a change in the repertoire of expressed genes. To effect this, the complement of nuclear proteins, such as transcription factors and chromatin remodelling components must change. Current knowledge of the nuclear proteins and nucleocytoplasmic transport machinery relevant to spermatogenesis is consolidated in this review, and their functional linkages are highlighted not only as a means of regulating nuclear protein composition, but also as a key mechanism regulating gene transcription and hence cell fate. Through this, we hypothesize that male germ cell differentiation is mediated through regulation of nuclear transport machinery components, and thereby of the access of critical factors to the nucleus. The importance of nucleocytoplasmic trafficking to male germ cell differentiation is discussed, using the sex-determining factors Sry and SOX9, cell cycle regulators, CREM and cofactors and the Smads as specific examples, together with the roles in gametogenesis for particular nuclear transport factors in Caenorhabditis elegans and Drosophila. (c) 2005 Wiley Periodicals, Inc. Publication Types: Review Review, Tutorial PMID: 16163727 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Hered. 2005 Sep 8; [Epub ahead of print] Lack of the SOX9 Gene Polymorphism in Sex Reversal Dogs (78,XX; SRY negative). Nowacka J, Nizanski W, Klimowicz M, Dzimira S, Switonski M. the Department of Animal Genetics and Breeding, Agricultural University of Poznan, Wolynska 33, 60-637 Poznan, Poland. The molecular background of the most frequent intersexuality syndrome in dogs (female-to-male sex reversal with the female karyotype and a lack of the SRY gene) is unknown. In this article, new cases of this syndrome are described in two unrelated American Staffordshire terrier dogs and one miniature pinscher dog subjected to cytogenetic and molecular analysis due to the presence of an enlarged clitoris. One dog was operated on and histological studies of the gonads revealed a testicular structure without signs of spermatogenesis, but the uterus wall appeared to be normal. All three dogs had female chromosome complements and lacked the Y-linked genes SRY and ZFY. Eight fragments, representing the vast majority of the coding sequence of the SOX9 gene, and two fragments of the 5' flanking region of this gene were analyzed. The studied fragments had identical DNA sequences when comparing the intersexual dogs with GenBank sequences (AY237827; NW139883). Thus a mutation in the coding sequence as well as the promoter region of the SOX9 gene might be excluded as a cause of this type of intersexuality. The importance of further studies of the 5' flanking region of this gene is discussed. PMID: 16150950 [PubMed - as supplied by publisher] --------------------------------------------------------------- 8: J Biol Chem. 2005 Oct 21;280(42):35203-8. Epub 2005 Aug 18. Sox9 and p300 cooperatively regulate chromatin-mediated transcription. Furumatsu T, Tsuda M, Yoshida K, Taniguchi N, Ito T, Hashimoto M, Ito T, Asahara H. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. Chromatin structure is a fundamental component of gene regulation, expression, and cellular differentiation. We have previously reported that the multifunctional coactivator p300 is a member of the Sox9 (Sry-type high mobility group box 9)-related transcriptional apparatus and activates Sox9-dependent transcription during chondrogenesis. However, the mechanism of synergy between Sox9 and p300 in chromatin-mediated transcription has not been elucidated. In the present study we investigated the activity of Sox9 and p300 on chromatinized templates in vitro. Recombinant Sox9 was shown to be associated with several transcriptional cofactors including p300. In vitro transcription assays revealed that p300 potentiated Sox9-dependent transcription on chromatinized DNA and, importantly, was associated with hyperacetylated histones. Consistent with these results, the histone deacetylase inhibitor trichostatin A stimulated the expression of Sox9-regulated cartilage matrix genes and induced histone acetylation around the enhancer region of the collagen alpha1 (II) gene in chondrocytes. These findings suggest that Sox9 interacts with chromatin and activates transcription via regulation of chromatin modification. PMID: 16109717 [PubMed - in process] --------------------------------------------------------------- 9: J Biochem (Tokyo). 2005 Jul;138(1):13-9. From SRY to SOX9: mammalian testis differentiation. Kanai Y, Hiramatsu R, Matoba S, Kidokoro T. Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657. aykanai@mail.ecc.u-tokyo.ac.jp Sry (sex-determining region on the Y chromosome) is a master gene that initiates testis differentiation of the bipotential indifferent gonad in mammals. In mice, Sry expression is transiently activated in a center-to-pole wave along the anteroposterior (AP) axis of developing XY gonads. Shortly after the onset of Sry activation, Sox9 (Sry-related HMG box-9), a fundamental testis-differentiation gene common to all vertebrates, is also activated in a center-to-pole pattern similar to the initial Sry expression profile. Several male-specific cellular events, such as glycogenesis, coelomic epithelium proliferation, mesonephric migration and vasculogenesis, are induced in XY gonads following the onset of Sry and Sox9 expression. This paper mainly focuses on recent advances in elucidating the regulatory mechanisms of Sry and Sox9 expression and male-specific cellular events immediately downstream of SRY action during the initial phases of testis differentiation. Publication Types: Review PMID: 16046443 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mamm Genome. 2005 Apr;16(4):281-9. Characterization of Pisrt1/Foxl2 in Ellobius lutescens and exclusion as sex-determining genes. Baumstark A, Hameister H, Hakhverdyan M, Bakloushinskaya I, Just W. Department of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany. The rodent Ellobius lutescens is an exceptional mammal which determines male sex constitutively without the SRY gene and, therefore, may serve as an animal model for human 46,XX female-to-male sex reversal. It was suggested that other factors of the network of sex-determining genes determine maleness in these animals. However, some sex-determining genes like SOX9 and SF1 have already been excluded by segregation analysis as primary sex-determining factors in E. lutescens. In this work, we have cloned and characterized two genes of the PIS (polled intersex syndrome) gene interval, which were reported as candidates in female-to-male sex reversal in hornless goats recently. The genes Foxl2 and Pisrt1 from that interval were identified in E. lutescens DNA and mapped to Chromosome 8. We have excluded linkage of Foxl2 and Pisrt1 loci with the sex of the animals. Hence, the involvement of this gene region in sex determination may be specific for goats and is not a general mechanism of XX sex reversal or XX male sex determination. PMID: 15965789 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Development. 2005 Jul;132(13):3045-54. Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells. Bouma GJ, Albrecht KH, Washburn LL, Recknagel AK, Churchill GA, Eicher EM. The Jackson Laboratory, Bar Harbor, ME 04609, USA. jbouma@jax.org The nuclear receptor transcription factor Dax1 is hypothesized to play a role in testicular development, although the mechanism of its action is unknown. Here, we present evidence that Dax1 plays an early essential role in fetal testis development. We hypothesize that upregulation of Sox9 expression in precursor somatic cells, a process required for their differentiation as Sertoli cells, depends on the coordinated expression of Dax1, Sry and another gene, Tda1. Our conclusion and model are based on the following experimental findings: (1) presence of a mutant Dax1 allele (Dax1-) results in complete gonadal sex reversal in C57BL/6JEi (B6) XY mice, whereas testes develop in DBA/2J (D2) and (B6xD2)F1 XY mice; (2) B6-DAX1 sex reversal is inherited as a complex trait that includes the chromosome 4 gene Tda1; (3) B6 Dax1-/Y fetal gonads initiate development as ovaries, even though Sry expression is activated at the correct time and at appropriate levels; (4) upregulation of Sox9 does not occur in B6 Dax1-/Y fetal gonads in spite of apparently normal Sry expression; and (5) overexpression of Sry in B6 Dax1-/Y fetal gonads upregulates Sox9 and corrects testis development. PMID: 15944188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: EMBO J. 2005 May 18;24(10):1798-809. Epub 2005 May 5. Prostaglandin D2 induces nuclear import of the sex-determining factor SOX9 via its cAMP-PKA phosphorylation. Malki S, Nef S, Notarnicola C, Thevenet L, Gasca S, Mejean C, Berta P, Poulat F, Boizet-Bonhoure B. Institut de Genetique Humaine, CNRS UPR1142, 141 rue de la Cardonille, Montpellier Cedex 5, France. During mammalian gonadal development, nuclear import/export of the transcription factor SOX9 is a critical step of the Sry-initiated testis-determining cascade. In this study, we identify a molecular mechanism contributing to the SOX9 nuclear translocation in NT2/D1 cells, which is mediated by the prostaglandin D2 (PGD2) signalling pathway via stimulation of its adenylcyclase-coupled DP1 receptor. We find that activation of cAMP-dependent protein kinase A (PKA) induces phosphorylation of SOX9 on its two S64 and S181 PKA sites, and its nuclear localization by enhancing SOX9 binding to the nucleocytoplasmic transport protein importin beta. Moreover, in embryonic gonads, we detect a male-specific prostaglandin D synthase expression and an active PGD2 signal at the time and place of SOX9 expression. We thus propose a new step in the sex-determining cascade where PGD2 acts as an autocrine factor inducing SOX9 nuclear translocation and subsequent Sertoli cell differentiation. PMID: 15889150 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Genome. 2005 Apr;48(2):181-6. SRY-related (Sox) genes in the genome of European Atlantic sturgeon (Acipenser sturio). Hett AK, Ludwig A. Institute for Zoo and Wildlife Research, Department of Evolutionary Genetics, Berlin, Germany. The Sox-gene family represents an ancient group of transcription factors involved in numerous developmental processes and sex determination in vertebrates. SOX proteins are characterized by a conserved high mobility group (HMG)-box domain, which is responsible for DNA binding and bending. We studied Sox genes in sturgeon, one of the most primitive groups of fishes characterized by a high chromosome number. Male and female genomes were screened for Sox genes using highly degenerate primers that amplified a broad range of HMG boxes. A total of 102 clones, representing 22 different sequences coding for 8 Sox genes, was detected and classified according to their orthologues. Sox2, Sox3, Sox4, Sox9, Sox11, Sox17, Sox19, and Sox21 were found in sturgeon; these genes represent Sox groups B, C, E, and F. In a phylogenetic analysis (neighbor-joining, maximum likelihood, maximum parsimony), these genes clustered with their mouse orthologues. In the case of Sox4, Sox17, and Sox21, we found evidence of gene duplication. PMID: 15838539 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Dev Genes Evol. 2005 Jun;215(6):297-305. Epub 2005 Apr 8. Divergent expression patterns of Sox9 duplicates in teleosts indicate a lineage specific subfunctionalization. Kluver N, Kondo M, Herpin A, Mitani H, Schartl M. Department of Physiological Chemistry I, University of Wurzburg, 97074, Wurzburg, Germany, phch1@biozentrum.uni-wuerzburg.de. Sry-related HMG-box genes are key regulators of several developmental processes. Sox9 encodes a transcription factor required for cartilage formation and testis determination in mammals. In zebrafish (Danio rerio) and stickleback (Gasterosteus aculeatus) two co-orthologs of Sox9 are present. To date, only one Sox9 had been identified in medaka (Oryzias latipes). We have now isolated the second Sox9 gene. Sequence analysis, phylogenetic data, linkage mapping as well as expression pattern all together suggest that the medaka Sox9a and Sox9b are co-orthologs. During embryogenesis, the expression pattern of Sox9a and Sox9b are distinct but overlap considerably in craniofacial cartilage elements. Comparing the zebrafish Sox9a and Sox9b expression patterns with medaka Sox9a and Sox9b expression domains revealed that some are identical but others are clearly different. We conclude that Sox9 regulatory subfunctions were not partitioned before divergence of the teleosts and evolved to lineage-specific expression domains. PMID: 15818483 [PubMed - in process] --------------------------------------------------------------- 15: Hum Mol Genet. 2005 May 1;14(9):1221-9. Epub 2005 Mar 24. Sox9 is sufficient for functional testis development producing fertile male mice in the absence of Sry. Qin Y, Bishop CE. Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030, USA. In the dominant mouse mutant Odd Sex, XXOds/+ mice develop as phenotypic, sterile males due to male-pattern expression of Sox9 in XXOds/+ embryonic gonads. To test whether SOX9 was sufficient to generate a fully fertile male in the absence of Sry, we constructed an XY(Sry(-))Ods/+ male mouse, in which the male phenotype is controlled autosomally by the Ods mutation. Mice were initially fertile, but progressively lost fertility until 5-6 months when they were sterile with very few germ cells in the testis. XY(Sry-)Ods/+ males also failed to establish the correct male-specific pattern of vascularization at the time of sex determination, which could be correlated to an inability of XY(Sry-),Ods/+ males to fully down-regulate Wnt4 expression in the embryonic gonad. Increasing the amount of SOX9 by producing homozygous XY(Sry-)Ods/Ods males was able to completely rescue the phenotype and restore correct vascular patterning and long-term fertility. These data indicate that activation of SOX9 in the gonad is sufficient to trigger all the downstream events needed for the development of a fully fertile male and provide evidence that Sox9 may down-regulate Wnt4 expression in the gonad. PMID: 15790596 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: J Biol Chem. 2005 Apr 29;280(17):17562-71. Epub 2005 Mar 1. Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling. Qiao B, Padilla SR, Benya PD. Orthopaedic Hospital, Los Angeles, J. Vernon Luck, Sr., M.D. Research Center and UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California 90095, USA. Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2. PMID: 15743758 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Dev Biol. 2005 Mar 15;279(2):481-90. Sox9 and Nkx2.5 determine the pyloric sphincter epithelium under the control of BMP signaling. Theodosiou NA, Tabin CJ. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. The organs of the digestive tract are specified by coordinated signaling between the endoderm and mesoderm during development. These epithelial-mesenchymal interactions lead to the organ-specific morphogenesis and differentiation of regions along the gut tube. In this paper, we show that in the chick, the SRY-related transcription factor Sox9 is a marker for the posterior gizzard. Viral misexpression of Sox9 in the gizzard mesoderm is sufficient to specify epithelium characteristic of the pyloric sphincter. Sox9 expression is normally limited to the region of the posterior gizzard under the regulation of BMP signaling from the adjacent midgut. Misexpression of an activated form of BMPR1b in the gizzard upregulates Sox9 expression, while the BMP antagonist noggin down-regulates Sox9 expression in the gizzard mesoderm. Previously, Nkx2.5 was identified as a marker for the mesoderm of the pyloric sphincter. As with Sox9, BMP signaling appears to regulate Nkx2.5 and its ability to determine the pyloric epithelium. Despite these similarities, our evidence suggests that Sox9 and Nkx2.5 are regulated independently by BMP signaling, and act coordinately to specify the pyloric sphincter. PMID: 15733673 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Am J Hum Genet. 2005 Apr;76(4):663-71. Fine mapping of chromosome 17 translocation breakpoints > or = 900 Kb upstream of SOX9 in acampomelic campomelic dysplasia and a mild, familial skeletal dysplasia. Hill-Harfe KL, Kaplan L, Stalker HJ, Zori RT, Pop R, Scherer G, Wallace MR. Division of Genetics, Department of Pediatrics, University of Florida College of Medicine, Gainesville, USA. Previously, our group reported a five-generation family in which a balanced t(13;17) translocation is associated with a spectrum of skeletal abnormalities, including Robin sequence, hypoplastic scapulae, and a missing pair of ribs. Using polymerase chain reaction (PCR) with chromosome-specific markers to analyze DBA from somatic cell hybrids containing the derivative translocation chromosomes, we narrowed the breakpoint on each chromosome. Subsequent sequencing of PCR products spanning the breakpoints identified the breaks precisely. The chromosome 17 breakpoint maps approximately 932 kb upstream of the sex-determining region Y (SRY)-related high-mobility group box gene (SOX) within a noncoding transcript represented by two IMAGE cDNA clones. A growing number of reports have implicated chromosome 17 breakpoints at a distance of up to 1 Mb from SOX9 in some cases of campomelic dysplasia (CD). Although this multigeneration family has a disorder that shares some features with CD, their phenotype is significantly milder than any reported cases of (nonmosaic) CD. Therefore, this case may represent an etiologically distinct skeletal dysplasia or may be an extremely mild familial example of CD, caused by the most proximal translocation breakpoint from SOX9 reported to date. In addition, we have refined the breakpoint in a acampomelic CD case described elsewhere and have found that it lies approximately 900 kb upstream of SOX9. PMID: 15717285 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Acta Histochem. 2005;106(6):427-37. Epub 2005 Jan 13. Immunohistochemical analysis of Sox9 expression in periosteum of tibia and calvaria after surgical release of the periosteum. Fujii T, Ueno T, Kagawa T, Sugahara T, Yamamoto T. Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan. gmd13131@cc.okayama-u.ac.jp Sox9 is a transcription factor belonging to the SRY family of high-mobility box proteins, and plays a major role in endochondral ossification. Sox9 is a potent activator of the type-2 collagen pheno-type marker of articular cartilage. Regulation of osteogenic molecular signals in periosteal bone formation has not yet been elucidated yet. The purpose of the present study was to analyze histologically the bone formation in surgically released and repositioned periosteum, and to determine expression of Sox9 and type-2 collagen in periosteal bone formation of tibia and calvaria. After surgery, the released tibial periosteum formed ectopic cartilage. At 7 days, a combination of endochondral and intramembranous ossification was apparent. Some fibroblasts derived from the released periosteum showed Sox9 expression. Chondrocytes and cartilage matrix both displayed type-2 collagen expression. At 7 days, an additional new bone was formed on the calvaria. Osteoblasts and fibroblasts derived from released calvarial periosteum did not express Sox9 or type-2 collagen. Sox9 was not expressed throughout the process periosteal bone formation on the calvaria. It is concluded that we revealed Sox9 and type-2 collagen expression in periosteal cells after periosteum release and that the generative potential of periosteal cells of calvaria is different from that of tibia. PMID: 15707652 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2414-9. Epub 2005 Feb 7. Transcriptional coactivator PGC-1alpha regulates chondrogenesis via association with Sox9. Kawakami Y, Tsuda M, Takahashi S, Taniguchi N, Esteban CR, Zemmyo M, Furumatsu T, Lotz M, Belmonte JC, Asahara H. Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Chondrogenesis is a multistep pathway in which multipotential mesenchymal stem cells (MSC) differentiate into chondrocytes. The transcription factor Sox9 (SRY-related high mobility group-Box gene 9) regulates chondrocyte differentiation and cartilage-specific expression of genes, such as Col2a1 (collagen type II alpha1). However, Sox9 expression is detected not only in chondrogenic tissue but also in nonchondrogenic tissues, suggesting the existence of a molecular partner(s) required for Sox9 to control chondrogenesis and chondrogenic gene expression. Here, we report identification of peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) as a coactivator for Sox9 during chondrogenesis. Expression of PGC-1alpha is induced at chondrogenesis sites during mouse embryonic limb development and during chondrogenesis in human MSC cultures. PGC-1alpha directly interacts with Sox9 and promotes Sox9-dependent transcriptional activity, suggesting that PGC-1alpha acts as a transcriptional coactivator for Sox9. Consistent with this finding, PGC-1alpha disruption in MSC by small interfering RNA inhibits Col2a1 expression during chondrogenesis. Furthermore, overexpression of both PGC-1alpha and Sox9 induced expression of chondrogenic genes, including Col2a1, followed by chondrogenesis in the MSC and developing chick limb. Together, our results suggest a transcriptional mechanism for chondrogenesis that is coordinated by PGC-1alpha. PMID: 15699338 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Dev Biol. 2005 Feb 15;278(2):511-25. Influence on spatiotemporal patterns of a male-specific Sox9 activation by ectopic Sry expression during early phases of testis differentiation in mice. Kidokoro T, Matoba S, Hiramatsu R, Fujisawa M, Kanai-Azuma M, Taya C, Kurohmaru M, Kawakami H, Hayashi Y, Kanai Y, Yonekawa H. Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan. Testis induction is associated with gonadal Sry and Sox9 expression in mammals. This study investigated whether Sry expression directly induces male-specific Sox9 activation during early phases of testis differentiation. We have established an XX sex-reversal mouse line carrying the Sry transgene driven by a weak basal promoter of the Hsp70.3 gene (Hsp-Sry), whereby the transgene was activated in the gonads along the entire anteroposterior axis from earlier stages. The effects of misexpression and overexpression of Sry on the spatiotemporal pattern of Sox9 expression were examined using both XX and XY gonads of Hsp-Sry transgenic embryos. It was shown that ectopic expression of Sry transcripts in the entire gonadal area from earlier stages promotes neither any advance in the timing nor any appreciable ectopic activation of endogenous Sox9 expression. Immediately after the onset of Sox9 activation, however, both the level of Sox9 expression and the number of SOX9-positive cells were significantly enhanced in Hsp-Sry/XY gonads, as compared with those in wild-type/XY and Hsp-Sry/XX gonads. These findings suggest that, although Sry is capable of up-regulating Sox9 expression dose-dependently, Sry mRNA expression alone is not likely to provide positional or timing information needed for male-specific Sox9 activation in developing XY gonads. PMID: 15680367 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Dev Biol. 2005 Feb 15;278(2):473-81. Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal. Bullejos M, Koopman P. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. The phenomenon of B6-Y(DOM) sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (B6) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y(DOM) sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y(DOM) sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway. PMID: 15680364 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: J Biol Chem. 2005 Mar 4;280(9):8343-50. Epub 2004 Dec 28. Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding protein/p300 recruitment. Furumatsu T, Tsuda M, Taniguchi N, Tajima Y, Asahara H. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor beta (TGF-beta) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-beta-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of alpha1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-beta-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis. PMID: 15623506 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Genet Sel Evol. 2005;37 Suppl 1:S55-64. Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation. Pailhoux E, Vigier B, Schibler L, Cribiu EP, Cotinot C, Vaiman D. Laboratoire de biologie du developpement et reproduction, Institut national de la recherche agronomique, 78352 Jouy-en-Josas Cedex, France. pailhoux@jouy.inra.fr In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4-5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation. PMID: 15601595 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Dev Biol. 2004 Dec 15;276(2):431-40. Wnt4 is required for proper male as well as female sexual development. Jeays-Ward K, Dandonneau M, Swain A. Section of Gene Function and Regulation, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 -/- gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development. PMID: 15581876 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Biol Reprod. 2005 Feb;72(2):492-501. Epub 2004 Oct 20. Profiling gene expression during the differentiation and development of the murine embryonic gonad. Small CL, Shima JE, Uzumcu M, Skinner MK, Griswold MD. Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA. The application of microarray technology to the study of mammalian organogenesis can provide greater insights into the steps necessary to elicit a functionally competent tissue. To this end, a temporal profile of gene expression was generated with the purpose of identifying changes in gene expression occurring within the developing male and female embryonic gonad. Gonad tissue was collected from mouse embryos at 11.5, 12.5, 14.5, 16.5, and 18.5 days postcoitum (dpc) and relative steady-state levels of mRNA were determined using the Affymetrix MGU74v2 microarray platform. Statistical analysis produced 3693 transcripts exhibiting differential expression during male and/or female gonad development. At 11.5 dpc, the gonad is morphologically indifferent, but at 12.5 dpc, transitions to a male or female phenotype are discernible by the appearance of testicular cords. A number of genes are expressed during this period and many share similar expression profiles in both sexes. As expected, the expression of two well-known sex determination genes, specifically Sry and Sox9, is unique to the testis. Beyond 12.5 dpc, differential gene expression becomes increasingly evident as the male and female tissue morphologically and physiologically diverges. This is evident by two unique waves of transcriptional activity occurring after 14.5 dpc in the male and female. With this study, a large number of transcripts comprising the murine transcriptome can be examined throughout male and female embryonic gonad development and allow for a more complete description of gonad differentiation and development. PMID: 15496517 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Dev Biol. 2004 Oct 15;274(2):271-9. SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors. Sekido R, Bar I, Narvaez V, Penny G, Lovell-Badge R. Division of Developmental Genetics, MRC National Institute for Medical Research, London NW7 1AA, UK. The Y chromosome gene Sry encodes a transcription factor required to initiate testis development. The related autosomal gene Sox9 is up-regulated shortly after the onset of Sry transcription and is thought essential for the differentiation of Sertoli cells. The lineage that gives rise to Sertoli cells has its origins within the coelomic epithelium (CE) of the genital ridge, but from cells also able to give rise to an interstitial cell type. It was not known at what point SRY acts in the derivation of this lineage or how the two genes interact. To investigate the identity of the cells expressing Sry, we designed two transgenes driven by the Sry promoter: one gives expression of a stable reporter, human placental alkaline phosphatase (hPLAP), while the second gives expression of a functional Myc-epitope tagged SRY protein (SRYMYC). Taking advantage of lasting hPLAP activity after transcription of the reporter gene has ceased, we could show that SryhPLAP was expressed exclusively in all cells fated to become Sertoli cells. SRYMYC-single-positive cells were first observed in the gonad and not in the CE. Subsequently, they became SRYMYC/SOX9-double-positive, but only for a few hours before turning into SOX9-single-positive cells. After the coelomic epithelial cells migrate into the gonad, there is first a decision to become interstitial or supporting cells, and then the transient expression of SRY in the latter determines their fate as Sertoli cells by up-regulating Sox9. PMID: 15385158 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Dev Dyn. 2004 Oct;231(2):387-95. Sox8 is expressed at similar levels in gonads of both sexes during the sex determining period in turtles. Takada S, DiNapoli L, Capel B, Koopman P. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. A critical gene involved in mammalian sex determination and differentiation is the Sry-related gene Sox9. In reptiles, Sox9 resembles that of mammals in both structure and expression pattern in the developing gonad, but a causal role in male sex determination has not been established. A closely related gene, Sox8, is conserved in human, mouse, and trout and is expressed in developing testes and not developing ovaries in mouse. In this study, we tested the possibility of Sox8 being important for sex determination or sex differentiation in the red-eared slider turtle Trachemys scripta, in which sex is determined by egg incubation temperature between stages 15 and 20. We cloned partial turtle Sox8 and anti-Mullerian hormone (Amh) cDNAs, and analyzed the expression patterns of these genes in developing gonads by reverse transcriptase-polymerase chain reaction and whole-mount in situ hybridization. While Amh is expressed more strongly in males than in females at stage 17, Sox8 is expressed at similar levels in males and females throughout the sex-determining period. These observations suggest that differential transcription of Sox8 is not responsible for regulation of Amh, nor responsible for sex determination in turtle. 2004 Wiley-Liss, Inc. PMID: 15366016 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Eur J Hum Genet. 2004 Sep;12(9):706-12. Mutation analysis of five candidate genes in Chinese patients with hypospadias. Wang Y, Li Q, Xu J, Liu Q, Wang W, Lin Y, Ma F, Chen T, Li S, Shen Y. Institute of Basic Medical Sciences, PUMC&CAMS, Beijing, 100005, China. Hypospadias is the displacement of the urethral meatus from the tip of the glans to the ventral side of the phallus. During fetal development, SRY, SOX9, WT1, SRD5A2 and AR are important at different stages in the differentiation and development of the male genital system. Mutations in these genes impair masculinization and may be associated with hypospadias. In order to explore these possibilities, we employed polymerase chain reaction and direct sequencing to analyze the coding regions of these five genes in 90 Chinese hypospadias patients. We found a total of 16 different mutations in SRD5A2, AR and WT1 in 24 of these 90 patients. Seven mutations are novel. No mutation was found in SRY or SOX9. SNP V89L found in SRD5A2 was statistically significant between patients and controls. Our results indicated that mutations in SRD5A2, AR and WT1 were associated with hypospadias. In conclusion, mutations are frequently found in genes that control androgen action and metabolism, but are seldom found in genes active in the early phase of sex determination and differentiation. Mutations in AR, SRD5A2 or WT1 seem to be associated not only with hypospadias but also with micropenis. PMID: 15266301 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Development. 2004 Aug;131(15):3627-36. Epub 2004 Jun 30. Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination. Schmahl J, Kim Y, Colvin JS, Ornitz DM, Capel B. Department of Cell Biology, Duke University, Durham, NC 27710, USA. Recently, we demonstrated that loss of Fgf9 results in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. We now show that Fgf9 is necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate. PMID: 15229180 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Endocrinology. 2004 Aug;145(8):3950-60. Epub 2004 May 13. The effects of estrogen on the expression of genes underlying the differentiation of somatic cells in the murine gonad. Britt KL, Stanton PG, Misso M, Simpson ER, Findlay JK. Prince Henry's Institute of Medical Research, Monash Medical Centre Clayton, Clayton, Victoria 3168, Australia. kara.britt@phimr.monash.edu.au. Estrogen (17beta-estradiol, E2)-deficient aromatase knockout (ArKO) mice develop Sertoli and Leydig cells at puberty. We hypothesized that estrogen, directly or indirectly, regulates genes responsible for somatic cell differentiation and steroidogenesis. ArKO ovaries expressed estrogen receptors alpha and beta, and LH receptor, indices of estrogen responsiveness in the ovary. Wild-type (Wt) and ArKO mice received either E2 or placebo for 3 wk, from 7-10 wk of age. E2 decreased serum FSH and LH and increased uterine weights of 10-wk-old ArKO mice. We measured mRNA expression of Sertoli cell, Sry-like HMG box protein 9 (Sox9); three upstream transcription factors, liver receptor homolog-1 (Lrh-1), steroidogenic factor 1, and dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1; and one downstream factor, Mullerian-inhibiting substance. Placebo-treated ArKO ovaries have increased Sox9 (15-fold; P < 0.001), Mullerian-inhibiting substance (2.9-fold), Lrh-1 (7.7-fold), and dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1 (12-fold) expression compared with Wt at 10 wk. Steroidogenic factor 1 was similar to Wt. Consistent with increased serum T levels and Leydig cells in their ovaries, placebo-treated ArKO ovaries had increased 17alpha-hydroxylase, 17beta-hydroxysteroid dehydrogenase type-3, and 17beta-hydroxysteroid dehydrogenase type-1 expression compared with Wt at 10 wk. E2 treatment for 3 wk improved the ovarian phenotype, decreased development of Sertoli cells, decreased the expression of Sox9, Lrh-1, and the steroidogenic enzymes in ArKO ovaries, and induced ovulation in some cases. In conclusion, the expression of the genes regulating somatic cell differentiation is directly or indirectly responsive to estrogen. PMID: 15142992 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Theor Biol. 2004 Jun 7;228(3):359-65. The elusive action of sex-determining genes: mitochondria to the rescue? Mittwoch U. Galton Laboratory, Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK. u.mittwoch@ucl.ac.uk According to the accepted dogma of mammalian sex determination, the Y-linked gene SRY initiates male development by inducing hitherto uncommitted somatic cells of the fetal gonad to develop into Sertoli cells. However, it has become evident that the correct functioning of an increasing number of genes on other chromosomes is required for testicular organogenesis. They include the SRY-related gene, SOX9, which plays important roles in both sex determination and chondrogenesis, as well as genes responsible for the production of growth factors, i.e. fibroblast growth factor 9, platelet derived growth factor A, and the members of the insulin-receptor family of genes. It is known, moreover, that differences between the sexes begin to develop long before the differentiation of Sertoli cells, including an increase in gonadal size and cell proliferation, and accelerated development of XY embryos at early pre-implantation stages. There is also evidence of transcription of Y-linked, and of X-linked, genes and of an enhanced metabolic rate in XY embryos. Furthermore, the condition of true hermaphroditism does not fit into a simple genotype/phenotype relationship. The proposal that "testis-determining" genes act by increasing metabolic rates rather than directly determining Sertoli cell differentiation can account for a number of observations that do not fit the current model, including pregonadal sex differences, the activity of the same gene in different organ systems, and the frequent co-existence of sexual and somatic abnormalities. It also sheds light on the pervasive differences between metabolic rates of mammalian males and females, while the facts of true hermaphroditism can be viewed as remnants of temperature-dependent sex determination in ectothermic vertebrates. Growing interest in mitochondria, which play a central role in the provision of energy to eukaryotic cells, makes a shift of paradigm from gonadal histology to energy metabolism timely, particularly since new techniques have become available for testing the hypothesis, and for widening the experimental approach to sex determination. If the hypothesis is correct, it would mean that male sex is determined by nuclear genes inherited from the father regulating the activity of maternally derived mitochondria. Publication Types: Review Review, Tutorial PMID: 15135034 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Reprod Dev. 2004 Apr;50(2):227-35. Effects of trichostatin a, a histone deacetylase inhibitor, on mouse gonadal development in vitro. Mizukami T, Fujisawa M, Kanai Y, Kurohmaru M, Hayashi Y. Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan. aa17134@mail.ecc.u-tokyo.ac.jp Sry, Sox9 and M33 are thought to act as architectural transcription factors or as a chromatin regulator in gonadal development. However, the direct relationship between chromatin structure and sex determination has not yet been revealed. To clarify the effect of chromatin structural change on gonadal development, we examined the effects of trichostatin A, a histone deacetylase inhibitor, on mouse gonadal development in vitro. In the 0.1 microM treated testicular explants, the size of the gonad was significantly decreased, although the testicular cord formation occurred normally. In the 1.0 microM treated explants, the gonads revealed one or two large testicular cords. Sox9 and MIS expressions suggest that Sertoli cell differentiation is induced normally within the testicular cord, while Dnmt3b expression suggests that several immature Sertoli cells are located on the outside of the testicular cord. The 3beta-hsd expression indicates that Leydig cell differentiation occurs normally. On the other hand, germ cell loss was observed in the treated testicular explants. In the treated ovarian explants, the number of premeiotic germ cells was reduced without gonadal size change. Thus, trichostatin A affects the development of germ cells, but does not affect sex determination. PMID: 15118250 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Hum Mol Genet. 2004 Jun 15;13(12):1213-8. Epub 2004 Apr 28. Long-range activation of Sox9 in Odd Sex (Ods) mice. Qin Y, Kong LK, Poirier C, Truong C, Overbeek PA, Bishop CE. Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030, USA. The Odd Sex mouse mutation arose in a transgenic line of mice carrying a tyrosinase minigene driven by the dopachrome tautomerase (Dct) promoter region. The minigene integrated 0.98 Mb upstream of Sox9 and was accompanied by a deletion of 134 kb. This mutation causes female to male sex reversal in XX Ods/+ mice, and a characteristic eye phenotype of microphthalmia with cataracts in all mice carrying the transgene. Ods causes sex reversal in the absence of Sry by upregulating Sox9 expression and maintaining a male pattern of Sox9 expression in XX Ods/+ embryonic gonads. This expression, which begins at E11.5, triggers downstream events leading to the formation of a testis. We report here that the 134 kb deletion, in itself, is insufficient to cause sex reversal. We demonstrate that in Ods, the Dct promoter is capable of acting over a distance of 1 Mb to induce inappropriate expression of Sox9 in the retinal pigmented epithelium of the eye, causing the observed microphthalmia. In addition, it induces Sox9 expression in the melanocytes where it causes pigmentation defects. We propose that Ods sex reversal is due to the Dct promoter element interacting with gonad-specific enhancer elements to produce the observed male pattern expression of Sox9 in the embryonic gonads. PMID: 15115764 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Int J Gynecol Pathol. 2004 Apr;23(2):180-1. Expression of a transcription factor, SOX9, in Sertoli-stromal cell tumors of the ovary. Kato N, Fukase M, Motoyama T. Department of Pathology, Yamagata University School of Medicine, Yamagata, Japan. Two cases of ovarian Sertoli-stromal cell tumor were examined for expression of a transcription factor, SOX9, which plays an essential role in Sertoli cell differentiation, downstream of Sry, as in the testis. By reverse transcriptase polymerase chain reaction, SOX9 mRNA was expressed in both tumors, despite the absence of Sry. These findings suggest that SOX9 expression probably plays some role in Sry-independent testicular differentiation in ovarian Sertoli-stromal cell tumors. PMID: 15084848 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Development. 2004 May;131(9):1891-901. Epub 2004 Mar 31. Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Chaboissier MC, Kobayashi A, Vidal VI, Lutzkendorf S, van de Kant HJ, Wegner M, de Rooij DG, Behringer RR, Schedl A. INSERM U470, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 2, France. Sex determination in mammals directs an initially bipotential gonad to differentiate into either a testis or an ovary. This decision is triggered by the expression of the sex-determining gene Sry, which leads to the activation of male-specific genes including the HMG-box containing gene Sox9. From transgenic studies in mice it is clear that Sox9 is sufficient to induce testis formation. However, there is no direct confirmation for an essential role for Sox9 in testis determination. The studies presented here are the first experimental proof for an essential role for Sox9 in mediating a switch from the ovarian pathway to the testicular pathway. Using conditional gene targeting, we show that homozygous deletion of Sox9 in XY gonads interferes with sex cord development and the activation of the male-specific markers Mis and P450scc, and leads to the expression of the female-specific markers Bmp2 and follistatin. Moreover, using a tissue specific knock-out approach, we show that Sox9 is involved in Sertoli cell differentiation, the activation of Mis and Sox8, and the inactivation of Sry. Finally, double knock-out analyses suggest that Sox8 reinforces Sox9 function in testis differentiation of mice. PMID: 15056615 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Nippon Rinsho. 2004 Feb;62(2):247-54. [Sex differentiation and sex chromosomes] [Article in Japanese] Shinka T, Nakahori Y. Department of Human Genetics and Public Health, Graduate School of Proteomics, Faculty of Medicine, University of Tokushima. The mechanisms for sex differentiation and the genes on the sex chromosomes are varied among different species. For human, SRY is the only testis-determining factor on the Y chromosome and triggers the cascade for male sex-determination. However, even if normal SRY exists, the haploinsufficienty of SOX9 or KTS+ splicing form of WT-1 can cause male-to-female sex reversal. Furthermore, the duplication of the partial region on the X chromosome including DAX-1 gene can also cause male-to-female sex reversal. The sex-determining system seems to be sensitive for the gene dosage or the gene expression level. Publication Types: Review Review, Tutorial PMID: 14968529 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Cytogenet Genome Res. 2003;101(3-4):242-9. Molecular characterization of three gonad cell lines. Beverdam A, Wilhelm D, Koopman P. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce SOX9 transcription upon expression of SRY, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate SOX9 expression upon transfection of SRY-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of SOX9 expression during testis determination. Copyright 2003 S. Karger AG, Basel PMID: 14684990 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Cytogenet Genome Res. 2003;101(3-4):219-23. Cell aggregation precedes the onset of Sox9-expressing preSertoli cells in the genital ridge of mouse. Moreno-Mendoza N, Harley V, Merchant-Larios H. Department of Cell Biology and Physiology, Institute of Biomedical Investigations, National University Autonomous of Mexico, Mexico City, Mexico. SOX9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that SRY, the testis determining gene, turns SOX9 on in male embryos because it is turned off in female embryos. Spatial expression of SRY follows a cranio-caudal pattern. Here, we asked if SOX9 is expressed in the same cell lineage and with a similar pattern as SRY. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of SOX9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first SOX9-positive cells appear. Although SOX9-positive cells are located among loose mesenchymal cells by stages of 8-14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first SOX9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of SOX9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that SOX9 is expressed only in loose mesenchymal cells in both sexes and that expression of SOX9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of SOX9 and SRY pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges. Copyright 2003 S. Karger AG, Basel PMID: 14684986 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Cytogenet Genome Res. 2003;101(3-4):199-205. Expression studies of the PIS-regulated genes suggest different mechanisms of sex determination within mammals. Pannetier M, Servel N, Cocquet J, Besnard N, Cotinot C, Pailhoux E. Biologie du Developpement et Reproduction, INRA Bat J. Poly, Jouy-en-Josas, France. In mammals, the Y-located SRY gene is known to induce testis formation from the indifferent gonad. A related gene, SOX9, also plays a critical role in testis differentiation in mammals, in birds and reptiles. It is now assumed that SRY acts upstream of SOX9 in the sex determination cascade, but the regulatory link which should exist between these two genes remains unknown. Studies on XX sex reversal in polled goats (PIS mutation: Polled Intersex Syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, which share a common transcriptional regulatory region, PIS. In this review, we present the expression pattern of these PIS-regulated genes in mice. The FOXL2 expression profile of mice is similar to that described in goats in accordance with a conserved role of this ovarian differentiating gene in mammals. On the contrary, the PISRT1 expression profile is different between mice and goats, suggesting different mechanisms of the primary switch in the testis determination process within mammals. A model based on two different modes of SOX9 regulation in mice and other mammals is proposed in order to integrate our results into the current scheme of gonad differentiation. Copyright 2003 S. Karger AG, Basel Publication Types: Review Review, Tutorial PMID: 14684983 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Cytogenet Genome Res. 2003;101(3-4):185-98. Turning on the male--SRY, SOX9 and sex determination in mammals. Knower KC, Kelly S, Harley VR. Human Molecular Genetics Laboratory, Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia. The decision of the bi-potential gonad to develop into either a testis or ovary is determined by the presence or absence of the Sex-determining Region gene on the Y chromosome (SRY). Since its discovery, almost 13 years ago, the molecular role that SRY plays in initiating the male sexual development cascade has proven difficult to ascertain. While biochemical studies of clinical mutants and mouse genetic models have helped in our understanding of SRY function, no direct downstream targets of SRY have yet been identified. There are, however, a number of other genes of equal importance in determining sexual phenotype, expressed before and after expression of SRY. Of these, one has proven of central importance to mammals and vertebrates, SOX9. This review describes our current knowledge of SRY and SOX9 structure and function in the light of recent key developments. Copyright 2003 S. Karger AG, Basel Publication Types: Review Review, Tutorial PMID: 14684982 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Nature. 2003 Nov 20;426(6964):291-5. Comment in: Nature. 2003 Nov 20;426(6964):241. Testis determination requires insulin receptor family function in mice. Nef S, Verma-Kurvari S, Merenmies J, Vassalli JD, Efstratiadis A, Accili D, Parada LF. Center for Developmental Biology, University of Texas, Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-9133, USA. In mice, gonads are formed shortly before embryonic day 10.5 by the thickening of the mesonephros and consist of somatic cells and migratory primordial germ cells. The male sex-determining process is set in motion by the sex-determining region of the Y chromosome (Sry), which triggers differentiation of the Sertoli cell lineage. In turn, Sertoli cells function as organizing centres and direct differentiation of the testis. In the absence of Sry expression, neither XX nor XY gonads develop testes, and alterations in Sry expression are often associated with abnormal sexual differentiation. The molecular signalling mechanisms by which Sry specifies the male pathway and models the undifferentiated gonad are unknown. Here we show that the insulin receptor tyrosine kinase family, comprising Ir, Igf1r and Irr, is required for the appearance of male gonads and thus for male sexual differentiation. XY mice that are mutant for all three receptors develop ovaries and show a completely female phenotype. Reduced expression of both Sry and the early testis-specific marker Sox9 indicates that the insulin signalling pathway is required for male sex determination. PMID: 14628051 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Biol Reprod. 2004 Jan;70(1):114-22. Epub 2003 Sep 17. Disturbed expression of Sox9 in pre-sertoli cells underlies sex-reversal in mice b6.Ytir. Moreno-Mendoza N, Torres-Maldonado L, Chimal-Monroy J, Harley V, Merchant-Larios H. Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, UNAM. Mexico, D.F. Mexico 04510. Sry in some varieties of Mus musculus domesticus fails to form normal testis when introduced into the C57BL/6J (B6) strain. We studied the developmental pattern of pre-Sertoli cells that express Sox9 by immunofluorescence and the profile levels of Sox9 transcripts by semiquantitative reverse transcriptase polymerase chain reaction and in situ hybridization in developing gonads of B6-Ytir mice. Sox9-positive cells (pre-Sertoli cells) appeared in all B6.Ytir genital ridges at 11.5 and 12.5 days postcoitum (dpc). However, at 13.5 dpc, Sox9-positive cells were not detected only in 50% of the B6.Ytir gonads compared with 100% of B6 gonads. Although pre-Sertoli cells formed the seminiferous cords after 14.5 dpc in the medial region of the B6.Ytir gonad, the cranial and caudal regions formed ovarian tissue. Further, B6.Ytir ovaries have lower levels of Sox9 than ovotestes at all fetal stages. These results suggest that although the pre-Sertoli cell lineage forms in B6.Ytir genital ridges, its further differentiation into Sertoli cells is apparently prevented. The cause may be the low levels of Sox9 and down-regulation of its product. Results suggest that inhibitory signals of Sox9 acting along the whole genital ridge or only at its cranial and/or caudal regions underlie formation of B6.Ytir ovaries or ovotestes, respectively. Furthermore, our results suggest that infertility of B6.Ytir females may be due to the abnormal presence of Sox9 transcripts in their ovaries. PMID: 13679320 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Biol Reprod. 2004 Jan;70(1):160-7. Epub 2003 Sep 17. Marsupial anti-Mullerian hormone gene structure, regulatory elements, and expression. Pask AJ, Whitworth DJ, Mao CA, Wei KJ, Sankovic N, Graves JA, Shaw G, Renfree MB, Behringer RR. Department of Zoology, University of Melbourne, Victoria 3010, Australia. During male sexual development in reptiles, birds, and mammals, anti-Mullerian hormone (AMH) induces the regression of the Mullerian ducts that normally form the primordia of the female reproductive tract. Whereas Mullerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Mullerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms. PMID: 13679313 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: J Med Invest. 2003 Aug;50(3-4):192-8. Roles of estrogen receptor alpha (ER alpha) in the regulation of the human Mullerian inhibitory substance (MIS) promoter. Chen G, Shinka T, Kinoshita K, Yan HT, Iwamoto T, Nakahori Y. Department of Human Genetics and Public Health, Graduate School of Proteomics, Faculty of Medicine, The University of Tokushima, Tokushima, Japan. Sex differentiation consists of multi-step pathway that involves expression of many different genes. Mullerian duct inhibitory substance (MIS) has a key role for regression of the Mullerian duct during male sex differentiation. Recently, endocrine disruptors (EDs), which often have estrogen-like activities, have caused concern over worldwide. It has been reported that estrogen regulates the MIS expression. Therefore, we tested whether ER alpha and ER beta influence the MIS promoter activity in the NT2/D1 cell line which expresses many sex differentiation-related genes such as SRY, SOX9, and DAX-1. RT-PCR analysis revealed that the NT2/D1 cells express both ER alpha and ER beta in addition to MIS. Under the low concentration of 17beta-estradiol (E2), the over-expression of exogenous ER alpha increased the MIS promoter activity 3.3-fold compared with the control. However, as E2 concentration was increased, the MIS promoter activity was decreased. For ER beta, we could not observe alterations of the MIS promoter activity. Furthermore, the over-expression of the exogenous SF-1 inhibited the activation of the MIS promoter with ER alpha. Although it remains unclear whether the effects of ER alpha on the MIS promoter are mediated through the genomic or the no-genomic actions, the present results suggest that ER alpha up-regulates the MIS promoter activity in the NT2/D1 cells under low concentrations of E2, and that the two ERs may work in different manners for the MIS promoter activation. The present findings may be useful to understand the molecular mechanisms by which EDs or estrogens affect the MIS expression. PMID: 13678390 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Poult Sci. 2003 Sep;82(9):1462-7. Profiles of mRNA expression of genes related to sex differentiation of the gonads in the chicken embryo. Yamamoto I, Tsukada A, Saito N, Shimada K. Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan. Sex is determined genetically in birds. The homogametic sex is male (ZZ), whereas the heterogametic sex is female (ZW). According to the genetic sex, gonads develop into testes or ovary. In this study, we performed experiments to reveal mRNA expression patterns in the gonad between d 5.5 and 8.5 of incubation and examined a possible role of Dss-Ahc critical region on the X chromosome 1 (Dax1), Steroidogenic factor 1 (Sf1), P450aromatase (P450arom), Estrogen receptor alpha (ER alpha), doublesex and mab3 related transcription factor 1 (Dmrt1), Sry-related HMG box gene 9 (Sox9), Gata binding protein 4 (Gata4), and anti-mullerian hormone (Amh) in sex differentiation in chicken embryonic gonads using RNase protection assay. In embryonic chicken gonads, Dax1 mRNA was expressed in both sexes but was higher in females than in males at d 6.5 and 7.5 of incubation. The Sf1 mRNA was expressed in both sexes, but it was expressed more in males at d 5.5 than in females but more in females than in males at d 7.5 and 8.5 of incubation. The P450arom mRNA was expressed only in female gonads from d 5.5 of incubation. The ER alpha mRNA was expressed in both sexes, but it did not show a sex difference. On the other hand, the Dmrt1 mRNA was expressed in both sexes, but it showed a male-specific expression pattern. The male-specific expression pattern was observed in Sox9 mRNA, but it was not expressed in female gonads. The Gata4 mRNA was expressed in both sexes, and sex differences were not revealed throughout the observational period. Amh mRNA was expressed in both sexes, but it had male-specific mRNA expression pattern at d 6.5 to 8.5 of incubation. These results indicate that Dax1, Sf1, and P450arom have possible roles in ovary formation, whereas Dmrt1, Sox9, and Amh are related to testis formation in differentiating chicken gonads at d 5.5 to 8.5 of incubation. PMID: 12967261 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Endocr Rev. 2003 Aug;24(4):466-87. The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Harley VR, Clarkson MJ, Argentaro A. Prince Henry's Institute of Medical Research, Clayton 3168, Victoria, Australia. Vincent.harley@med.monash.edu.au Despite 12 yr since the discovery of SRY, little is known at the molecular level about how SRY and the SRY-related protein, SOX9 [SRY-related high-mobility group (HMG) box 9], initiate the program of gene expression required to commit the bipotential embryonic gonad to develop into a testis rather than an ovary. Analysis of SRY and SOX9 clinical mutant proteins and XX mice transgenic for testis-determining genes have provided some insight into their normal functions. SRY and SOX9 contain an HMG domain, a DNA-binding motif. The HMG domain plays a central role, being highly conserved between species and the site of nearly all missense mutations causing XY gonadal dysgenesis. SRY and SOX9 are architectural transcription factors; their HMG domain is capable of directing nuclear import and DNA bending. Whether SRY and SOX9 activate testis-forming genes, repress ovary-forming genes, or both remains speculative until downstream DNA target genes are identified. However, factors that control SRY and SOX9 gene expression have been identified, as have a dozen sex-determining genes, allowing some of the pieces in this molecular genetic puzzle to be connected. Many genes, however, remain unidentified, because in the majority of cases of XY females and in all cases of XX males lacking SRY, the mutated gene is unknown. Publication Types: Review PMID: 12920151 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Cytogenet Genome Res. 2002;99(1-4):236-44. Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation. Mizuno S, Kunita R, Nakabayashi O, Kuroda Y, Arai N, Harata M, Ogawa A, Itoh Y, Teranishi M, Hori T. Department of Agricultural and Biological Chemistry, College of Bioresource Sciences, Nihon University, Fujisawa, Japan. s-mizuno@brs.nihon-u.ac.jp Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination. Copyright 2002 S. Karger AG, Basel Publication Types: Review Review, Tutorial PMID: 12900570 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Mol Reprod Dev. 2003 Aug;65(4):373-81. Sry and Sox9 expression during canine gonadal sex determination assayed by quantitative reverse transcription-polymerase chain reaction. Meyers-Wallen VN. James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA. Testis induction is associated with gonadal Sry and Sox9 expression in mammals, and with Sox9 expression in vertebrates where Sry is absent. In mammals, Sry might initiate testis induction by upregulating Sox9 expression; however, direct evidence supporting this hypothesis is lacking. Models of Sry-negative XX sex reversal (XXSR), in which testes develop in the absence of Sry, could provide the link between Sry and Sox9 in testis induction. To define the stages at which testis determination occurs in the canine model, Sry and Sox9 expression were measured in normal urogenital ridges (UGR) and gonads by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Testicular Sry expression rose continuously during canine developmental ages comparable to human carnegie stages (CS) 16-18, with maximal expression at CS 18. Sox9 was expressed in both male and female canine UGR up to CS 17, at which time testis expression became tenfold greater than in the ovary. Although Sox9 was detected by qRT-PCR in ovaries and mesonephroi of both sexes, expression was detected only in canine testes by whole mount in situ hybridization (WMISH). The timing of Sry and Sox9 expression is consistent with a role in testis determination: Sry expression begins at CS 16 in testes, followed by upregulation of Sox9 expression at CS 17. The quantity and temporal and spatial patterns of Sry and Sox9 expression in normal canine gonads are similar to those in humans, sheep, and pigs. These studies should provide the basis for understanding the mechanism of testis induction in the canine model of Sry-negative XXSR. Copyright 2003 Wiley-Liss, Inc. PMID: 12840810 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Hum Mol Genet. 2003 Jul 15;12(14):1755-65. Dimerization of SOX9 is required for chondrogenesis, but not for sex determination. Bernard P, Tang P, Liu S, Dewing P, Harley VR, Vilain E. UCLA Department of Human Genetics, Gonda Center, Room 6357, 695 Charles Young Drive South, Los Angeles, CA 90095-7088, USA. The SRY-related SOX9 gene is involved in both chondrogenesis and the early steps of mammalian sex determination. Mutations in the human SOX9 gene cause campomelic dysplasia, a severe skeletal malformation syndrome associated with male-to-female sex reversal in most, but not all, XY individuals. Here we show that SOX9 contains a dimerization domain, and binds co-operatively as a dimer in the presence of the DNA enhancer element in genes involved in chondrocyte differentiation, such as Col11a2 and Col9a2, but binds as a monomer to the regulatory region of the sex-determining gene SF1. Frameshift SOX9 mutations truncate its two activation domains, while all missense mutations reported to date lie in the high mobility group (HMG) DNA-binding domain. We identify a missense mutation (A76E), the first outside the HMG domain, in an XY patient presenting with campomelic dysplasia but without sex reversal. This mutation disrupts the dimerization capability of SOX9, interfering with both the DNA binding and consequent transactivation of both the Col11a2 and Col9a2 enhancers. Consistent with the patient's phenotype, the A76E mutation does not affect DNA binding and activation of the SF1 enhancer. DNA-dependent cooperative dimerization could represent a novel mechanism to achieve tissue-specific regulation of gene expression by a SOX transcription factor. These results establish that SOX9 cooperative dimerization is required for chondrogenesis but not for sex determination and may explain why campomelic dysplasia need not be associated with XY sex reversal. PMID: 12837698 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: J Endocrinol Invest. 2003;26(3 Suppl):23-8. Sexual differentiation. Sinisi AA, Pasquali D, Notaro A, Bellastella A. Endocrinology and Medical Andrology Section, Department of Clinical and Experimental Medicine and Surgery, Second University of Naples, Naples, Italy. antonio.sinisi@unina2.it In humans, like as in other mammals, the gonads, the internal genital ducts, and the external genital structures all develop from bipotential embryologic tissues. Male or female phenotype develops through a cascade of processes which initiate with sex determination and follow with sex differentiation. The karyotype (46, XY or 46, XX) of the embryo (genetic sex) determines whether primordial gonad differentiates into a testis or an ovary, respectively (gonadal differentiation). A Y-related gene, SRY, acts as a switch signal for testis differentiation. Testis development process involves several steps controlled by other non-OY-linked genes, such as Wilms tumor gene 1 (WT1), EMX2, LIM1, steroidogenic factor 1(SF-1), SRY box-related gene 9 (SOX9). Since other genes, such as Wnt-4 and DAX-1, are necessary for the initiation of female pathway in sex determination, female development cannot be considered a default process. Hormonal production of differentiated gonads is relevant for differentiation of the internal and external genitalia during fetal life, and for the development of secondary sex characteristics at puberty. Antimullerian hormone (AMH) secreted by Sertoli cells inhibits the development of female internal genitalia (tube, uterus, upper part of vagina); testosterone secreted by Leydig cells induces stabilization of wolffian ducts and development of internal male genitalia. Differentiation of external male genitalia requires the transformation of testosterone to dihydrotestosterone by 5alpha reductase type 2 expressed in genital skin and urogenital sinus. The effects of androgens occur in presence of functional androgen receptor (AR) protein. Mutations of genes coding for steroidogenic enzymes, AMH, AMH receptor, AR and 5alpha reductase are all associated with impairment of sex differentiation and result in genital ambiguity. Publication Types: Review Review, Tutorial PMID: 12834017 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: Trans Am Clin Climatol Assoc. 2003;114:51-63; discussion 64-5. Battle of the sexes: new insights into genetic pathways of gonadal development. Jameson JL, Achermann JC, Ozisik G, Meeks JJ. Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Sex determination is governed by a series of genetic switches that influence cell fate and differentiation during critical periods of gonadal development. Remarkably, the primordial fetal gonad is bipotential. Therefore, gonadal development provides an excellent opportunity to identify genes involved in differential organogenesis. The identification of the testis-determining gene, SRY (Sex-reversed on the Y), was a pivotal first step towards unraveling this genetic pathway. It is now clear that numerous other genes, in addition to SRY, are necessary for normal testis development. For example, human mutations in a variety of genes (SOX9, WT1, SF1) impair testis development. Murine models provide evidence for additional genes (Lhx9, Emx2, M33, Dmrt, Fgf9). This lecture will highlight insights gleaned from human mutations in the nuclear receptors, SF1 (Steroidogenic Factor1) (NR5A1) and DAX1 (Dosage-sensitive sex reversal, Adrenal hypoplasia congenita, X chromosome) (NR0B1). These studies reveal the exquisite sensitivity of SF1-dependent developmental pathways to gene dosage and function in humans. Publication Types: Review Review, Tutorial PMID: 12813911 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: Mol Endocrinol. 2003 Jul;17(7):1332-43. Epub 2003 Apr 3. Induction of the Sry-related factor SOX6 contributes to bone morphogenetic protein-2-induced chondroblastic differentiation of C3H10T1/2 cells. Fernandez-Lloris R, Vinals F, Lopez-Rovira T, Harley V, Bartrons R, Rosa JL, Ventura F. Departament de Ciencies Fisiologiques II, Campus de Bellvitge, Universitat de Barcelona, 08907 L' Hospitalet de Llobregat, Spain. Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis. PMID: 12677004 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Biol Reprod. 2003 Apr;68(4):1098-106. Epub 2002 Oct 31. Porcine SRY promoter is a target for steroidogenic factor 1. Pilon N, Daneau I, Paradis V, Hamel F, Lussier JG, Viger RS, Silversides DW. Centre de recherche en reproduction animale, Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada J2S 7C6. To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated a 4.5-kilobase (kb) pig SRY 5' flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at -661 base pairs (bps) 5' from the translation initiation site. Deletion studies of the SRY 5' flanking sequences in PGR 9E11 cells demonstrated that -1.4 kb of 5' flanking sequences retained full transcriptional activity compared with the -4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, because deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the -1.4 kb fragment identified two potential binding sites for SF-1, at -1369 and at -290 from the ATG. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse SF-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at -1369 bp and at -290 bp, and that the site at -1369 bp is quantitatively the most important. PMID: 12606467 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: Cytogenet Genome Res. 2002;96(1-4):161-8. Sex chromosomes and sex determination in weird mammals. Marshall Graves JA. Research School of Biological Science, Australian National University, Canberra, Australia. graves@rsbs.anu.edu.au Weird mammals are of two types. Highly divergent mammals, such as the marsupials and monotremes, have informed us of the evolutionary history of the Y chromosome and sex-determining gene, and the recently specialized rodents can help us predict its future. The Y chromosome has had a short but eventful history, and is already heading briskly for oblivion. It originated as a homologous partner of the X when it acquired a sex-determining gene (not necessarily SRY). Most of the genes on the Y, even those with a male-specific function, evolved from genes now on the X. At the mercy of a high rate of variability and the forces of drift and selection, the Y has lost genes at a rate of 3-6 genes/million years, sparing those that acquired critical male-specific functions. Even these genes have disappeared from one mammalian lineage or another as their functions were usurped by genes elsewhere in the genome. The mammalian testis-determining gene, SRY, is a typical Y-borne gene. It arose by truncation of a gene (SOX3) on the X that is expressed in brain development, and it may work by interacting with (inhibiting?) related genes, including SOX9. Variant sex-determining systems in rodents show that the action of SRY can change, as it evidently has in the mouse, and SRY can be inactivated, as in akodont rodents, or even completely superseded, as in mole voles. Copyright 2002 S. Karger AG, Basel PMID: 12438793 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Semin Reprod Med. 2002 Aug;20(3):189-98. Disorders of gonadal development. Dewing P, Bernard P, Vilain E. Department of Human Genetics, UCLA School of Medicine, 695 Charles E. Young Drive South, Los Angeles, CA 90095, USA. The molecular mechanisms of gonadal development are a complex process, which involves the tightly regulated differentiation of a bipotential embryonic gonad into either testes or ovary. Once this has occurred, the phenotypic and gonadal sex of an individual has been genetically determined. This process, however, may not always be so straightforward. By studying individuals who are sex reversed or who have ambiguous genitalia, the discovery of the handful of sex-determining genes that we know of today was made possible. It was not until recently that the transcription factors SRY, DAX1, SOX9, SF-1, and WT1 were recognized to be involved in gonadal development. Dissecting the molecular pathway of mammalian sex determination will be crucial in understanding the development of the gonads and the pathophysiology of human disorders of sex determination. Publication Types: Review Review, Tutorial PMID: 12428199 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Semin Reprod Med. 2002 Aug;20(3):157-68. Molecular genetics of sex determination. Cotinot C, Pailhoux E, Jaubert F, Fellous M. Unite de Biologie du Developpement et Biotechnologies, INRA, Jouy en Josas, France. In humans, the choice between male or female development is genetically determined. Sex determination take place when the bipotential embryonic gonad becomes either testis or ovary. This process is directed by genes that have been discovered by genetic analysis of sex-reversed patients and confirmed by knockout experiments in mice. The testis-determining pathway is better known than the ovary pathway. SRY, a gene located on the Y chromosome, triggers a complex genetic cascade leading to testicular differentiation. In this cascade, two genes play a crucial role in male differentiation, SOX9 and FGF9, which contribute to testicular cord formation. However, only a minority of 46,XY sex-reversed patients can be explained by mutations in known genes such as SRY, SOX9, WTI, and SF1, suggesting that other genes influencing sex determination are yet to be discovered. In females, some rare genes that induce ovarian failure or female-to-male sex reversal have been found through gene-targeted inactivation in mice or positional cloning of mutations in humans and goats. In both sexes, genetic analysis of sex-reversed individuals (XX males, XX and XY hermaphrodites, and XY with complete or partial dysgenesis) remains an approach of choice to isolate new genes involved in sex determination. Publication Types: Review Review, Tutorial PMID: 12428196 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Gen Comp Endocrinol. 2002 Oct 15;129(1):20-6. Expression profiles of Dax1, Dmrt1, and Sox9 during temperature sex determination in gonads of the sea turtle Lepidochelys olivacea. Torres Maldonado LC, Landa Piedra A, Moreno Mendoza N, Marmolejo Valencia A, Meza Martinez A, Merchant Larios H. Department of Cell Biology and Phisiology, Instituto de Investigaciones Biomedicas, Ciudad Universitaria, UNAM Apartado Postal 70228, C.P. 04510, Mexico, DF, Mexico. Sex determination is controlled either by genetic or environmental factors. In mammals Sry initiates determination but no homologue of this gene exists in non-mammalian species. Other genes of the mammalian sex-determining pathway have been identified in gonads of different vertebrates. Sox9, Dax1, and Dmrt1 are expressed at the onset of gonadal development in birds and reptiles. In the sea turtle Lepidochelys olivacea, a species with temperature sex determination (TSD), Sox9 is expressed in undifferentiated gonads at male- (MPT) or female-promoting temperatures (FPT). At MPT, Sox9 remains expressed in male gonads, but at FPT it is downregulated coinciding with the onset of the ovarian morphologic differentiation and female sex determination. At MPT however, male sex is determined early than at FPT in still undifferentiated gonads suggesting that other genes maintain Sox9 expression in testis. Here we used RT-PCR to study the expression profiles of Dax1, Dmrt1, and Sox9 in gonads of embryos of L. olivacea incubated at MPT or at FPT. The profiles were correlated with sex determination during and after the temperature-sensitive period (TSP). Dax1 maintained similar levels at both temperatures during the TSP. The Dax1 expression level increased significantly in ovaries compared to testes at stage 27, once they were morphologically distinct. The expression levels of Dmrt1 were higher at MPT than at FPT at all stages, in contrast with Sox9 levels which were similar at both temperatures at stages 23-25. Together, current results suggest that, whereas Dax1 is not involved in TSD in L. olivacea, upregulation of Dmrt1 and downregulation of Sox9 may play a role in male and female sex determination, respectively. PMID: 12409092 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: J Biol Chem. 2003 Jan 3;278(1):117-23. Epub 2002 Oct 23. Regulation of human COL9A1 gene expression. Activation of the proximal promoter region by SOX9. Zhang P, Jimenez SA, Stokes DG. Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA. The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter activity. In transient-transfection analyses with promoter deletion constructs, it was found that full promoter activity in RCS cells depended on the region from -560 bp to +130 bp relative to the transcriptional start site (+1). Sequence analysis of the region from -890 bp to the transcriptional start predicted five putative SOX/Sry-binding sites. Mutation analysis revealed that two of three putative SOX/Sry binding sites within the -560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection experiments with a SOX9 expression plasmid revealed that a construct containing the five putative SOX/Sry-binding sites was transactivated 20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within the -560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from -560 to -357 bp, which does not contain any other conspicuous SOX9 sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a specific complex with one of the SOX/Sry-binding sites with in the -560 to +130 region. PMID: 12399468 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Reproduction. 2002 Oct;124(4):447-57. Vertebrate sex determination: many means to an end. Morrish BC, Sinclair AH. Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Rd, Melbourne, Victoria 3052, Australia. The differentiation of a testis or ovary from a bipotential gonadal primordium is a developmental process common to mammals, birds and reptiles. Since the discovery of SRY, the Y-linked testis-determining gene in mammals, extensive efforts have failed to find its orthologue in other vertebrates, indicating evolutionary plasticity in the switch that triggers sex determination. Several other genes are known to be important for sex determination in mammals, such as SOX9, AMH, WT1, SF1, DAX1 and DMRT1. Analyses of these genes in humans with gonadal dysgenesis, mouse models and using in vitro cell culture assays have revealed that sex determination results from a complex interplay between the genes in this network. All of these genes are conserved in other vertebrates, such as chickens and alligators, and show gonad-specific expression in these species during the period of sex determination. Intriguingly, the sequence, sex specificity and timing of expression of some of these genes during sex determination differ among species. This finding indicates that the interplay between genes in the regulatory network leading to gonad development differs between vertebrates. However, despite this, the development of a testis or ovary from a bipotential gonad is remarkably similar across vertebrates. Publication Types: Review Review, Tutorial PMID: 12361462 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Development. 2002 Oct;129(19):4627-34. Gonadal differentiation, sex determination and normal Sry expression in mice require direct interaction between transcription partners GATA4 and FOG2. Tevosian SG, Albrecht KH, Crispino JD, Fujiwara Y, Eicher EM, Orkin SH. Division of Hematology and Oncology, Children's Hospital, Harvard Medical School, Boston, MA, USA. In mammals, Sry expression in the bipotential, undifferentiated gonad directs the support cell precursors to differentiate as Sertoli cells, thus initiating the testis differentiation pathway. In the absence of Sry, or if Sry is expressed at insufficient levels, the support cell precursors differentiate as granulosa cells, thus initiating the ovarian pathway. The molecular mechanisms upstream and downstream of Sry are not well understood. We demonstrate that the transcription factor GATA4 and its co-factor FOG2 are required for gonadal differentiation. Mouse fetuses homozygous for a null allele of Fog2 or homozygous for a targeted mutation in Gata4 (Gata4(ki)) that abrogates the interaction of GATA4 with FOG co-factors exhibit abnormalities in gonadogenesis. We found that Sry transcript levels were significantly reduced in XY Fog2(-/-) gonads at E11.5, which is the time when Sry expression normally reaches its peak. In addition, three genes crucial for normal Sertoli cell function (Sox9, Mis and Dhh) and three Leydig cell steroid biosynthetic enzymes (p450scc, 3betaHSD and p450c17) were not expressed in XY Fog2(-/-) and Gata(ki/ki) gonads, whereas Wnt4, a gene required for normal ovarian development, was expressed ectopically. By contrast, Wt1 and Sf1, which are expressed prior to Sry and necessary for gonad development in both sexes, were expressed normally in both types of mutant XY gonads. These results indicate that GATA4 and FOG2 and their physical interaction are required for normal gonadal development. PMID: 12223418 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: Mol Reprod Dev. 2002 Sep;63(1):5-16. Sox9 in a teleost fish, medaka (Oryzias latipes): evidence for diversified function of Sox9 in gonad differentiation. Yokoi H, Kobayashi T, Tanaka M, Nagahama Y, Wakamatsu Y, Takeda H, Araki K, Morohashi K, Ozato K. Division of Biological Science, Graduate School of Science, and Laboratory of Freshwater Fish Stocks, Bioscience Center, Nagoya University, Nagoya, Japan. hayokoi@lab.nig.ac.jp Sox9 is a transcription factor containing the Sry-related high-mobility-group (HMG) box. Mutations in human SOX9 gene cause skeletal defects and male-to-female sex reversal, indicating its essential roles in chondrogenesis and testis development. Comparative studies have shown that Sox9 is expressed in chondrogenic tissues and testis in other vertebrates. Therefore, it was suggested that roles of Sox9 in cartilage and male gonad development are conserved among vertebrates. To investigate the evolutional significance of Sox9 in the gonad and cartilage development of teleost fish, we isolated medaka sox9 and analyzed its expression. Two kinds of transcripts (sox9 and sox9lf) were isolated by cDNA library screening. The sox9 encoded 487 amino acids and showed approximately 70% amino acid identity with known vertebrate SOX9 proteins. The sox9lf was a longer form of the sox9, which was transcribed from an additional exon in the 5' upstream region. Interestingly, the expression of medaka sox9 was predominantly observed in the adult ovary by northern blot and in situ hybridization analyses, whereas in the testis, its expression was detectable only by RT-PCR. During medaka embryogenesis, its expression was observed in the cranial cartilage and pectoral fin endoskeleton. These observations suggest that the function of Sox9 in the cartilage is conserved among vertebrates, while that in the gonad is quite different in medaka. Copyright 2002 Wiley-Liss, Inc. PMID: 12211055 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: Genesis. 2002 Aug;33(4):170-80. The porcine SRY promoter is transactivated within a male genital ridge environment. Daneau I, Pilon N, Boyer A, Behdjani R, Overbeek PA, Viger R, Lussier J, Silversides DW. Faculty of Veterinary Medicine, University of Montreal, Saint Hyacinthe, Quebec, Canada. In mammals the SRY gene functions as a dominant genetic switch for testis determination (Gubbay et al.: Nature 346:1128-1135, 1990; Koopman et al.: Nature 351:117-121, 1991; Sinclair et al.: Nature 346:240-244, 1990). To study SRY transcriptional regulation within an evolutionary context, we have generated transgenic mice that express green fluorescent protein (GFP) under the control of 4.5 kb of pig SRY 5' flanking sequences (pSRYp-GFP). Autofluorescence was observed in the genital ridges of e11.5 male embryos (18-21 tail somites), and by e12.5 (27 tail somites) autofluorescence was observed within the testes cords. The expression of the transgene did not display the abrupt termination characteristic of endogenous mouse SRY, but rather showed a gradual reduction in expression characteristic of human, pig and sheep SRY. Surprisingly, no autofluorescence was observed in normal XX genital ridges, although more sensitive RT-PCR analysis detected transgene transcription. When the transgene was bred into a constitutively male line of mice (Odsex; Bishop et al.: Nat Genet 26:490-494, 2000), autofluorescence was visible in genital ridges of XX animals, in the genetic absence of Sry protein. Via RT-PCR analysis, purified autofluorescent cells from e12.5 gonadal ridges expressed mouse SRY but not Oct4 transcripts, whereas autofluorescent cells from e14.5 gonadal ridges expressed MIS but not Oct4 transcripts, in each case consistent with a pre-Sertoli cell phenotype. In vitro expression studies performed in CV-1 cells demonstrated that pig SOX9 cDNA transactivated the pig SRY promoter but that pig SRY cDNA did not. When a SOX9 potential binding site identified at -205 of the pig SRY 5' flanking sequences was mutated, the SOX9 transactivation effect was reduced by 70%. This site is conserved in the 5' flanking sequences of bovine and human SRY genes but not in the mouse gene. Gel retardation assays using this binding site showed specific binding to SOX9-enriched nuclear extracts that was competed by excess unlabelled binding site but not by mutated binding site. We suggest that pig SRY gene is responsive to a testicular environment and propose a model of feedback amplification of pig SRY transcription by SOX9. Copyright 2002 Wiley-Liss, Inc. PMID: 12203914 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11199-204. Epub 2002 Aug 8. A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination. Gasca S, Canizares J, De Santa Barbara P, Mejean C, Poulat F, Berta P, Boizet-Bonhoure B. Human Molecular Genetics Group, Institut de Genetique Humaine, Centre National de la Recherche Scientifique Unite Propre de Recherche 1142, 34396 Montpellier Cedex 5, France. In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Mullerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Mullerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation. PMID: 12169669 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: J Cell Biochem. 2002;86(2):277-89. Sox9 transactivation and testicular expression of a novel human gene, KIAA0800. Zhao LJ, Zhang S, Chinnadurai G. Institute for Molecular Virology, St. Louis University Health Sciences Center, 3681 Park Avenue, Missouri 63110, USA. zhaol@slu.edu The Sry and Sox9 sex-determination factors initiate and promote testis differentiation by gene transactivation through similar promoter elements. However, knowledge is limited concerning what genes are regulated by Sry/Sox9 in the testis. Identification and characterization of Sry/Sox9-regulated genes are critical for understanding sexual differentiation. We now demonstrate that a novel human gene, KIAA0800, is preferentially expressed in the testis and is transactivated by Sox9. The KIAA0800 promoter is repressed by an upstream element involving a polyT track and two Alu repeats. Two specific Sox9-bindings sites have been identified in the KIAA0800 promoter by using DNaseI footprinting assays and gel electrophoretic mobility shift assays. Sox9 transactivation of the KIAA0800 promoter appears to be exerted mainly through the relief of promoter repression. Genes homologous to the human KIAA0800 exist in organisms with differentiated sex tissues including mouse, Drosophila, and C. elegans, but not in unicellular organisms, including yeast and bacteria. Further, our recent sequence analysis shows that KIAA0800 protein is 97% identical between human and mouse. Thus, KIAA0800 is a novel Sox9-activated gene that is evolutionarily conserved and potentially involved in sexual differentiation. Copyright 2002 Wiley-Liss, Inc. PMID: 12111997 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Novartis Found Symp. 2002;244:57-66; discussion 66-7, 79-85, 253-7. The molecular action of testis-determining factors SRY and SOX9. Harley VR. Prince Henry's Institute of Medical Research, Monash Medical Centre, Melbourne, VIC, Australia. Despite 10 years of work since the discovery of SRY, little is known about its biochemical function. The HMG domain, a DNA-binding and DNA-bending motif, plays a central role, being the only region conserved between species and the site of almost all clinical mutations causing XY gonadal dysgenesis. By contrast, SOX9 harbours a number of highly conserved regions, including two domains required for maximal transactivation. The beat shock protein HSP70 recognizes a specific region of SOX9 hitherto of unknown function which may facilitate the assembly of multi-protein complexes at promoter enhancer regions. The SRY and SOX9 HMG domains carry two nuclear localization signals (NLSs), one at each end which function independently and by distinct mechanisms. The N-terminal NLS is bound by calmodulin while the C-terminal NLS is bound by importin beta. Four XY gonadal dysgenesis patients with mutations in SRY NLS regions showed reduced nuclear import accompanied in some cases by reduced importin beta recognition. A campomelic dysplasia patient with SOX9 mutation outside the NLS regions also showed defective SOX9 nuclear import implying that nuclear import defects could be a common explanation for SRY and SOX9 HMG domain mutations. Publication Types: Review Review, Tutorial PMID: 11990798 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: Novartis Found Symp. 2002;244:43-53; discussion 53-6, 79-85, 253-7. Anomalies of human sexual development: clinical aspects and genetic analysis. Vilain E. UCLA Department of Human Genetics, CA, USA. Disorders of human sex determination result in malformations of the external and internal genitalia. These malformations may vary from sexual ambiguity to complete sex reversal (XY female, XX male). Most of the knowledge of the molecular mechanisms involved in the mammalian sex determination pathway has been derived from the genetic analysis of intersex patients. Clinical management of these conditions critically depends on a precise understanding of their pathophysiology. Until recently, only transcription factors such as SRY, SOX9, DAX1, WT1 and SF1 were known to be responsible for abnormal gonadal development and sexual ambiguity. Gonadal dysgenesis may be isolated, as in the case of SRY mutations, or associated with abnormal development of other organs, such as bone or adrenals, consistent with the spatial expression profile of the disrupted genes (SOX9 or SF1). WNT4 is a new sex-determining signalling molecule. Deletions of Wnt4 were shown to be responsible for the masculinization of XX mouse pups while its duplication and overexpression in humans leads to XY sex reversal. Similarly, duplications of loci containing DAX1 or SOX9 have also been shown to cause sex reversal. These results support the emerging concept that mammalian sex determination is dosage sensitive at multiple steps of its pathway. Publication Types: Review Review, Tutorial PMID: 11990797 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Novartis Found Symp. 2002;244:4-18; discussion 18-22, 35-42, 253-7. Sex-determining genes in mice: building pathways. Lovell-Badge R, Canning C, Sekido R. Division of Developmental Genetics, MRC National Institute for Medical Research, London, UK. Sry is active in the mouse for a very brief period in somatic cells of the genital ridge to initiate Sertoli cell differentiation. SRY protein must act within the context of other gene products required for gonadal development and must itself act on one or more target genes that will ensure the further differentiation and maintenance of Sertoli cells. Over the last few years several genes have been found that have important roles in gonadal development and sex determination. These include genes encoding transcription factors such as Lhx9, Wt1, Sf1, Dax1, Gata4, Dmrt1 and Sox9, and some involved in cell cell signalling, including Amh, Wnt4 and Dhh. While more await discovery, it is now possible to start putting some of the known genes into pathways or networks. Sox9 probably occupies a critical role in mammals for both the initiation and maintenance of Sertoli cell differentiation. Data will be presented that are consistent with SRY acting directly on Sox9 to ensure its up-regulation. SF1 is also central to gonadal differentiation. Our results imply that it contributes to transcriptional activation of several relevant genes, not just those required for male development, including Sox9 and Amh, but also those that can have an antagonistic effect on Sertoli cell differentiation, such as Dax1. Progress in establishing other regulatory interactions will also be discussed. Publication Types: Review Review, Tutorial PMID: 11990796 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Novartis Found Symp. 2002;244:23-31; discussion 31-42, 253-7. Early gonadal development: exploring Wt1 and Sox9 function. Guo JK, Hammes A, Chaboissier MC, Vidal V, Xing Y, Wong F, Schedl A. Human Molecular Genetics Unit, University of Newcastle upon Tyne, UK. Prior to sex determination the gonadal anlage is formed as a bipotential primordium with the capacity to differentiate into either testes or ovaries depending on the presence or absence of the Sry gene. Knockout experiments have implicated five genes in the formation or survival of the gonadal primordium: Wt1, Sf1, Lim1, Lhx9 and Emx2. We are particularly interested in the Wilms tumour suppressor, WT1, which is characterized by complex posttranscriptional modifications. Here we will focus on published in vitro evidence suggesting distinct functions for the various isoforms and present our own results from in vivo experiments. Our data suggest that WT1 is an important regulator of the transcription or stability of the sex-determining gene Sry. One of the first genes expressed after the initial male sex-determining signal is the Sox9 gene. Human SOX9 has been implicated in male-to-female sex reversal. To analyse Sox9 function in mouse development we have performed transgenic experiments and ectopically expressed this gene in XX gonads. Our data indicate that Sox9 is sufficient to induce testis formation in mice. Here we will discuss our new data and present an updated model for Wt1 and Sox9 function in gonad formation and sex determination. Publication Types: Review Review, Tutorial PMID: 11990794 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Novartis Found Symp. 2002;244:225-36; discussion 236-9, 253-7. The molecular genetic jigsaw puzzle of vertebrate sex determination and its missing pieces. Scherer G. Institute of Human Genetics and Anthropology, University of Freiburg, Germany. Since the identification of SRY as the mammalian Y-chromosomal testis-determining gene a decade ago, more than a dozen additional genes essential for early gonadal development in mammals and other vertebrate classes have been identified. The location of these known pieces of the puzzle in the sex determination pathway, and how they interact, is briefly outlined. Two insights emerge: except for SRY, the same basic set of genes appears to operate during early gonadal development in all vertebrate classes, despite the difference in mechanisms; and vertebrate sex determination results from a complex network of regulatory interactions and not from a simple hierarchical cascade of gene actions. However, important pieces of the puzzle are still missing, such as the molecular nature of the sex switch in marsupials, monotremes and non-mammalian vertebrates; the target of SRY; the upstream regulators of SOX9; and the genes in the ovarian pathway. The enigma of SRY-positive XY gonadal dysgenesis females and SRY-negative XX males also indicates that the picture is still far from complete. Filling in these missing pieces is the challenge for the future. Publication Types: Review Review, Tutorial PMID: 11990793 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Reprod Suppl. 2001;58:65-80. Mammalian gonadal differentiation: the pig model. Pailhoux E, Mandon-Pepin B, Cotinot C. Unite de Biologie du Developpement et Biotechnologies, Batiment J.Poly. INRA, 78350 Jouy en Josas, France. pailhoux@biotec.jouy.inra.fr In mammals, testicular differentiation is initiated by SRY (the sex-determining region of the Y chromosome) gene expression in Sertoli cell precursors, followed by upregulation of the SOX9 gene (SRY-related HMG box gene 9). Subsequently, differentiated testis produces two hormones that induce sexual differentiation of the internal and external genital tract. Knowledge of the molecular mechanisms involved in gonadal differentiation has increased greatly over the past decade. Several genes are involved in genital ridge formation in both sexes, and others act specifically in testicular or ovarian developmental pathways. As for other mammals, relatively few data are available on the first steps of ovarian differentiation in pigs. In this review, the expression profiles of most genes known to be involved in gonadal differentiation in pigs will be presented and compared with those observed in mice. The main feature of gonadal differentiation in the pig is fetal steroidogenesis, especially cytochrome P450 aromatase gene organization and expression. Another specific feature of gonadal differentiation in pigs is the appearance of numerous cases of XX sex-reversed animals. This intersex condition occurs as early as day 50 after coitus, during embryogenesis, and appears to be triggered genetically. It leads to a wide range of phenotypes, strikingly similar to those observed in humans. Identification of the genes involved in this pathology will improve our knowledge of mammalian gonadal differentiation and may allow the eradication of this genetic disease in pigs. Publication Types: Review Review, Tutorial PMID: 11980203 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Genet Sel Evol. 2002 Jan-Feb;34(1):129-37. SRY-related genes in the genome of the rice field eel (Monopterus albus). Zhou R, Cheng H, Zhang Q, Guo Y, Cooper RK, Tiersch TR. Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China. rjzhou@public.wh.hb.cn The mammalian sex determining gene, SRY, is the founding member of the new growing family of Sox (SRY-like HMG-box gene) genes. Sox genes encode transcription factors with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the existence of Sox genes in the rice field eel, Monopterus albus, and DNA sequence information of the HMG box region of five Sox genes. The Sox1, Sox4 and Sox14 genes do not have introns in the HMG box region. The Sox9 gene and Sox17 gene, which each have an intron in the conserved region, show strong identity at the amino acid level with the corresponding genes of mammals and chickens. Similar structure and identity of the Sox9 and Sox17 genes among mammals, chickens and fish suggest that these genes have evolutionarily conserved roles, potentially including sex determination and differentiation. PMID: 11929629 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Trends Endocrinol Metab. 2002 Apr;13(3):106-11. Sex with two SOX on: SRY and SOX9 in testis development. Clarkson MJ, Harley VR. Prince Henry's Institute of Medical Research, PO 5152, Clayton, Victoria 3168, Australia. Although gonads are not required for development or survival, defects in gonadal development undoubtedly have a profound influence on affected individuals. Recent complementary studies in the fields of cytology, biochemistry and molecular genetics have revealed that normal gonad development involves an exquisitely regulated network of gene expression and protein-protein interactions. The initial event of gonadogenesis, in both males and females, involves the formation of a bipotential primordium. A Y chromosome then activates the male-specific pathway. The demonstration that mutations in the SOX proteins, SRY and SOX9, are responsible for disorders associated with male-to-female sex reversal showed dramatically that SRY and SOX9 have an essential role in male sex differentiation. This was emphasized when it was shown that female mice carrying transgenes that encode these proteins developed as males. SRY and SOX9 proteins have been characterized extensively and aspects of their function and regulation are now known. Publication Types: Review Review, Tutorial PMID: 11893523 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1146-51. Epub 2002 Jan 29. A direct role of SRY and SOX proteins in pre-mRNA splicing. Ohe K, Lalli E, Sassone-Corsi P. Institut de Genetique et de Biologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale, Universite Louis Pasteur, B.P. 163, 67404 Illkirch-Strasbourg, France. The mammalian testis determining factor SRY and its related Sox factors are critical developmental regulators. They share significant similarity in their high mobility group (HMG) domain and display discrete patterns of tissue-specific expression. Here we show that SRY and the Sox protein SOX6 colocalize with splicing factors in the nucleus and are dynamically redistributed following the blockage of splicing in living cells. Anti-SOX6 antibodies supershift the spliceosomal complex from assembled splicing reactions and inhibit splicing in vitro of multiple pre-mRNA substrates. Most importantly, SOX6-depleted nuclear extracts have impaired splicing activity, which is efficiently restored by addition of the recombinant SOX6 HMG domain and also by recombinant SRY and the SOX9 HMG domain. These results reveal an unexpected biological function of the SRY, SOX6, and SOX9 gene products and provide a functional link to the biochemical mechanisms operating in mammalian sex determination and in other developmental processes regulated by Sox genes. PMID: 11818535 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: Am J Med Genet. 2001 Dec 1;104(3):239-45. Acampomelic campomelic syndrome. Moog U, Jansen NJ, Scherer G, Schrander-Stumpel CT. Department of Clinical Genetics, University of Maastricht, PO Box 1475, 6201 BL Maastricht, The Netherlands. ute.moog@gen.unimaas.nl Campomelic syndrome (or campomelic dysostosis, CD; MIM *114290) is an autosomal dominant skeletal malformation syndrome characterized by shortness and bowing of long bones, especially of the lower limbs. Additional radiological and clinical findings are 11 pairs of ribs and a bell-shaped thorax, hypoplastic scapulae, narrow iliac wings, non-mineralized thoracic pedicles, clubbed feet, Robin sequence, typical facial anomalies and tracheomalacia. The disorder is frequently lethal due to respiratory distress. Sex reversal occurs in most patients with an XY karyotype. CD is caused by heterozygous mutations in the SOX9 gene, an SRY-related gene at 17q24.3-q25.1 with pleiotropic effects on the skeletal and genital systems. In addition, cases with chromosomal rearrangements involving 17q have been described that are most likely caused by disturbing one or more cis-regulatory elements from an extended control region. Campomelia (bowed limbs) is seen in most but not all patients, defining a so-called acampomelic campomelic dysostosis (ACD). Half of the CD cases with 17q rearrangements have no or mild campomelia. Furthermore, campomelia is absent or only mildly present in a small subgroup of cases with a normal karyotype. We present a chromosomally normal boy with ACD and his clinical follow-up up to the age of 2 years, in whom a heterozygous SOX9 missense mutation (H165Y) was identified. A SOX9 missense mutation was published in two other patients with ACD. Although up to now a general genotype-phenotype correlation could not be established for CD, a correlation emerges for the ACD variant that needs further confirmation. Copyright 2001 Wiley-Liss, Inc. Publication Types: Case Reports PMID: 11754051 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: J Exp Zool. 2001 Nov 1;290(6):567-73. Identifying genes for male sex determination in humans. Ostrer H. Human Genetics Program, Department of Pediatrics, New York University School of Medicine, New York, New York 10016, USA. harry.ostrer@med.nyu.edu The convergence of genetic and molecular technologies has led to the identification of a number of genes for male sex determination. The observation of chromosomal translocations, deletions, and duplications in sex reversed individuals was instrumental for the positional cloning of SRY, SOX9, WT1, and DAX1. Cloning by protein-DNA interaction was required for the identification of SF1. The observation of an extended phenotype for the alpha thalassemia-mental retardation syndrome assigned a role for XH2 in the testicular determining process. Over the next several years, new sex determining genes will be identified by linkage analysis in large families with multiple sex reversed members, comparative genomic hybridization of sex reversed individuals, and database searches for genes that encode interacting proteins or paralogs of other species. Given the apparent differences in the sex determining mechanisms of even closely related species, the roles of all of these genes will require confirmation by demonstrating expression in human gonadal ridge at the critical time, and that mutations result in sex reversal. Copyright 2001 Wiley-Liss, Inc. Publication Types: Review Review, Tutorial PMID: 11748605 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Dev Dyn. 2001 Nov;222(3):328-40. Time course of female-to-male sex reversal in 38,XX fetal and postnatal pigs. Pailhoux E, Parma P, Sundstrom J, Vigier B, Servel N, Kuopio T, Locatelli A, Pelliniemi LJ, Cotinot C. Unite de Biologie du Developpement et Biotechnologies, INRA, Jouy en Josas, France. pailhoux@biotec.jouy.inra.fr In an attempt to understand the etiology of intersexuality in pigs, we thoroughly analyzed the gonads of 38,XX (SRY negative) female to male sex-reversed animals at different developmental stages: during fetal life [50 and 70 days postcoitum (dpc)], just after birth [35 days postpartum (dpp)] and during adulthood. For each animal studied, we performed parallel histological and ultrastructural analyses on one gonad and RT-PCR analysis on the other gonad in order to define the expression profiles of sexually regulated genes: SOX9, 3beta-HSD, P450 aromatase, AMH, FOXL2, and Wnt4. Light and electron microscopic examination showed that testicular cords differentiated in XX sex-reversed gonads but were hypoplastic. Although the testicular cords contained gonia at the fetal stages, the germ cells had all died through apoptosis within a few weeks after birth. Ultrastructurally normal Leydig cells also differentiated, but later, and enclosed whorl-like residual bodies. At the fetal stages, three of the six genes studied in the intersex gonads presented, as early as 50 dpc, a modified expression profile corresponding to an elevated expression of SOX9 and the beginning of AMH and P450 aromatase gene transcription. In addition to genes involved in the testicular pathway, the same gonads expressed FOXL2, an ovarian-specific factor. The ovaries of true hermaphrodites were ineffective in ensuring correct folliculogenesis and presented abnormal expression profiles of ovarian specific genes after birth. These results indicate that the genes involved in this pathology act very early during gonadogenesis and affect the ovary-differentiating pathway with variable expressivity from ovarian germ cell depletion through to trans-differentiation into testicular structures. Copyright 2001 Wiley-Liss, Inc. PMID: 11747069 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Biochem J. 2001 Dec 1;360(Pt 2):461-70. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Stokes DG, Liu G, Dharmavaram R, Hawkins D, Piera-Velazquez S, Jimenez SA. Department of Medicine, Division of Rheumatology, 233 S. 10th Street, Thomas Jefferson University, Philadelphia, PA 19107, USA. During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata. PMID: 11716775 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: In Vitro Cell Dev Biol Anim. 2001 Oct;37(9):581-8. Establishment and characterization of immortalized ovine Sertoli cell lines. Merhi RA, Guillaud L, Delouis C, Cotinot C. Unite de Biologie du developpement et Biotechnologies, INRA, Jouy-en-Josas, France. The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein ISGP-2], follicle-stimulating hormone [rFSH], alpha-inhibin, anti-Mullerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, alpha-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents. PMID: 11710435 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: Osteoarthritis Cartilage. 2001;9 Suppl A:S69-75. L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway. Lefebvre V, Behringer RR, de Crombrugghe B. Department of Molecular Genetics, The University of Texas, MD Anderson Cancer Center, Houston 77030, USA. lefebvr@bme.ri.ccf.org OBJECTIVE: This work was carried out to identify transcription factors controlling the differentiation of mesenchymal cells into chondrocytes. DESIGN: We delineated a cartilage-specific enhancer in the collagen type 2 gene (Col2a1) and identified transcription factors responsible for the activity of this enhancer in chondrocytes. We then analyzed the ability of these transcription factors to activate specific genes of the chondrocyte differentiation program and control cartilage formation in vivo. RESULTS: A 48-bp sequence in the first intron of Col2a1 drove gene expression specifically in cartilage in transgenic mouse embryos. The transcription factors L-Sox5, Sox6, and Sox9 bound and cooperatively activated this enhancer in vitro. They belong to the Sry-related family of HMG box DNA-binding proteins, which includes many members implicated in cell fate determination in various lineages. L-Sox5, Sox6, and Sox9 were coexpressed in all precartilaginous condensations in mouse embryos and continued to be expressed in chondrocytes until the cells underwent final hypertrophy. Whereas L-Sox5 and Sox6 are highly homologous proteins, they are totally different from Sox9 outside the HMG box domain. The three proteins cooperatively activated the Col2a1- and aggrecan genes in cultured cells. Heterozygous mutations in SOX9 in humans lead to campomelic dysplasia, a severe and generalized skeletal malformation syndrome. Embryonic cells with a homozygous Sox9 mutation were unable to form cartilage in vivo and activate essential chondrocyte marker genes. Preliminary data indicated that the mutation of Sox5 and Sox6 in the mouse led to severe skeletal malformations. CONCLUSIONS: L-Sox5, Sox6, and Sox9 play essential roles in chondrocyte differentiation and, thereby, in cartilage formation. Their discovery will help to understand further the molecular mechanisms controlling chondrogenesis in vivo, uncover genetic mechanisms underlying cartilage diseases, and develop novel strategies for cartilage repair. PMID: 11680692 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Dev Dyn. 2001 Oct;222(2):228-37. Expression of AMH, SF1, and SOX9 in gonads of genetic female chickens during sex reversal induced by an aromatase inhibitor. Vaillant S, Magre S, Dorizzi M, Pieau C, Richard-Mercier N. Institut Jacques Monod, UMR 7592 CNRS et Universites Paris VI et Paris VII, 2 place Jussieu, 75251 Paris Cedex 05, France. Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti-Mullerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sex-reversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular-like cords, sex-reversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular-like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli-cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole. Copyright 2001 Wiley-Liss, Inc. PMID: 11668600 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: Genome Biol. 2001;2(9):REVIEWS1025. Epub 2001 Aug 29. New clues to the puzzle of mammalian sex determination. Bowles J, Koopman P. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. p.koopman@imb.uq.edu.au WT1 and SOX9 are transcription factors with critical roles in mammalian sex determination and gonadal development. Recent studies in vivo clarify the roles of two alternative splice isoforms of WT1, and demonstrate that SOX9 can induce male sex determination. Publication Types: Review Review, Tutorial PMID: 11574062 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: J Exp Zool. 2001 Sep 15;290(5):523-8. Structural and regulatory macromolecules in sex differentiation of gonads. Pelliniemi LJ, Frojdman K. Laboratory of Electron Microscopy, University of Turku, FIN-20520 Turku, Finland. LJPELMI@utu.fi The manifestations of sex determination were studied in vivo by detection and localization of structural and regulatory macromolecules (type IV collagen alpha 1, alpha 2, alpha 3, alpha 4, and alpha 5; laminin alpha 5, beta 1, and beta 2; cytokeratins 18 and 19, desmin, vimentin; integrin alpha(6;) anti-Mullerian hormone (AMH); and SOX9 in developing male and female gonads by light and electron microscopy, immunocytochemistry, and protein analysis. The goal has been to find sex-related differences and on this basis to offer new molecules to be tested further for a possible role in sex determination. Specific antibodies for each molecule or for a defined subchain were used to allow tentative correlation with specific genes. Sex-dependent differences in timing and localization were found in laminin alpha 5; collagen, alpha 3, alpha 4, and alpha 5; cytokeratin 19; AMH; and SOX9. On this basis we hypothesize that the transcription factors for the mentioned structural proteins must be directly or indirectly involved in the regulatory chain of gonadal sex differentiation. Especially promising is the finding in the rat that laminin alpha 5 chain disappears from the basement membrane of embryonic testicular cords (Sertoli cells) when AMH secretion by Sertoli cells starts, and that the same chain reappears as the AMH disappears two weeks after birth. Via AMH as an intermediary factor, we now have for the first time a putative cascade of regulatory molecules from SRY, SF1, and SOX9 to a component of a structural protein (laminin alpha 5 chain) which directly participates in the formation of the basement membrane of the testicular cords. J. Exp. Zool. 290:523-528, 2001. Copyright 2001 Wiley-Liss, Inc. PMID: 11555860 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: J Exp Zool. 2001 Sep 15;290(5):498-503. Timing of SOX9 downregulation and female sex determination in gonads of the sea turtle Lepidochelys olivacea. Torres-Maldonado L, Moreno-Mendoza N, Landa A, Merchant-Larios H. Departamento de Biologia Celular, Instituto de Investigaciones Biomedicas, UNAM, Mexico 04510. The SRY-related gene SOX9 is involved in the differentiation of Sertoli cells in male gonads of vertebrates with different kinds of sex determination. In the olive ridley Lepidochelys olivacea, a species with temperature sex determination (TSD), the SOX9 protein is expressed at stages 21-24 in medullary cells in gonads of embryos incubated at both male-(MPT) or female-promoting temperatures (FPT). However, at FPT the expression of SOX9 protein decreases at stage 25 and disappears at stage 26, suggesting this as the critical period for SOX9 regulation by temperature. Here, we used reverse transcriptase polymerase chain reaction (RT-PCR) to detect SOX9 transcripts in gonads of embryos switched from MPT to FPT at stage 23 and sampled at days 6-14. Simultaneously, groups of embryos were switched back to MPT and gonadal sex was established. SOX9 transcripts were detected at days 6-12 of switching, when embryos reached stage 25 and were no longer detected at day 14, when the embryos were at stage 26. Embryos switched back to MPT at days 6 or 8 formed testes, whereas embryos switched at days 10 or 14 developed ovaries. Results suggest that at MPT the male sex-determining pathway that maintains SOX9 expression in male gonads is established at stage 24. In contrast, at FPT, the female sex-determining pathway involved in downregulation of SOX9 in female gonads occurs within two days at stage 25. J. Exp. Zool. 290:498-503, 2001. Copyright 2001 Wiley-Liss, Inc. PMID: 11555857 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: J Exp Zool. 2001 Sep 15;290(5):463-74. Regulation of male sexual development by Sry and Sox9. Koopman P, Bullejos M, Bowles J. Centre for Molecular and Cellular Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. p.koopman@lmb.uq.edu.au Sry, a gene from the Y chromosome, is known to initiate testis formation and subsequent male differentiation in mammals. A related gene, Sox9, also plays a critical role in testis determination, possibly in all vertebrates. A number of models have been presented regarding the molecular modes of action of these two genes. However, details regarding their regulation, regulatory target genes, and interacting protein factors and co-factors have not been established with any certainty. In this review, we examine new evidence and re-examine existing evidence bearing on these issues, in an effort to build up an integrative model of the network of gene activity centred around Sry and Sox9. J. Exp. Zool. 290:463-474, 2001. Copyright 2001 Wiley-Liss, Inc. Publication Types: Review PMID: 11555853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Nat Genet. 2001 Jul;28(3):216-7. Sox9 induces testis development in XX transgenic mice. Vidal VP, Chaboissier MC, de Rooij DG, Schedl A. MDC for Molecular Medicine, Robert-Rossle-Strasse 10, 13092 Berlin, Germany. Mutations in SOX9 are associated with male-to-female sex reversal in humans. To analyze Sox9 function during sex determination, we ectopically expressed this gene in XX gonads. Here, we show that Sox9 is sufficient to induce testis formation in mice, indicating that it can substitute for the sex-determining gene Sry. PMID: 11431689 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Brain Res. 2001 Jul 6;906(1-2):143-8. Localisation of the SRY-related HMG box protein, SOX9, in rodent brain. Pompolo S, Harley VR. Howard Florey Institute of Experimental Physiology and Medicine, Parkville 3052, Victoria, Australia. sueli.pompolo@med.monash.edu.au Human mutations in the transcription factor gene, SOX9, cause campomelic dysplasia (CD), a severe dwarfism associated with brain abnormalities including dilation of lateral ventricles, hypoplasia of the corpus callosum and cerebellum defects. To improve our understanding of how SOX9 contributes to the molecular genetic pathway of brain development we sought to investigate the distribution of SOX9 protein in rat and mouse brain. The regions of SOX9 expression identified in this study correlated with the sites of reported brain abnormalities in CD patients. SOX9 immunoreactivity was observed in nuclei of scattered cells throughout the brain, in the ependymal layer and cells of the choroid plexus. In the forebrain most SOX9-immunoreactive nuclei co-localised with the glial astrocyte marker S-100. In the cerebellum, SOX9 was observed mostly in cells surrounding Purkinje cells, which were identified, by electron microscopy, as Golgi epithelial cells, also known as Bergmann glia. Using SOX9 antibody as a marker for the precursors of Bergmann glia, we traced their origin during mouse development. At embryonic day (E)14.5 and E16.5, SOX9 immunoreactivity was present mainly in the primordial choroid plexus, and ventricular zone. By E18.5, SOX9 was observed in the granular cell and Purkinje cell layers but no labelling was detectable in the external granular layer. These results suggest that SOX9 immunoreactivity is a marker for Bergmann cells during development and favour the proposed origin of the secondary glial scaffold arising from Bergmann cells derived exclusively from the ventricular zone. PMID: 11430871 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Mol Cell Endocrinol. 2001 Jun 20;179(1-2):3-16. Testis determination in mammals: more questions than answers. Veitia RA, Salas-Cortes L, Ottolenghi C, Pailhoux E, Cotinot C, Fellous M. Immunogenetique Humaine, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France. rvietia@pasteur.fr In humans, testis development depends on a regulated genetic hierarchy initiated by the Y-linked SRY gene. Failure of testicular determination results in the condition termed 46,XY gonadal dysgenesis (GD). Several components of the testis determining pathway have recently been identified though it has been difficult to articulate a cascade with the known elements of the system. It seems, however, that early gonadal development is the result of a network of interactions instead of the outcome of a linear cascade. Accumulating evidence shows that testis formation in man is sensitive to gene dosage. Haploinsufficiency of SF1, WT1 and SOX9 is responsible for 46,XY gonadal dysgenesis. Besides, data on SRY is consistent with possible dosage anomalies in certain cases of male to female sex reversal. 46,XY GD due to monosomy of distal 9p and 10q might also be associated with an insufficient gene dosage effect. Duplications of the locus DSS can lead to a failure of testicular development and a duplication of the region containing SOX9 has been implicated in XX sex reversal. Transgenic studies in mouse have shown, however, that this mammal is less sensitive to gene dosage than man. Here, we will try to put in place the known pieces of the jigsaw puzzle that is sex determination in mammals, as far as current knowledge obtained from man and animal models allows. We are certain that from this attempt more questions than answers will arise. Publication Types: Review Review, Tutorial PMID: 11420125 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Gene. 2001 May 30;270(1-2):161-9. Molecular evolution of Sry and Sox gene. Nagai K. Tokyo Medical University, Department of Biochemistry, 160-8402, Tokyo, Japan. kozo-nag@tokyo-med.ac.jp The mammalian Sry on the short arm of the Y chromosome encodes a nuclear factor-like protein harboring a DNA-binding domain known as the HMG box. The Sox genes encode similar factor like proteins, but the sequence similarity of the HMG box to that of Sry is variable as being at least 60%. The functional relationship of Sox to Sry genes with special reference to sex determination is unclear except for a few items such as human autosomal Sox9. Thus, it is significant to know more about the evolutionary in addition to the functional relationship between Sry and Sox genes for deepening and broadening our understanding concerning primary sex determination. Therefore, to clarify the ancestry and molecular evolution of the mammalian sex determining gene Sry with its evolutionary relationships to the Sox gene, a molecular phylogenetic tree for the HMG box superfamily was constructed and analyzed, and the following conclusions were reached: (1) The nuclear non histone HMG proteins are supposedly the oldest, appearing at least more than one billion years ago, before the divergence of animals and plants. They diverged into two subgroups: one contains HMG14 and HMG17, and the other one contains HMG1 and HMG2 with various other genes. Subsequent divergences include the nucleolar UBF, nuclear SSRP as well as fungal mating protein Mc, MAT and Ste11. (2) The Sox and Sry genes diverged following the diversification of lymphoid transcription factors TCF and LEF. The Sry gene might have definitely evolved from the Sox gene cluster a few hundred million years ago. Additionally, the marsupial Sry, e.g. from Wallabie's and Dunnart's, is distinguished by being distant from eutherian Sry, but being closely related to the Sox gene cluster. (3) Molecular evolutionary rates estimated in mammalian Sry as the divergent rate per 100 million years are much higher than in Sox genes or other genes from the HMG box superfamily. This rapid evolution of Sry might agree with the fact that the Srys are present not on the pseudoautosomal region but on the distal region with no recombination of the Y chromosomal short arm. PMID: 11404013 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: J Endocrinol. 2001 Jun;169(3):573-9. Dexamethasone enhances SOX9 expression in chondrocytes. Sekiya I, Koopman P, Tsuji K, Mertin S, Harley V, Yamada Y, Shinomiya K, Nifuji A, Noda M. Department of Molecular Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan. SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox- consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9. PMID: 11375127 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: J Exp Zool. 2001 Apr 15;291(1):85-91. Characterisation and expression of Sox9 in the Leopard gecko, Eublepharis macularius. Valleley EM, Cartwright EJ, Croft NJ, Markham AF, Coletta PL. Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, United Kingdom. Since the discovery of the sex-determining gene, Sry, a number of genes have been identified which are involved in sex determination and gonadogenesis in mammals. Although Sry is known to be the testis-determining factor in mammals, this is not the case in non-mammalian vertebrates. Sox9 is another gene that has been shown to have a male-specific role in sex determination, but, unlike Sry, Sox9 has been shown to be involved in sex determination in mammals, birds, and reptiles. This is the first gene to be described that has a conserved role in sex determination in species with either chromosomal or environmental sex-determining mechanisms. Many reptiles do not have sex chromosomes but exhibit temperature-dependent sex determination (TSD). Sox9 has been shown to be expressed in both turtle and alligator during gonadogenesis. To determine if Sox9 also has a role in a gecko species with TSD, we studied gonadal expression of Sox9 during embryonic development of the Leopard gecko (Eublepharis macularius). Gecko Sox9 was found to be highly conserved at the nucleotide level when compared to other vertebrate species including human, chick, alligator, and turtle. Sox9 was found to be expressed in embryos incubated at the male-producing temperature (32.5 degrees C) as well as in embryos incubated at the female-producing temperatures (26 and 34 degrees C), Northern blot analysis showed that Sox9 was expressed at both temperatures from morphological stages 31 to 37. mRNA in situ hybridisation on isolated urogenital systems showed expression at both female- and male-producing temperatures up to stage 36. After this stage, no expression was seen in the female gonads but expression remained in the male. These data provide further evidence that Sox9 is an essential component of a testis-determining pathway that is conserved in species with differing sex-determining mechanisms. PMID: 11335918 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: EXS. 2001;(91):25-56. Sry, Sox9 and mammalian sex determination. Koopman P. Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, QLD 4072, Australia. Sry is the Y-chromosomal gene that acts as a trigger for male development in mammalian embryos. This gene encodes a high mobility group (HMG) box transcription factor that is known to bind to specific target sequences in DNA and to cause a bend in the chromatin. DNA bending appears to be part of the mechanism by which Sry influences transcription of genes downstream in a cascade of gene regulation leading to maleness, but the factors that cooperate with, and the direct targets of, Sry remain to be identified. One gene known to be downstream from Sry in this cascade in Sox9, which encodes a transcription factor related to Sry by the HMG box. Like Sry, mutations in Sox9 disrupt male development, but unlike Sry, the role of Sox9 is not limited to mammals. This review focuses on what is known about the two genes and their likely modes of action, and draws together recent data relating to how they might interconnect with the network of gene activity implicated in testis determination in mammals. Publication Types: Review PMID: 11301599 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: EXS. 2001;(91):1-10. The one-to-four rule and paralogues of sex-determining genes. Ohno S. Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010-3000, USA. Because of two successive rounds of tetraploidization at their inception, the vertebrates contain four times more protein-coding genes in their genome than the invertebrates: 60,000 vs. 15,000. Consequently, each invertebrate gene has been amplified to the maximum of four paralogous genes in vertebrates: the one-to-four rule. When this rule is applied to genes pertinent to gonadal development and differentiation, the following emerged: (i) Two closely related zinc-finger transcription factor genes in invertebrates have been amplified to two paralogous groups in vertebrates. One consisted of EGR1, EGR2, EGR3 and EGR4, whereas the only known paralogue of the other is WT1, which controls the developmental fate of the entire nephric system, and therefore of gonads. Interestingly, EGR1 and WT1 act as antagonists of each other in nephroblastic cells. (ii) SF-1, which controls the fate of two steroid hormone-producing organs, adrenals and gonads, is descended from the invertebrate Ftz-F1 gene, and its only known paralogue is GCNF-1. (iii) The Y-linked SRY, the mammalian testis-determining gene, is a paralogue neither of SOX3 (SRX) nor of SOX9. Its ancient origin suggests that SRY once became extinct in earlier vertebrates, only to revive itself in the mammalian ancestor. (iv) Inasmuch as four paralogues of one invertebrate nuclear receptor gene have differentiated to receptors of androgen, mineralocorticoid, glucocorticoid and progesterone, there should at most be four paralogous estrogen-receptor genes in the vertebrate genome. It is likely that one of them plays a pivotal role in the estrogen-dependent sex-determining mechanism so commonly found among reptiles, amphibians and fish. Publication Types: Review Review, Tutorial PMID: 11301594 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Semin Reprod Med. 2000;18(1):41-9. Sexual differentiation. Ostrer H. Department of Pediatrics, New York University School of Medicine, New York, New York, USA. Sexual differentiation in humans is genetically and hormonally controlled. In response to a signal from a dominant-acting gene on the Y chromosome, primordial cells in the embryonic gonad ridge differentiate into Sertoli cells and affect newly migrated germ cells to differentiate as spermatogonia, thus creating a testis. The cells of the embryonic testis secrete hormones that lead to the development of most, if not all, male secondary sexual characteristics. The Sertoli cells secrete mullerian inhibitory factor (MIF), causing regression of the mullerian ducts and of stray oogonia. The Leydig cells secrete testosterone, causing differentiation and growth of the wolffian duct structures. Dihydrotestosterone, created by metabolism of testosterone, causes growth of the prostate and phallus and fusion of the labioscrotal folds. In the absence of SRY, Sertoli cell differentiation does not occur. Rather germ cells migrating into the primordial gonad differentiate as oogonia and cause interstitial cells to differentiate as granulosa cells. In the absence of MIF and testosterone, the mullerian ducts differentiate and grow as female internal genitalia and the external genitalia are feminized. Several genes have been identified that control testis determination. These include SRY, WT1, SOX9, SF1, XH2, and DAX1. Most of these genes were discovered by analysis of rare cases of sex reversal (genetic sex of one type, gonadal sex of the other type). Publication Types: Review Review, Tutorial PMID: 11299518 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Clin Genet. 2001 Apr;59(4):207-15. Sex determination: lessons from families and embryos. Ostrer H. Human Genetics Program, Department of Pediatrics, New York University School of Medicine, New York, NY 10016, USA. harry.ostrer@med.nyu.edu Genetic studies in familial cases of sex reversal and in human embryos have contributed to the understanding of human sex determination and its disorders. For some heritable disorders of sex reversal, the gonadal phenotype was frequently overlooked until sex reversal was discovered fortuitously by chromosome analysis, often resulting in preventable complications. Within families, the phenotypes are variable and, in some instances, these can be explained by known genetic mechanisms. When a novel molecular marker is shared by family members affected with sex reversal, the level of confidence is higher that this marker may play a role in the development of the phenotype. The identification of pedigrees with sufficient power to generate significant linkage of disorder (lod) scores from genomewide screens can now lead to the identification of novel sex-determining genes. Studies of the gonads of 46,XY human embryos have shown that SOX9 expression follows a pattern similar to that of SRY and, in both instances, stands in contrast to the expression observed in the mouse. Differences between human and mouse embryonic gonads have also been observed for the temporal expression of DAX1, suggesting that the mechanisms of action of SRY, SOX9, and DAX1 may vary between these and other species. Publication Types: Review Review, Tutorial PMID: 11298673 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: Am J Med Genet. 2001 Jan 15;98(2):176-81. Genetic study of SOX9 in a case of campomelic dysplasia. Giordano J, Prior HM, Bamforth JS, Walter MA. Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada. Campomelic dysplasia (CD) is a sporadic autosomal dominant syndrome that results in skeletal malformation and developmental abnormalities. Death usually occurs neonatally as a result of respiratory insufficiencies, but life expectancy varies depending on the severity of the phenotype. XY sex reversal is common in CD, and a range of genital defects is observed in males and females. CD is due to mutations in SOX9, a member of the SOX (SRY-related HMG box) gene family. SOX9 is a transcription factor involved in chondrogenesis and sex determination. We present a CD patient with a normal 46,XX karyotype and female phenotype. Single-stranded conformation polymorphism analysis of DNA from this CD patient demonstrated a single-stranded conformation polymorphism shift in the C-terminal region of SOX9. DNA sequencing showed a frameshift mutation resulting from the insertion of a single guanine residue in nucleotide region 1,453-1,456. This insertion mutation creates a mutant SOX9 open reading frame that is 201 nucleotides longer than the normal gene. It has been shown that the C-terminal region of SOX9 is responsible for the transactivating ability of the protein. The frameshift identified here affects approximately half of the protein region needed for full transactivating function. We hypothesize that residual SOX9 function may explain why this patient survived infancy. Copyright 2001 Wiley-Liss, Inc. PMID: 11223854 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: Cell Mol Life Sci. 1999 Dec;56(11-12):883-93. Whatever happened to SRY? O'Neill MJ, O'Neill RJ. Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269, USA. The mammalian sex-determining gene, SRY, was identified by positional cloning approximately 10 years ago. Since its discovery, intense research into this gene has been directed on two main fronts: elucidation of its function in development of the testis and examination of its singular evolutionary history. The role or SRY as the testis-determining factor (TDF) places it at a crucial point in the highly conserved morphogenetic process of vertebrate gonadogenesis. None of the genes that directly activate SRY nor any of its immediate downstream targets have yet been positively identified. Several genes, however, such as SF1, DAX1, and SOX9, whose spatial and temporal expression profiles overlap with that of SRY, are strongly implicated as co-regulators of gonadogenesis. Molecular genetic manipulation of these genes in mice has shown that they are indispensable to sexual development. Remarkably, its key position in this cascade of gene action has not protected SRY from strong yet poorly understood selective forces that have caused it to evolve rapidly in mammals. The evolution of SRY has been characterized not only by rapid sequence divergence within mammals, but also by structural changes such as intron insertion, gene amplification, and deletion. Publication Types: Review Review, Tutorial PMID: 11212323 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: Mol Genet Metab. 2001 Jan;72(1):61-6. Exclusion of SOX9 as the testis determining factor in Ellobius lutescens: evidence for another testis determining gene besides SRY and SOX9. Baumstark A, Akhverdyan M, Schulze A, Reisert I, Vogel W, Just W. Department of Human Genetics, University of Ulm, Germany. In mammals the initiation of testis determination usually depends on the Y-chromosomal gene SRY. A few species, however, escape from this rule with a testis determination that is independent of SRY. The mole vole Ellobius lutescens is one of these species. It is not known how testis determination is initiated in this species but it has been suggested that a gene from the sex determination cascade usually acting downstream of SRY is mutated and has taken over the testis-determining function. At present SOX9 is the only candidate gene for which a testis-determining function in the absence of SRY has been observed. To test the hypothesis that testis differentiation in E. lutescens is initiated by SOX9, segregation analysis of SOX9 alleles was performed in an E. lutescens family. As there is no marker data available in this species we screened both Ellobius SOX9 introns for polymorphisms suitable for segregation studies. A biallelic polymorphism was found in the second intron of the SOX9 gene and analysis of this marker in the Ellobius family revealed an inheritance pattern completely independent of the sex of the animals. Thus, SOX9 can be excluded from being the testis-determining factor in E. lutescens. These results provide evidence for another possibly yet unknown gene besides SRY and SOX9 able to exert testis-determining function. Copyright 2001 Academic Press. PMID: 11161830 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: J Biol Chem. 2000 Apr 14;275(15):10738-44. SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6. Sekiya I, Tsuji K, Koopman P, Watanabe H, Yamada Y, Shinomiya K, Nifuji A, Noda M. Department of Molecular Pharmacology, Medical Research Institute, Tokyo 101, Japan. SOX9 is a transcription factor that plays a key role in chondrogenesis. Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated. TC6 is a clonal chondrocytic cell line derived from articular cartilage. The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by approximately 3-fold the transcriptional activity of the AgCAT-8 construct containing 8-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron. In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines. Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells. PMID: 10753864 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: Hum Genet. 2000 Dec;107(6):650-2. Epub 2000 Nov 14. Erratum in: Hum Genet. 2004 Aug;115(3):263. Mutation analysis of subjects with 46, XX sex reversal and 46, XY gonadal dysgenesis does not support the involvement of SOX3 in testis determination. Lim HN, Berkovitz GD, Hughes IA, Hawkins JR. Department of Paediatrics, University of Cambridge, Addenbrooke's Hospital, UK. HL215@mole.bio.cam.ac.uk Despite the identification of an increasing number of genes involved in sex determination and differentiation, no cause can be attributed to most cases of 46, XY gonadal dysgenesis, approximately 20% of 46, XX males and the majority of subjects with 46, XX true hermaphroditism. Perhaps the most interesting candidate for involvement in sexual development is SOX3, which belongs to the same family of proteins (SOX) as SRY and SOX9, both of which are involved in testis differentiation. As SOX3 is the most likely evolutionary precursor to SRY, it has been proposed that it has retained a role in testis differentiation. Therefore, we screened the coding region and the 5' and 3' flanking region of the SOX3 gene for mutations by means of single-stranded conformation polymorphism and heteroduplex analysis in eight subjects with 46, XX sex reversal (SRY negative) and 25 subjects with 46, XY gonadal dysgenesis. Although no mutations were identified, a nucleotide polymorphism (1056C/T) and a unique synonymous nucleotide change (1182A/C) were detected in a subject with 46, XY gonadal dysgenesis. The single nucleotide polymorphism had a heterozygosity rate of 5.1% (in a control population) and may prove useful for future X-inactivation studies. The absence of SOX3 mutations in these patients suggests that SOX3 is not a cause of abnormal male sexual development and might not be involved in testis differentiation. PMID: 11153920 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: Am J Hum Genet. 2001 Jan;68(1):275-80. Epub 2000 Dec 7. Primate DAX1, SRY, and SOX9: evolutionary stratification of sex-determination pathway. Patel M, Dorman KS, Zhang YH, Huang BL, Arnold AP, Sinsheimer JS, Vilain E, McCabe ER. Department of Physiological Science, University of California Los Angeles, Los Angeles, CA 90095, USA. The molecular evolution of DAX1, SRY, and SOX9, genes involved in mammalian sex determination, was examined in six primate species. DAX1 and SRY have been added to the X and Y chromosomes, respectively, during mammalian evolution, whereas SOX9 remains autosomal. We determined the genomic sequences of DAX1, SRY, and SOX9 in all six species, and calculated K(a), the number of nonsynonymous substitutions per nonsynonymous site, and compared this with the K(s), the number of synonymous substitutions per synonymous site. Phylogenetic trees were constructed by means of the DAX1, SRY, and SOX9 coding sequences, and phylogenetic analysis was performed using maximum likelihood. Overall measures of gene and protein similarity were closer for DAX1 and SOX9, but DAX1 exhibited nonsynonymous amino acid substitutions at an accelerated frequency relative to synonymous changes, similar to SRY and significantly higher than SOX9. We conclude that, at the protein level, DAX1 and SRY are under less selective pressure to remain conserved than SOX9, and, therefore, diverge more across species than does SOX9. These results are consistent with evolutionary stratification of the mammalian sex determination pathway, analogous to that for sex chromosomes. PMID: 11112659 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: Genesis. 2000 Nov-Dec;28(3-4):111-24. Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination. Bergstrom DE, Young M, Albrecht KH, Eicher EM. The Jackson Laboratory, Bar Harbor, ME 04609, USA. Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000. Copyright 2000 Wiley-Liss, Inc. PMID: 11105052 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: Nat Genet. 2000 Dec;26(4):490-4. A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse. Bishop CE, Whitworth DJ, Qin Y, Agoulnik AI, Agoulnik IU, Harrison WR, Behringer RR, Overbeek PA. Department of Obstetrics & Gynecology, Baylor College of Medicine, Houston, Texas, USA. bishop@bcm.tmc.edu In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals. PMID: 11101852 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: Poult Sci. 2000 Nov;79(11):1650-68. Spermiogenesis in commercial poultry species: anatomy and control. Thurston RJ, Korn N. Department of Animal and Veterinary Sciences, Clemson University, South Carolina 29634-0361, USA. ront@clemson.edu Spermatogenesis is a complicated process dependent upon several factors. Formation of a testis requires the interaction of gene-products and hormones (androgens) on pluripotent tissue. In birds, the female is the heterogametic (ZW) sex, but W chromosomal genes do not influence gonadal development in a way similar to the SRY gene on the mammalian Y chromosome. However, autosomal genes such as SRY-like HMG box gene 9 (SOX9) may influence gonadal development. Hormones affect development; male gonads subjected to estrogen form an ovotestis, whereas ovaries exposed to aromatase inhibitors form an atypical testis. Sertoli cell numbers are set early in spermiogenesis, possibly under the influence of follicle-stimulating hormone and thyroid hormone, and this may determine the number of gonial cells that can be supported. Sertoli cells make a number of substances that affect testicular development and function, particularly anti-Mullerian hormone, which inhibits female oviduct formation from the Mullerian anlage, inhibits aromatase activity to stop estrogen production, and possibly stimulates androgen production by Leydig cells. Undifferentiated primordial germ cells (PGC) migrate to the testis and are converted to spermatogonia by factors from gonadal ridge tissue and androgens. The PGC of males in the ovary form oocytes of Z genotype, whereas the female PGC in males form mostly Z sperm (with a few of W genotype). Transmission electron microscopy micrographs of turkey testis are presented, and control of spermatogenesis by hormones and cytokines is discussed. This discussion includes follicle-stimulating hormone, luteinizing hormone, inhibin, activin, follistatin, tumor necrosis factor-alpha, growth factors such as transforming growth factor-beta, interleukins, and interferon. Although information concerning paracrine and autocrine regulation of the avian testis by these substances is sparse, much can be learned from mammalian studies, in which putative roles of each of these substances have been established. How Sertoli cells cause directed apoptosis of spermatogonia using the Fas-ligand, Fas-receptor pathway is reviewed, as well as ways to circumvent this process. A possible role for ubiquitin concerning prevention of heat-induced damage to the testis is presented. Publication Types: Review PMID: 11092341 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Clin Genet. 2000 Sep;58(3):224-7. A novel mutation (296 del G) of the SOX90 gene in a patient with campomelic syndrome and sex reversal. Ninomiya S, Yokoyama Y, Teraoka M, Mori R, Inoue C, Yamashita S, Tamai H, Funato M, Seino Y. Department of Pediatrics, Okayama University Medical School, Japan. shin1@cc.okayama-u.ac.jp The human SOX9 gene is responsible for the campomelic syndrome (CMPS) and sex reversal. This gene encodes a transcription factor containing a DNA binding domain homologous to the SRY high mobility group (HMG) domain. A novel mutation of SOX9, i.e. a single G deletion in one allele at nt 296 from A of the first ATG in the open reading frame, was identified in a patient with CMPS with sex reversal. The deletion resulted in a frameshift mutation upstream of the HMG box and a stop codon 30 bp downstream of the HMG box. The predicted truncated SOX9 protein contained 108 amino acids instead of the 509 amino acids of the normal SOX9 protein, removing nearly 80% of the SOX9 protein, including the HMG and the C-terminal transactivation domain. Most patients with CMPS reported previously died within the neonatal period. Our findings that the patient has survived, although has been in daily need of mechanical ventilation support for 5 years and 3 months despite a severely impaired SOX9 protein, do not support a linear relationship between the type of mutation and severity of the clinical outcome. Publication Types: Case Reports PMID: 11076045 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: Trends Genet. 2000 Nov;16(11):488-94. Mammalian sex reversal and intersexuality: deciphering the sex-determination cascade. Vaiman D, Pailhoux E. Laboratoire de Genetique Biochimique et de Cytogenetique, Departement de Genetique Animale, INRA Centre de Recherches de Jouy-en-Josas, 78352, Jouy-en-Josas, France. vaiman@biotec.jouy.inra.fr The sex-determination cascade constitutes a model of the exquisite mechanisms of gene regulation that lead to the development of mammalian embryos. The discovery of the sex-determining region of the Y chromosome (SRY) in the early 1990s was the first crucial step towards a general understanding of sex determination. Since then, several genes that encode proteins with a role in this cascade, such as WT1, SF-1, SOX9, DAX-1 and WNT4, have been identified. Many of the interactions between these proteins have still to be elucidated, while, no-doubt, others are still to be identified. The study of mammalian intersexes forms a promising way towards the identification of the still-missing genes and a comprehensive view of mammalian sex determination. Intersexuality in the goat, studied for over a century, will, presumably, bring to light new genes involved in the female sex-determination pathway. Publication Types: Review Review, Tutorial PMID: 11074290 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 107: Mech Dev. 2000 Nov;98(1-2):51-70. Large-scale screen for genes involved in gonad development. Wertz K, Herrmann BG. Max-Planck-Institut fur Immunbiologie, Abt. Entwicklungsbiologie, Stubeweg 51, 79108, Freiburg, Germany. The vertebrate gonad develops from the intermediate mesoderm as an initially bipotential organ anlage, the genital ridge. In mammals, Sry acts as a genetic switch towards testis development. Sox9 has been shown to act downstream of Sry in testis development, while Dax1 appears to counteract Sry. Few more genes have been implicated in early gonad development. However, the genetic networks controlling early differentiation events in testis and ovary are still far from being understood. In order to provide a broader basis for the molecular analysis of gonad development, high-throughput gene expression analysis was utilized to identify genes specifically expressed in the gonad. In total, among 138 genes isolated which showed tissue specific expression in the embryo, 79 were detected in the developing gonad or sex ducts. Twenty-seven have not been functionally described before, while 40 represent known genes and 12 are putative mouse orthologues. Forty-five of the latter two groups (86%) have not been described previously in the fetal gonad. In addition, 21 of the gonad specific genes showed sex-dimorphic expression suggesting a role in sex determination and/or gonad differentiation. Eighteen of the latter (86%) have not been described previously in the fetal gonad. In total we provide new data on 72 genes which may play a role in gonad or sex duct development and/or sex determination. Thus we have generated a large gene resource for the investigation of these processes, and demonstrate the suitability of high-throughput gene expression screening for the genetic analysis of organogenesis. PMID: 11044607 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 108: Genesis. 2000 Aug;27(4):145-52. Absence of SOX3 in the developing marsupial gonad is not consistent with a conserved role in mammalian sex determination. Pask AJ, Harry JL, Renfree MB, Marshall Graves JA. Department of Zoology, The University of Melbourne, Victoria, Australia. a.pask@zoology.unimelb.edu.au Expression of Sox3 has been detected in the testes of humans and of developing and adult mice at the same time as Sox9 and Sry. The co-expression of these three related Sox genes in the mouse indifferent gonadal ridge led to the hypothesis that these three genes, encoding transcription factors with similar DNA target binding sites, may interact with each other in initiating testis differentiation. The location of SOX3 on the marsupial Dunnart X chromosome also makes it a candidate for the marsupial X-linked gene responsible for the SRY- and hormone-independent initiation of scrotum or mammary gland development. Here we show that although marsupial SOX3 is highly conserved at the genetic level and appears to have a conserved role in CNS development, its expression during sexual differentiation differs from that of mice and humans. SOX3 expression is absent from the developing marsupial genital ridge and from the scrotal and mammary primordia during the critical time of differentiation and throughout the time that SRY is expressed. The absence of expression in the developing gonad strongly suggests that SOX3 does not have a conserved role in mammalian sexual determination or differentiation. Copyright 2000 Wiley-Liss, Inc. PMID: 10992324 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 109: Hum Genet. 2000 Mar;106(3):269-76. SOX14 is a candidate gene for limb defects associated with BPES and Mobius syndrome. Wilmore HP, Smith MJ, Wilcox SA, Bell KM, Sinclair AH. Department of Paediatrics and Centre for Hormone Research, The University of Melbourne, Royal Children's Hospital, Australia. Members of the SOX gene family encode proteins with homology to the HMG box DNA-binding domain of SRY, the Y-linked testis-determining gene. SOX genes are expressed during embryogenesis and are involved in the development of a wide range of different tissues. Mutations in SRY, SOX9 and SOX10 have been shown to be responsible for XY sex reversal, campomelic dysplasia and Waardenburg-Hirschsprung disease, respectively. It is likely that mutations in other SOX genes are responsible for a variety of human genetic diseases. SOX14 has been identified from a human genomic library and the mouse and chicken sequences obtained by polymerase chain reaction amplification. The SOX14 amino acid sequence is highly conserved across these species, suggesting an important role for this protein in vertebrate development. SOX14 is expressed in the neural tube and apical ectodermal ridge of the developing chicken limb. This is the only SOX gene known to be expressed in the apical ectodermal ridge, a structure that directs outgrowth of the embryonic limb bud. Human SOX14 is localised to a 1.15-Mb yeast artificial chromosome on chromosome 3q23, close to loci for BPES (blepharophimosis, ptosis, epicanthus inversus syndrome) and Mobius syndrome. Although SOX14 maps outside these loci, its expression pattern and chromosomal localisation suggest that it is a candidate gene for the limb defects frequently associated with these syndromes. PMID: 10798354 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 110: Dev Dyn. 2000 Mar;217(3):293-8. Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development. de Santa Barbara P, Moniot B, Poulat F, Berta P. Human Molecular Genetics Group, Institut de Genetique Humaine, Montpellier, France. Many transcription factors have been identified and implicated in male sex determination pathway. Specifically involved in Sertoli cell differentiation and subsequent anti-Mullerian hormone (AMH) secretion in eutherian mammals, they include steroidogenic factor-1 (SF-1), SOX9 (SRY HMG box related gene 9), WT1 (Wilms' tumor 1), and GATA-4 (a zinc finger transcription factor). These factors have been described to execute their function in the male sex determination pathway by controlling AMH transcriptional expression. To understand the hierarchies of these factors and their involvement in the developing testis, for the first time we show the expression and subcellular localization of these factors by immunohistochemistry in the early human testis during embryogenesis compared with AMH expression. If these studies do not refute their possible synergistic interaction to control AMH expression in human embryo, they also reveal a new sexual dimorphism in SOX9 expression during the sex determination process. We show that SOX9 sex specifically shifts from the cytoplasmic to the nuclear compartment at the time of testis differentiation and AMH expression. Putative models for this subcellular distribution are discussed. PMID: 10741423 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 111: Am J Med Genet. 1999 Dec 29;89(4):176-85. Sex determination and the Y chromosome. McElreavey K, Fellous M. Immunogenetique Humaine, Institut Pasteur, 25 rue du Dr Roux 75724 Paris Cedex 15, France. kenmce@pasteur.fr Although SRY was first identified 10 years ago, we still know remarkably little about its mode of action or downstream target genes. Recently, potential protein partners have been identified and there has been considerable activity to understand the roles of WT1, SF-1, DAX-1 and SOX9 in gonadogenesis. The emerging picture is one of complex interactions, involving both positive and negative regulatory signals that, depending on the cellular and promoter context, drive the expression of male-specific genes. Despite recent advances, however, we are still unable to explain the genetic cause of most cases of 46,XY gonadal dysgenesis or even a single case of Y-chromosome-negative 46,XX maleness. Copyright 2000 Wiley-Liss, Inc. Publication Types: Review Review, Tutorial PMID: 10727993 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 112: Mech Dev. 2000 Mar 1;91(1-2):403-7. SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. Hanley NA, Hagan DM, Clement-Jones M, Ball SG, Strachan T, Salas-Cortes L, McElreavey K, Lindsay S, Robson S, Bullen P, Ostrer H, Wilson DI. School of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, UK. nhanle@mednet.swmed.edu SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated (SRY, SOX9) or over-expressed (DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination. PMID: 10704874 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 113: Nucleic Acids Res. 2000 Mar 15;28(6):1473-80. Cloning and characterisation of the Sry-related transcription factor gene Sox8. Schepers GE, Bullejos M, Hosking BM, Koopman P. Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, Queensland 4072, Australia. SOX proteins form a large family of transcription factors related by a DNA-binding domain known as the HMG box. Some 30 Sox genes have been identified in mammals and orthologues have been found in a wide range of other metazoans. Sox genes are highly conserved and are known to play important roles in embryonic development, including roles in gonadal, central nervous system, neural crest and skeletal development. Several SOX genes have been implicated in human congenital diseases. We report here the isolation of Sox8 and its characterisation in mice and humans. This gene has a remarkably similar primary structure and genomic organisation to the campomelic dysplasia gene SOX9 and the Waardenburg-Shah syndrome gene SOX10. SOX8 protein is able to bind to canonical SOX target DNA sequences and activate transcription in vitro through two separate trans -activation regions. Further, Sox8 is expressed in the central nervous system, limbs, kidneys, gonads and craniofacial structures during mouse embryo development. Sox8 maps to the t complex on mouse chromosome 17 and to human chromosome 16p13.3, a region associated with the microphthalmia-cataract syndrome CATM and the alpha-thalassemia/mental retardation syndrome ATR-16. PMID: 10684944 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 114: FEBS Lett. 2000 Jan 28;466(2-3):249-54. Two Sox9 messenger RNA isoforms: isolation of cDNAs and their expression during gonadal development in the frog Rana rugosa. Takase M, Noguchi S, Nakamura M. Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Japan. Sox is a family of SRY-related testis-determining genes. We have isolated two different mRNA isoforms of the frog Sox9 gene from adult frog testis cDNAs. One form (Sox9 alpha) encodes a 482 amino acid protein containing the HMG box, whereas the other form (Sox9 beta), which completely lacks the HMG box, is a truncated 265 amino acid protein of Sox9 alpha. Sox9 alpha is 82% similar to mouse, 86% to chicken, and 77% to trout Sox9 at the amino acid level. Sox9 expression was upregulated in embryos after stage 16, and was seen in both developing testes and ovaries. The size of Sox9 transcripts was determined to be 7.8 knt by Northern blot analysis. In addition, Sox9 alpha expression was found prominently in the testis and brain among various tissues of adult frogs examined, and was considerably higher than Sox9 beta. The fact that Sox9 is expressed in both sexes suggests that this gene is involved in gonadal development of male and female frogs. This is dissimilar to the pattern in birds and mammals, in which Sox9 expression is male-specific. PMID: 10682837 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 115: Dev Dyn. 1999 Dec;216(4-5):411-9. Temperature-dependent sex determination in the American alligator: AMH precedes SOX9 expression. Western PS, Harry JL, Graves JA, Sinclair AH. Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Victoria, Australia. Gonadal morphogenesis is very similar among mammals, birds, and reptiles. Despite this similarity, each group utilises quite different genetic triggers for sex determination. In mammals, testis development is initiated by action of the Y-chromosome gene SRY. Current evidence suggests that SRY may act together with a related gene, SOX9, to activate another gene(s) in the pathway of testicular differentiation. A downstream candidate for regulation by SRY and SOX9 is AMH. In mouse, Sox9 is expressed in the Sertoli cells of the embryonic testis and it precedes the onset of Amh expression. During mouse gonadogenesis, Amh is confined to the embryonic testis, although it later shows postnatal expression in the ovary. Reptiles such as the American alligator, which exhibit temperature-dependent sex determination (TSD) do not have dimorphic sex chromosomes and apparently no SRY orthologue. SOX9 is expressed during testis differentiation in the alligator; however, it appears to be expressed too late to cause testis determination. Here we describe the cloning and expression of the alligator AMH gene and show that AMH expression precedes SOX9 expression during testis differentiation. This is the opposite to that observed in the mouse where SOX9 precedes AMH expression. The data presented here, as well as findings from recent expression studies in the chick, suggest that AMH expression is not regulated by SOX9 in the non-mammalian vertebrates. PMID: 10633860 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 116: Am J Med Genet. 1999 Dec 3;87(4):349-53. Autosomal XX sex reversal caused by duplication of SOX9. Huang B, Wang S, Ning Y, Lamb AN, Bartley J. Genzyme Genetics, Orange, California, USA. SOX9 is one of the genes that play critical roles in male sexual differentiation. Mutations of SOX9 leading to haploinsufficiency can cause campomelic dysplasia and XY sex reversal. We report here evidence supporting that SOX9 duplication can cause XX sex reversal. A newborn infant was referred for genetic evaluation because of abnormal male external genitalia. The infant had severe penile/scrotal hypospadias. Gonads were palpable. Cytogenetic analysis demonstrated a de novo mosaic 46,XX,dup(17)(q23.1q24.3)/46, XX karyotype. Fluorescent in situ hybridization (FISH) with a BAC clone containing the SOX9 gene demonstrated that the SOX9 gene is duplicated on the rearranged chromosome 17. The presence of SRY was ruled out by FISH with a probe containing the SRY gene and polymerase chain reaction with SRY-specific primers. Microsatellite analysis with 13 markers on 17q23-24 determined that the duplication is maternal in origin and defined the boundary of the duplication to be approximately 12 centimorgans (cM) proximal and 4 cM distal to the SOX9 gene. Thus, SOX9 duplication is the most likely cause for the sex reversal in this case because it plays an important role in male sex determination and differentiation. This study suggests that extra dose of SOX9 is sufficient to initiate testis differentiation in the absence of SRY. Other SRY-negative XX sex-reversed individuals deserve thorough investigation of SOX9 gene. Copyright 1999 Wiley-Liss, Inc. Publication Types: Case Reports PMID: 10588843 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 117: Biol Reprod. 1999 Sep;61(3):741-8. Reverse transcription-polymerase chain reaction analysis of genes involved in gonadal differentiation in pigs. Parma P, Pailhoux E, Cotinot C. Laboratoire de Biologie cellulaire et moleculaire, INRA, 78350 Jouy en Josas, France. In mammals, testis development is initiated in the embryo as a response to the expression of the sex-determining gene, SRY. The time course of SRY expression during gonadal differentiation in the male has been described in detail only in mice and sheep. In this study, we used reverse transcription-polymerase chain reaction analysis to define the SRY transcription profile in pig genital ridges. SRY transcripts were first detectable from 23 days postcoitum (dpc), then declined sharply after 35 dpc. None were detected at 60 dpc. In addition, we analyzed temporal expression of other genes known to be involved in mammalian sex determination: WT-1, SF-1, SOX9, and AMH. A key stage seems to be 28 dpc, in which SOX9 expression switches between the male and female, and AMH expression begins to attest to Sertoli cell differentiation and to correspond to seminiferous cord formation in the male. Expression of gonadotropin receptors and aromatase was also investigated in porcine gonads, and we showed that their transcripts were detected very early on, especially in the male: 25 dpc for the LH receptor (rLH) and aromatase, and 28 dpc for the FSH receptor (rFSH). In the female, aromatase transcripts were not detected until 70 dpc, and rFSH expression occurred later: at 45 dpc at the onset of meiosis. Moreover, no difference was observed between the sexes for the onset of rLH transcription at 25 dpc. Such a thorough study has never been performed on pigs; developmental analysis will be useful for investigating sex-reversed gonads and determining ontogeny in intersexuality, a common pathology in pigs. PMID: 10456852 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 118: Cell Mol Life Sci. 1999 Jun;55(6-7):839-56. Sry and Sox9: mammalian testis-determining genes. Koopman P. Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia. p.koopman@cmcb.uq.edu.au Sry is the Y-chromosomal gene that acts as a trigger for male development in mammalian embryos. This gene encodes a high mobility group (HMG) box transcription factor that is known to bind to specific target sequences in DNA and to cause a bend in the chromatin. DNA bending appears to be part of the mechanism by which Sry influences transcription of genes downstream in a cascade of gene regulation leading to maleness, but the direct targets of Sry remain to be positively identified. One gene known to be downstream from Sry in this cascade is Sox9, which encodes a transcription factor related to Sry by the HMG box. Like Sry, mutations in Sox9 disrupt male development, but unlike Sry, the role of Sox9 is not limited to mammals. This review focuses on what is known about the two genes and their likely modes of action, and draws together recent data relating to how they might interconnect with the network of gene activity implicated in testis determination in mammals. Publication Types: Review PMID: 10412367 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 119: Cell Mol Life Sci. 1999 Jun;55(6-7):824-30. The one-to-four rule and paralogues of sex-determining genes. Ohno S. Beckman Research Institute of the City of Hope, Duarte, California 91010-3000, USA. Because of two successive rounds of tetraploidization at their inception, the vertebrates contain four times more protein-coding genes in their genome than the invertebrates: 60,000 versus 15,000. Consequently, each invertebrate gene has been amplified to the maximum of four paralogous genes in vertebrates: the one-to-four rule. When this rule is applied to genes pertinent to gonadal development and differentiation, the following emerged: (i) Two closely related zinc-finger transcription factor genes in invertebrates have been amplified to two paralogous groups in vertebrates. One consisted of EGR1, EGR2, EGR3 and EGR4, whereas the only known paralogue of the other is WT1, which controls the developmental fate of the entire nephric system, and therefore of gonads. Interestingly, EGR1 and WT1 act as antagonists of each other in nephroblastic cells. (ii) SF-1, which controls the fate of two steroid hormone-producing organs, adrenals and gonads, is descended from the invertebrate Ftz-F1 gene, and its only known paralogue is GCNF-1. (iii) The Y-linked SRY, the mammalian testis-determining gene, is a paralogue neither of SOX3 (SRX) nor of SOX9. Its ancient origin suggests that SRY once became extinct in earlier vertebrates, only to revive itself in the mammalian ancestor. (iv) Inasmuch as four paralogues of one invertebrate nuclear receptor gene have differentiated to receptors of androgen, mineralocoticoid, glucocorticoid and progesterone, there should at most be four paralogous estrogen-receptor genes in the vertebrate genome. It is likely that one of them plays a pivotal role in the estrogen-dependent sex-determining mechanism so commonly found among reptiles, amphibians and fish. Publication Types: Review Review, Tutorial PMID: 10412365 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 120: J Mol Evol. 1999 May;48(5):517-27. Diversification pattern of the HMG and SOX family members during evolution. Soullier S, Jay P, Poulat F, Vanacker JM, Berta P, Laudet V. ERS155 du CNRS, Centre de Recherche en Biochimie Macromoleculaire, CNRS, BP5051, Route de Mende, 34293 Montpellier Cedex 5, France. From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans. PMID: 10198118 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 121: Annu Rev Physiol. 1999;61:417-33. Gene interactions in gonadal development. Parker KL, Schedl A, Schimmer BP. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235, USA. kparke@mednet.swmed.edu The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. Although the maturation of sexual function and reproduction occurs after birth, essentially all of the critical developmental steps take place during embryogenesis. Temporally, these steps can be divided into two different phases: sex determination, the initial event that determines whether the gonads will develop as testes or ovaries; and sexual differentiation, the subsequent events that ultimately produce either the male or the female sexual phenotype. A basic tenet of sexual development in mammals is that genetic sex--determined by the presence or absence of the Y chromosome--directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program leading to male sexual differentiation. In the absence of testicular hormones, the pathway of sexual differentiation is female. This chapter reviews the anatomic and cellular changes that constitute sexual differentiation and discusses SRY and other genes, including SF-1, WT1, DAX-1, and SOX9, that play key developmental roles in this process. Dose-dependent interactions among these genes are critical for sex determination and differentiation. Publication Types: Review Review, Tutorial PMID: 10099695 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 122: Dev Dyn. 1999 Mar;214(3):171-7. Temperature-dependent sex determination: upregulation of SOX9 expression after commitment to male development. Western PS, Harry JL, Graves JA, Sinclair AH. Department of Paediatrics and Centre for Hormone Research, University of Melbourne, Royal Children's Hospital, Victoria, Australia. In mammals, birds and reptiles the morphological development of the gonads appear to be conserved. This conservation is evident despite the different sex determining switches employed by these vertebrate groups. Mammals exhibit chromosomal sex determination (CSD) where the key sex determining switch is the Y-linked gene, SRY. Although SRY is the trigger for testis determination in mammals, it is not conserved in other vertebrate groups. However, a gene closely related to SRY, the highly conserved transcription factor, SOX9, plays an important role in the testis pathway of mammals and birds. In contrast to the CSD mechanism evident in mammals and birds, many reptiles exhibit temperature dependent sex determination (TSD) where the egg incubation temperature triggers sex determination. Here we examine the expression of SOX9 during gonadogenesis in the American alligator, (Alligator mississippiensis), a reptile that exhibits TSD. Alligator SOX9 is expressed in the embryonic testis but not in the ovary. However, the timing of SOX9 upregulation in the developing testis is not consistent with a role for this gene in the early stages of alligator sex determination. Since SOX9 upregulation in male embryos coincides with the structural organisation of the testis, SOX9 may operate farther downstream in the vertebrate sex differentiation pathway than previously postulated. PMID: 10090144 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 123: Nucleic Acids Res. 1999 Mar 1;27(5):1359-64. The DNA-binding specificity of SOX9 and other SOX proteins. Mertin S, McDowall SG, Harley VR. The Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia. SOX (SRY-related HMG box) proteins are transcription factors that have critical roles in the regulation of numerous developmental processes. They share at least 50% homology in their HMG domains, which bind the DNA element AACAAT. How different SOX proteins achieve specific regulation of target genes is not known. We determined the DNA-binding specificity of SOX9 using a random oligonucleotide selection assay. The optimal SOX9 binding sequence, AGAACAATGG, contained a core DNA-binding element AACAAT, flanked by 5' AG and 3' GG nucleotides. The specific interaction between SOX9 and AGAACAATGG was confirmed by mobility shift assays, DNA competition and dissociation studies. The 5' AG and 3' GG flanking nucleotides enhance binding by SOX9 HMG domain, but not by the HMG domain of another SOX factor, SRY. For SRY, different 5' and 3' flanking nucleotides are preferred. Our studies support the notion that SOX proteins achieve DNA sequence specificity through subtle preferences for flanking nucleotides and that this is likely to be dictated by signature amino acids in their HMG domains. Furthermore, the related HMG domains of SOX9 and Sox17 have similar optimal binding sites that differ from those of SRY and Sox5, suggesting that SOX factors may co-evolve with their DNA targets to achieve specificity. PMID: 9973626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 124: Baillieres Clin Endocrinol Metab. 1998 Apr;12(1):1-16. Genetic control of gonadal differentiation. Lim HN, Hawkins JR. Department of Paediatrics, University of Cambridge, Addenbrooke's Hospital, UK. The study of naturally occurring mutations in humans and induced mutations in mice that cause sex reversal has been instrumental in the cloning and functional analysis of genes involved in gonadal differentiation. Several genes required for this complex developmental process have now been identified. The genes LIM1, WT1 and FTZ-F1 have been demonstrated to be involved in the formation of the gonads prior to their differentiation as testes or ovaries. Subsequent sex-specific gonadal differentiation appears to be mediated by the SRY and SOX9 genes in the testis, and the DAX-1 gene in the ovary. Publication Types: Review Review, Tutorial PMID: 9890059 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 125: Int J Dev Biol. 1998 Nov;42(8):1143-51. Wilms' tumor suppressor gene (WT1) as a target gene of SRY function in a mouse ES cell line transfected with SRY. Toyooka Y, Tanaka SS, Hirota O, Tanaka S, Takagi N, Yamanouchi K, Tojo H, Tachi C. Developmental Biology Group, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan. With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells. PMID: 9879712 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 126: Bioessays. 1998 Sep;20(9):696-9. Mammalian sex determination: joining pieces of the genetic puzzle. Jimenez R, Burgos M. Departamento de Genetica, Facultad de Ciencias, Universidad de Granada, Spain. rjimenez@goliat.ugr.es Mammalian sex determination is controlled by the Y-linked gene SRY. Studies of sex-reversed patients and experimental data obtained with mice have identified other genes, such as DAX1, SOX9, SF1, and WT1, which take part in the process, and have suggested how these genes interact to determine the sex of a mammalian embryo. A recent paper in Nature by Swain et al. provides experimental data that basically confirm the previously proposed hypothesis that SRY acts by inhibiting the action of DSS/DAX1, which is a repressor of genes of the male pathway. Publication Types: Review Review, Tutorial PMID: 9819558 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 127: Mol Genet Metab. 1998 Oct;65(2):74-84. Mammalian sex determination: from gonads to brain. Vilain E, McCabe ER. Department of Human Genetics, UCLA School of Medicine and UCLA Children's Hospital, Los Angeles, California, 90095-1752, USA. In mammals, sex is determined by the Y chromosome, which encodes a testis-determining factor (TDF). This factor causes the undifferentiated embryonic gonads to develop as testes rather than ovaries. The testes subsequently produce the male sex hormones that are responsible for all male sexual characteristics. In 1990, the sex-determining gene, TDF, was identified and termed SRY in humans (Sry in mice). It encodes a protein containing a high mobility group (HMG) motif, which confers the ability to bind and to bend DNA. Genetic evidence supporting SRY as TDF came from the observation of a male phenotype in XX mice transgenic for a small genomic fragment containing Sry, and from the study of XY sex-reversed individuals who harbor de novo mutations in the SRY coding sequence. Other non-Y-linked genes involved in sex determination were subsequently found by genetic analysis of XY sex-reversed patients not explained by mutations in SRY. These genes are WT1, SF1, DAX1, and SOX9. A regulatory cascade hypothesis for mammalian sex determination, proposing that SRY represses a negative regulator of male development, was recently supported by observation of mice that expressed a DAX1 transgene and developed as XY sex-reversed females. The role of some sex-determining genes, such as DAX1 and SF1, in the development of the entire reproductive axis, a functionally integrated endocrine axis, leads to a new concept. Normal sexual development may result from the functional and developmental integration of a number of different genes that play roles in sex determination, sexual differentiation, and sexual behavior. Copyright 1998 Academic Press. Publication Types: Review Review, Tutorial PMID: 9787099 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 128: Mol Cell Biol. 1998 Nov;18(11):6653-65. Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Mullerian hormone gene. De Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M, Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P. Centre de Recherche de Biochime Macromoleculaire, CNRS UPR1142, 34293 Montpellier cedex 5, France. For proper male sexual differentiation, anti-Mullerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Mullerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis. PMID: 9774680 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 129: Hum Genet. 1998 Aug;103(2):115-23. The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor. Pusch C, Hustert E, Pfeifer D, Sudbeck P, Kist R, Roe B, Wang Z, Balling R, Blin N, Scherer G. Institute of Anthropology and Human Genetics, University of Tubingen, Germany. The SOX genes form a gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. We have cloned and sequenced the SOX10 and Sox10 genes from human and mouse, respectively. Both genes encode proteins of 466 amino acids with 98% sequence identity. Significant expression of the 2.9-kb human SOX10 mRNA is observed in fetal brain and in adult brain, heart, small intestine and colon. Strong expression of Sox10 occurs throughout the peripheral nervous system during mouse embryonic development. SOX10 shows an overall amino acid sequence identity of 59% to SOX9. Like SOX9, SOX10 has a potent transcription activation domain at its C-terminus and is therefore likely to function as a transcription factor. Whereas SOX9 maps to 17q, a SOX10 cosmid has previously been mapped by us to the region 22q13.1. Mutations in SOX10 have recently been identified as one cause of Waardenburg-Hirschsprung disease in humans, while a Sox10 mutation underlies the mouse mutant Dom, a murine Hirschsprung model. PMID: 9760192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 130: Am J Hum Genet. 1998 Sep;63(3):794-802. A gene involved in XY sex reversal is located on chromosome 9, distal to marker D9S1779. Flejter WL, Fergestad J, Gorski J, Varvill T, Chandrasekharappa S. Department of Pediatrics, University of Utah, Salt Lake City, Utah, USA. wflejter@wfubmc.edu The genetic mechanisms involved in sex differentiation are poorly understood, and progress in identification of the genes involved has been slow. The fortuitous finding of chromosomal rearrangements in association with a sex-reversed phenotype has led to the isolation of SRY and SOX9, both shown to be involved in the sex-determining pathway. In addition, duplications of the X chromosome, deletions of chromosomes 9 and 10, and translocations involving chromosome 17 have been reported to be associated with abnormal testicular differentiation, leading to male-to-female sex reversal in 46,XY individuals. We present the cytogenetic and molecular analyses of four sex-reversed XY females, each with gonadal dysgenesis and other variable malformations, and with terminal deletions of distal chromosome 9p, resulting from unbalanced autosomal translocations. PCR amplification and DNA sequence analysis of SRY revealed no mutations in the high-mobility-group domain (i.e., HMG box) in any of the four patients. Conventional and molecular cytogenetic analyses of metaphase chromosomes from each patient suggest that the smallest region of overlap (SRO) of deletions involves a very small region of distal band 9p24. Loss-of-heterozygosity studies using 17 highly polymorphic microsatellite markers, as well as FISH using YAC clones corresponding to the most distal markers on 9p, showed that the SRO lies distal to marker D9S1779. These results significantly narrow the putative sex-determining gene to the very terminal region of the short arm of chromosome 9. Publication Types: Case Reports PMID: 9718346 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 131: Dev Dyn. 1998 Aug;212(4):522-32. Early expression of AMH in chicken embryonic gonads precedes testicular SOX9 expression. Oreal E, Pieau C, Mattei MG, Josso N, Picard JY, Carre-Eusebe D, Magre S. Unite de Recherches sur l'Endocrinologie du Developpement, INSERM U493, Ecole Normale Superieure, Montrouge, France. In mammals, anti-Mullerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells only after birth. SOX9, a transcription factor related to the testis-determining factor SRY, is expressed in mouse testis 1 day before AMH. To determine the relationship between AMH and SOX9 in birds, we cloned the AMH promoter in search of SOX9 response elements, and we compared the expression of AMH and SOX9 in the gonads of chick embryos using in situ hybridization. Potential SOX response elements were found in the AMH promoter; however, AMH is expressed in both sexes at stage 25, 1 day before the first SOX9 transcripts appear in the male gonads. SOX9 is never expressed in the female. These results do not support the hypothesis that SOX9 could trigger the expression of testicular AMH in the chick but does not exclude a later role in testis development. PMID: 9707325 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 132: J Med Genet. 1998 Jul;35(7):597-9. Acampomelic campomelic dysplasia with de novo 5q;17q reciprocal translocation and severe phenotype. Savarirayan R, Bankier A. Victorian Clinical Genetics Service, Royal Children's Hospital, Parkville, Australia. Campomelic dysplasia (CD) is a rare skeletal malformation syndrome caused by mutations in the SRY related gene SOX9, mapped to 17q24.3-q25.1. A small proportion of cases are associated with structural rearrangements involving 17q and it has been proposed that this subgroup have a milder phenotype and better prognosis compared to those with mutations in the SOX9 gene. We report a severely affected infant with the acampomelic form of campomelic dysplasia, who died at 11 days and was found to have a de novo reciprocal translocation, 46,XX,t(5;17)(q15;q25.1). This is the second reported case of severe campomelic dysplasia associated with a structural rearrangement involving 17q and suggests that this subgroup of patients may not significantly differ from those without chromosomal rearrangements with regards to phenotype or prognosis. Publication Types: Case Reports PMID: 9678706 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 133: J Exp Zool. 1998 Aug 1;281(5):472-81. Evolution of the mammalian Y chromosome and sex-determining genes. Graves JA. School of Genetics and Human Variation, La Trobe University, Melbourne, Victoria, Australia. GENJMG@genome.latrobe.edu.au In mammals, male sex determination, as well as spermatogenesis, is controlled by genes on the Y chromosome. Evolutionary comparisons may be used to detect and test candidate genes for these functions, under the hypothesis that the rapid evolution of the mammalian Y chromosome causes it to contain few genes other than those with a critical function in male reproduction. Comparisons of the gene content of sex chromosomes from the three major groups of extant mammals (placentals, marsupials, and monotremes) show that part of the X chromosome, and a corresponding region of the Y, is shared by all mammals and must be very ancient, but part was added relatively recently. Evolution of the mammalian Y took place in several cycles of addition and attrition, as autosomal regions were added to the pseudoautosomal region of one sex chromosome, recombined onto the other, and degraded on the Y. This explains why most genes and pseudogenes on the Y chromosome have relatives on the X. The gene SRY itself is apparently no exception, being closely related to the highly conserved X-linked gene SOX3. Comparisons of SRY/SOX base sequence and gene location in the three groups of mammals suggest that SRY evolved from SOX3 relatively recently by mutation and loss of all sequences outside the HMG box. It is suggested here that, rather than acting as a transcriptional activator, the SRY gene acts to inhibit its paralogue SOX3, which in turn inhibits an ancient autosomal sex-determining gene SOX9. Publication Types: Review Review, Tutorial PMID: 9662834 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 134: Rev Invest Clin. 1998 Mar-Apr;50(2):171-6. [Genetic heterogeneity and phenotypic variability in 46,XY sex reversal] [Article in Spanish] Lopez-Lopez M, Zenteno JC, Mendez JP, Kofman-Alfaro S. Servicio de Genetica, Hospital General de Mexico-Facultad de Medicina UNAM, Mexico D.F. The discordance between the chromosomic and the gonadal-phenotypic sex is known as sex reversal (XX males and XY females). We review the XY pure gonadal dysgenesis characterized by female phenotype, primary amenorrhea and absence of secondary sexual development. Bilateral streak gonads are always present in the complete form of this syndrome, while variable degrees of virilization are found in the partial forms, depending on the severity of the testicular damage. A plausible explanation for this pathology are SRY mutations that interfere with the testicular differentiation. However, only 10-15% of the patients with the complete form show SRY mutations, particularly in the HMG box. The remaining cases are probably due to mutations in different autosomal or X-linked genes which are also involved in the sex differentiation cascade. Recently, it has been shown that mutations in several genes responsible of well known genetic entities as WT1, SOX9, DSS and SF1, result in sex reversal. These findings reveal the genetic heterogeneity and clinical variability of XY sex reversal and provide the basis establishing a hierarchy of genes and their participation in the sex determination pathway. Publication Types: Review Review, Tutorial PMID: 9658940 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 135: Bioessays. 1998 Mar;20(3):264-9. Interactions between SRY and SOX genes in mammalian sex determination. Graves JA. School of Genetics and Human Variation, LaTrobe University, Melbourne, Victoria, Australia. genjmg@genome.latrobe.edu.au The SRY gene on the mammalian Y chromosome undoubtedly acts to determine testis, but it is still quite unclear how. It was originally supposed that SRY acts directly to activate other genes in the testis-determining pathway. This paper presents an alternative hypothesis that SRY functions indirectly, by interacting with related genes SOX3 (from which SRY evolved) and SOX9 (which appears to be intimately involved in vertebrate gonad differentiation). Specifically, I propose that in females SOX3 inhibits SOX9 function, but in males, SRY inhibits SOX3 and permits SOX9 to enact its testis-determining role. This hypothesis makes testable predictions of the phenotypes of XX and XY individuals with deficiencies or overproduction of any of the three genes, and is able to account for the difficult cases of XX(SRY-) males and transdifferentiation in the absence of SRY. The hypothesis also suggests a way that the dominant SRY sex-determining system of present-day mammals may have evolved from an ancient system relying on SOX3 dosage. Publication Types: Review Review, Tutorial PMID: 9631654 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 136: J Biol Chem. 1998 Jun 12;273(24):14989-97. Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo. Zhou G, Lefebvre V, Zhang Z, Eberspaecher H, de Crombrugghe B. Department of Molecular Genetics, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-alpha1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1-3) that were essential for its chondrocyte-specific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bp Col2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as larger Col2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocyte-specific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage. PMID: 9614106 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 137: Matrix Biol. 1998 Mar;16(9):529-40. Toward understanding SOX9 function in chondrocyte differentiation. Lefebvre V, de Crombrugghe B. Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, USA. The transcription factors that trigger the determinative switch to chondrocyte differentiation in mesenchymal cells are still unknown. In humans, mutations in the gene for SOX9, a transcription factor with a DNA-binding domain similar to that of the mammalian testis-determining factor SRY, cause campomelic dysplasia, a severe dwarfism syndrome which affects all cartilage-derived structures. During mouse embryonic development, the Sox9 gene becomes active in all prechondrocytic mesenchymal condensations, and at later stages its expression is maintained at high levels in fully differentiated chondrocytes. A chondrocyte-specific enhancer in the gene for collagen type II (Col2a1), a characteristic marker of chondrocytes, is a direct target for SOX9, and ectopic expression of SOX9 in transgenic mouse embryos is sufficient to activate the endogenous Col2a1 gene in some tissues. These data suggest that SOX9 could have a major role in chondrogenesis. Studies are in progress to identify other target genes for SOX9 in chondrocytes and also other transcription factors that are believed to cooperate with SOX9 in the activation of chondrocyte-specific genes. Defining SOX9 function and the mechanisms that regulate SOX9 gene expression should contribute to a better understanding of chondrocyte differentiation. Publication Types: Review Review, Tutorial PMID: 9569122 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 138: Annu Rev Physiol. 1998;60:497-523. Sex in the 90s: SRY and the switch to the male pathway. Capel B. Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. B.Capel@cellbio.duke.edu In mammals the male sex determination switch is controlled by a single gene on the Y chromosome, SRY. SRY encodes a protein with an HMG-like DNA-binding domain, which probably acts as a local organizer of chromatin structure. It is believed to regulate downstream genes in the sex determination cascade, although no direct targets of SRY are clearly known. More genes in the pathway have been isolated through mutation approaches in mouse and human. At least three genes, SRY itself, SOX9, and DAX1, are dosage sensitive, providing molecular evidence that the sex determination step operates at a critical threshold. SRY initiates development of a testis from the bipotential cells of the early gonad. The dimorphic male and female pathways present a rare opportunity to link a pivotal gene in development with morphogenetic mechanisms that operate to pattern an organ and the differentiation of its cells. Mechanisms of testis organogenesis triggered downstream of SRY include pathways of cell signaling controlling cell reorganization, cell proliferation, cell migration, and vascularization. Publication Types: Review PMID: 9558474 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 139: Eur J Oral Sci. 1998 Jan;106 Suppl 1:112-6. Expression of the transcription factors Otlx2, Barx1 and Sox9 during mouse odontogenesis. Mitsiadis TA, Mucchielli ML, Raffo S, Proust JP, Koopman P, Goridis C. Laboratoire de Genetique et Physiologie du Developpement, IBDM, Campus de Luminy case 907, Marseilles, France. mitsiadi@ibdm.univ-mrs.fr The molecular mechanisms governing the decision between molariform and incisiform patterns of rodent dentition are not yet known. Transcription factors are regulators of regionally specific morphogenesis and key co-ordinators of gene activity during developmental processes. Here, we analysed the expression of several transcription factors during mouse tooth development. Otlx2/Rieg is a homeobox gene involved in Rieger syndrome, a human disorder characterized by dental hypoplasia. Otlx2/Rieg expression distinguishes stomatodeal epithelium well before tooth initiation, and thereafter its expression becomes restricted to the epithelia of both molar and incisor primordia. The recently identified homeodomain transcription factor Barx1 is first expressed in mesenchyme of the first branchial arch, but during advanced developmental stages the gene is exclusively expressed in the mesenchyme of molar primordia. Finally, the Sry-related transcription factor Sox9 is expressed in epithelial components and to a lesser degree in condensed mesenchyme of the developing teeth. These results suggest that Otlx2/Rieg, Barx1, and Sox9 participate in the hierarchical cascade of factors involved in the regulation of tooth morphogenesis. PMID: 9541211 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 140: Acta Paediatr Suppl. 1997 Nov;423:46-9. A molecular approach to sex determination in mammals. Swain A, Lovell-Badge R. Division of Developmental Genetics, MRC National Institute for Medical Research, London, UK. Mammalian sex determination occurs in the gonad of the developing embryo. This process is dependent on the Y-chromosome-encoded Sry gene that acts in the somatic cells of the genital ridge. The transient nature of Sry gene expression suggests that it acts as a switch from one cell fate to another. One of the roles of Sry is to initiate the differentiation of Sertoli cells, which are the first cell type of the testis to be formed. Two genes are thought to be important in Sertoli cell differentiation and function, Sox9, an Sry-related gene, and SF-1, a nuclear hormone receptor. Sox9 is expressed in Sertoli cells throughout development of the mouse embryo, and inactivating mutations in this gene in humans give rise to XY females. SF-1 is also expressed in Sertoli cells and is thought to activate the AMH gene--an early marker of these cells. DAX-1, an X-linked member of the nuclear hormone superfamily, is a candidate for a human condition in which duplication of regions of the X chromosome results in XY females. Expression of this gene during mouse development is associated with ovary development and is down-regulated in the differentiating testis. Mutations in DAX-1 in humans have shown that this gene is not necessary for testis development. The properties of the DAX-1 gene suggest that it is important in ovary determination and might therefore be antagonistic to the action of the Sry gene. Publication Types: Review Review, Tutorial PMID: 9401538 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 141: Nippon Rinsho. 1997 Nov;55(11):2809-15. [The genes in the molecular cascade of the mammalian sexual development] [Article in Japanese] Ikeda Y. Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience. The sex of an individual is established as a consequence of molecular events that occur in the complex genetic cascade. During sexual development, morphological changes are closely associated with the action of genes expression of which is tightly regulated according to the genetic program. Recently, a number of genes that are involved in this process have been isolated. WT-1 and SF-1 are required for the early formation of the gonadal primordium. SRY is the switch to start the male sexual differentiation by triggering the Sertoli cell lineage. An autosomal gene SOX9, which can cause the sex reversal, might also be involved in the male sex determination. SF-1 is a key factor for the male sexual differentiation, regulating the expression of MIS and the synthesis of testosterone in the fetal testis. DAX1 might be responsible for the female sexual development. The mutation or abnormal expression of these regulatory genes and their downstream genes can cause a variety types of the sex reversal and/or malformation of the gonad and the internal and external genitalia. Publication Types: Review Review, Tutorial PMID: 9396269 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 142: J Biol Chem. 1997 Oct 31;272(44):27848-52. Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9. Sudbeck P, Scherer G. Institute of Human Genetics, University of Freiburg, Breisacher Strasse 33, D-79106 Freiburg, Germany. SRY and SOX9, members of the family of high-mobility group (HMG) domain transcription factors, are both essential for testis formation during human embryonic development. The HMG domain is a DNA-binding and DNA-bending motif comprising about 80 amino acid residues. It has been shown that SRY and SOX9 are nuclear proteins. Using normal or mutant SRY-beta-galactosidase and SOX9-beta-galactosidase fusion proteins in transfection studies involving COS-7 cells, we have identified two nuclear localization signals (NLSs) within the HMG domains of both proteins that can independently direct the fusion proteins into the nucleus. Only mutational inactivation of both NLS motifs resulted in complete exclusion of the fusion proteins from the nucleus. The NLS sequences are located at the N and C termini of the HMG domain and are a bipartite NLS motif and a basic cluster NLS motif, respectively. Both NLS motifs are conserved in the HMG domains of other transcription factors. The implications of the present results are discussed regarding (a) the apparent dual function of certain basic amino acid residues in the HMG domain of SRY in both DNA binding and in nuclear localization and (b) the possible control of SOX9 in early gonadal differentiation at the level of nuclear translocation. PMID: 9346931 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 143: Mol Cell Biol. 1997 Apr;17(4):2336-46. SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene. Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B. Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies. PMID: 9121483 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 144: Dev Biol. 1997 Mar 1;183(1):108-21. SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse. Ng LJ, Wheatley S, Muscat GE, Conway-Campbell J, Bowles J, Wright E, Bell DM, Tam PP, Cheah KS, Koopman P. Department of Biochemistry, Hong Kong University. Two lines of evidence suggest that the Sry-related gene Sox9 is important for chondrogenesis in mammalian embryos. Sox9 mRNA is expressed in chondrogenic condensations in mice, and mutations in human SOX9 are known to cause skeletal dysplasia. We show here that mouse SOX9 protein is able to bind to a SOX/SRY consensus motif in DNA and contains a modular transcriptional activation domain, consistent with a role for SOX9 as a transcription factor acting on genes involved in cartilage development. One such gene is Col2a1, which encodes type II collagen, the major structural component of cartilage. We have compared, in detail, the expression of Sox9 and Col2a1 during mouse development. In chondrogenic tissues the expression profiles of the two genes were remarkably similar. Coexpression was detected in some nonchondrogenic tissues such as the notochord, otic vesicle, and neural tube, but others such as heart and lung differed in their expression of the two genes. Immunohistochemistry using an antibody specific for SOX9 revealed that expression of SOX9 protein mirrored the distribution of Sox9 mRNA. Our results suggest that SOX9 protein is involved in the regulation of Col2a1 during chondrogenesis, but that this regulation is likely to depend on additional cofactors. PMID: 9119111 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 145: Genes Cells. 1997 Feb;2(2):95-106. The ontogenesis of the steroidogenic tissues. Morohashi K. Laboratory of Gene Expression and Regulation, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Japan. moro@nibb.ac.jp Based on the common function of steroid hormone-producing tissues and homologous regulation by the hypothalamo-pituitary axis, the adrenal cortex and the gonads have been suggested to have an intimate ontogenic relationship. This assumption is also supported by the findings of common transcription factors implicated in the differentiation of both types of tissue and further supported by concomitant defects in such tissues due to the disruption of a single gene. Similarly, simultaneous anomalies in those tissues are also observed in some diseases caused by mutations of the genes encoding those transcription factors. A recent immunohistochemical study with one of the transcription factors, Ad4BP/SF-1, definitely demonstrated the presence of a particular cell population designated the 'adreno-genital primordium (AGP)' which gives rise to both the adrenal cortex and the gonads. In the process of differentiation from the AGP to the mature adrenal cortex and the gonads of the two sexes, several interesting issues can be raised as to the next targets for the study. To address these issues it is important to elucidate the upstream regulatory mechanisms and downstream target genes of such transcription factors as WT1, SRY, SOX9 and DAX1, in addition to Ad4BP/SF-1, all of which are implicated in steroidogenic tissue differentiation. Publication Types: Review PMID: 9167967 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 146: Cytogenet Cell Genet. 1997;76(1-2):109-14. Mapping in pig of genes involved in sexual differentiation: AMH, WT1, FTZF1, SOX2, SOX9, AHC, and placental and embryonic CYP19. Lahbib-Mansais Y, Barbosa A, Yerle M, Parma P, Milan D, Pailhoux E, Gellin J, Cotinot C. INRA, Laboratoire de Genetique Cellulaire BP27, Castanet-Tolosan, France. Lahbib@toulouse.inra.fr Intersexuality has been reported in pigs and investigations are in progress to identify and physically map the genes involved in sex-reversal. In this study we have mapped on porcine chromosomes seven genes which might be implicated in this developmental pathway. Four genes were mapped by radioactive in situ hybridization: AMH (Anti-Mullerian Hormone) and WT1 (Wilms' Tumor gene 1) were both mapped to pig chromosome 2 (SSC2) in the q14-->q21 and p14-->q11 regions, placental CYP19 (cytochrome P450, subfamily XIX) and FTZF1 (fushi tarazu factor [Drosophila] homolog 1, alias SF1 [steroidogenic factor 1]) to pig chromosome 1 (SSC1) in the q14-->q17 and q210-->q211 regions respectively. Four other genes were regionally located by PCR analysis on a cytogenetically characterized porcine somatic cell hybrid panel: a second CYP19 gene (expressed in embryo) was mapped to porcine 1q12-->q17, AHC (alias DAX1, adrenal hypoplasia congenital) to porcine Xp24, SOX2 and SOX9 (SRY sex determining region Y-box 2 and 9) to 13q23-->q41 and 12p13-->p11 respectively. These results are in global agreement with mapping data available in other mammalian species. PMID: 9154138 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 147: Hum Mutat. 1997;9(5):388-95. Mutations in SRY and SOX9: testis-determining genes. Cameron FJ, Sinclair AH. Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Victoria, Australia. PMID: 9143916 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 148: Clin Endocrinol (Oxf). 1997 Jan;46(1):101-8. Intersex disorders: shedding light on male sexual differentiation beyond SRY. MacLean HE, Warne GL, Zajac JD. Centre for Hormone Research, Royal Children's Hospital, Parkville, Vic, Australia. Male sexual differentiation involves a cascade of events initiated by the presence on the Y chromosome of the SRY gene, which causes the indifferent gonad to develop into a testis. Hormonal products of the testis, predominantly testosterone and Mullerian inhibiting substance (MIS), then control the sexual differentiation of the developing foetus. SRY is a transcription factor; however, target genes for its action have yet to be identified, because the DNA recognition sequence for SRY is found in many genes. Therefore the study of intersex disorders is being used to identify other genes active in the pathway of sexual differentiation. Genes identified as being important in the differentiation of the indifferent gonad include WT1 (abnormal in Denys Drash syndrome) and SF-1. The DSS locus may contain a gene that controls ovarian differentiation, and SOX9 (identified from campomelic dysplasia) is required for testis differentiation. In addition to playing a role in the development of the bipotential gonad, SF-1 may also activate MIS gene expression in the testis, causing regression of Mullerian structures. Luteinizing hormone and its receptor are required for Leydig cell differentiation, and the testosterone biosynthetic enzymes (P450scc, 3 beta-hydroxysteroid dehydrogenase, P45017 alpha and 17 beta-hydroxysteroid dehydrogenase) are all necessary for masculinization of external genitalia. 5 alpha-Reductase is required for the production of dihydrotestosterone, and the androgen receptor mediates the action of both testosterone and dihydrotestosterone. The identification of abnormal genes in other disorders of sexual differentiation is likely to provide further information about the factors required for testicular development and function. Publication Types: Review Review, Tutorial PMID: 9059565 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 149: Genomics. 1997 Jan 1;39(1):30-7. Isolation of a novel Sry-related gene that is expressed in high-metastatic K-1735 murine melanoma cells. Tani M, Shindo-Okada N, Hashimoto Y, Shiroishi T, Takenoshita S, Nagamachi Y, Yokota J. Biology Division, National Cancer Center Research Institute, Tokyo, Japan. To identify genes differentially expressed in association with metastatic potential of K-1735 mouse melanoma cells, the mRNA differential display method was applied to compare mRNAs from high- and low-metastatic K-1735 cells. A novel gene was identified as being expressed in high-metastatic cells but not in low-metastatic cells. Sequence analysis revealed that this gene had an open reading frame of 538 amino acid residues containing a Sry high-mobility group (HMG) box domain, known as a DNA binding motif, particularly in the Sox family genes. This gene showed the highest homology to the human SOX9 gene, which is the responsible gene for an inherited disease, campomelic dysplasia. Thus, this gene was designated the Sox21 gene (the 21st Sox family gene). The Sox21 gene was mapped to mouse Chromosome 15, to which neither genes containing the HMG box region nor the loci of hereditary diseases similar to campomelic dysplasia have been previously mapped. This gene was highly conserved and specifically expressed in the brain. These results suggest that expression of the Sox21 gene is involved in the development of nerve systems and may function to enhance the metastatic potential of K-1735 mouse melanoma cells of nerve cell origin. PMID: 9027483 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 150: Development. 1996 Sep;122(9):2813-22. A male-specific role for SOX9 in vertebrate sex determination. Kent J, Wheatley SC, Andrews JE, Sinclair AH, Koopman P. Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, Australia. Mutation analyses of patients with campomelic dysplasia, a bone dysmorphology and XY sex reversal syndrome, indicate that the SRY-related gene SOX9 is involved in both skeletal development and sex determination. To clarify the role SOX9 plays in vertebrate sex determination, we have investigated its expression during gonad development in mouse and chicken embryos. In the mouse, high levels of Sox9 mRNA were found in male (XY) but not female (XX) genital ridges, and were localised to the sex cords of the developing testis. Purified fetal germ cells lacked Sox9 expression, indicating that Sox9 expression is specific to the Sertoli cell lineage. Sex specificity of SOX9 protein expression was confirmed using a polyclonal antiserum. The timing and cell-type specificity of Sox9 expression suggests that Sox9 may be directly regulated by SRY. Male-specific expression of cSOX9 mRNA during the sex determination period was also observed in chicken genital ridges. The conservation of sexually dimorphic expression in two vertebrate classes which have significant differences in their sex determination mechanisms, points to a fundamental role for SOX9 in testis determination in vertebrates. Sox9 expression was maintained in the mouse testis during fetal and adult life, but no expression was seen at any stage by in situ hybridisation in the developing ovary. Male-specific expression was also observed in the cells surrounding the Mullerian ducts and in the epididymis, and expression in both sexes was detected in the developing collecting ducts of the metanephric kidney. These results suggest that SOX9 may have a wider role in the development of the genitourinary system. PMID: 8787755 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 151: Nat Genet. 1996 Sep;14(1):62-8. Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Morais da Silva S, Hacker A, Harley V, Goodfellow P, Swain A, Lovell-Badge R. Division of Developmental Genetics, MRC National Institute for Medical Research, Mill Hill, London, UK. Heterozygous mutations in SOX9 lead to a human dwarfism syndrome, Campomelic dysplasia. Consistent with a role in sex determination, we find that Sox9 expression closely follows differentiation of Sertoli cells in the mouse testis, in experimental sex reversal when fetal ovaries are grafted to adult kidneys and in the chick where there is no evidence for a Sry gene. Our results imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor, perhaps in all vertebrates. PMID: 8782821 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 152: Acta Paediatr Jpn. 1996 Aug;38(4):405-11. Mutations in SOX9 cause both autosomal sex reversal and campomelic dysplasia. Foster JW. Department of Genetics, University of Cambridge, UK. The human testis determining factor (SRY) has been cloned from the Y chromosome. This gene is a dominant inducer of male differentiation. Mutations in the SRY gene result in an XY individual developing as a sex reversed phenotypic female. Sex reversal in humans can also be caused by mutations located in autosomal or X-linked loci. One such sex-reversing locus (SRAI) is associated with the developmental disorder campomelic dysplasia (CD). Both these syndromes were mapped to human chromosome 17q by the identification of balanced reciprocal translocations in five unrelated patients. The translocation breakpoint of one such XY-female CD patient was mapped and the region surrounding it cloned. The closest distal marker used to map the translocation breakpoint was the SOX9 gene. Because of the close proximity of this gene to the breakpoint, it was subjected to mutation analysis in patients without overt chromosome rearrangements. Analysis of DNA from these patients and their parents identified de novo mutations in the SOX9 gene in patients with both autosomal sex reversal and CD. This showed that mutations in the SOX9 gene are responsible for both syndromes. Publication Types: Review Review, Tutorial PMID: 8840554 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 153: Mol Med. 1996 Jul;2(4):405-12. SOX genes: architects of development. Prior HM, Walter MA. Ocular Genetics Research Group, University of Alberta, Edmonton, Canada. Development in higher organisms involves complex genetic regulation at the molecular level. The emerging picture of development control includes several families of master regulatory genes which can affect the expression of down-stream target genes in developmental cascade pathways. One new family of such development regulators is the SOX gene family. The SOX genes are named for a shared motif called the SRY box a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene. Like SRY, SOX genes play important roles in chordate development. At least a dozen human SOX genes have been identified and partially characterized (Tables 1 and 2). Mutations in SOX9 have recently been linked to campomelic dysplasia and autosomal sex reversal, and other SOX genes may also be associated with human disease. Publication Types: Review Review, Tutorial PMID: 8827711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 154: Mamm Genome. 1996 Jul;7(7):481-5. Exclusion of Sox9 as a candidate for the mouse mutant tail-short. Uchida K, Koopman P, Mita A, Wakana S, Wright E, Kikkawa Y, Yonekawa H, Moriwaki K, Shiroishi T. Mammalian Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima, Shizuoka-ken 411, Japan. The Sry-related gene Sox9 has been proposed as the gene responsible for the mouse skeletal mutant Tail-short (Ts), on the basis of its expression in skeletogenic mesenchymal condensations in the mouse embryo and its chromosomal location in the region of Ts on distal Chromosome (Chr) 11. We present here detailed mapping of Ts locus relative to the Sox9, using an intersubspecific cross. Among 521 backcross progeny, 16 recombinants were detected between Sox9 and Ts, suggesting a separation of 3.5 +/- 0.01 cM, and excluding Sox9 as a candidate for Ts. A further nine recombinants were detected between Ts and the polycomb-like gene M33, suggesting that these loci are separated by 1.8 +/- 0.011 cM. Six microsatellite markers were co-localized to the Ts locus, providing reagents for positional cloning of Ts. PMID: 8672134 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 155: Curr Opin Genet Dev. 1996 Jun;6(3):316-21. Early steps in mammalian sex determination. Ramkissoon Y, Goodfellow P. Department of Genetics, University of Cambridge, UK. ydr@mole.bio.cam.sc.uk Since the cloning of the Y-located testis-determining factor, sex determining region Y (SRY), several other genes have been implicated in the process of mammalian sex determination. Mutations of an SRY-related gene, SRY-related high-mobility group box 9 (SOX9), result in autosomal sex reversal and campomelic dysplasia. The genes Steroidogenic factor 1 (SF1) and Wilms' tumour 1 (WT1) are required for early gonadal development as well as for the formation of adrenals and kidneys respectively. The gene responsible for adrenal hypoplasia congenita, DAX1, is a candidate for the X-linked dosage sensitive sex reversal gene (DSS). Publication Types: Review Review, Tutorial PMID: 8791510 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 156: Genomics. 1996 Jun 1;34(2):193-7. Construction of a mouse whole-genome radiation hybrid panel and application to MMU11. Schmitt K, Foster JW, Feakes RW, Knights C, Davis ME, Spillett DJ, Goodfellow PN. Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, United Kingdom. Whole-genome radiation hybrids have been used to construct human genome maps that integrate different types of markers. To investigate this methodology in mammalian species other than humans, a panel of 164 mouse x hamster whole-genome radiation hybrids was constructed. This set of hybrids was used to produce a high-resolution map of a region on MMU11 that included microsatellite markers and cDNA sequences. The mouse homologue of the human SRY-related gene SOX9 was mapped to an interval of approximately 1.1 cM flanked by the microsatellite markers D11Mit11 and D11Mit291. This interval includes the region containing the mouse Tail-short mutation, a possible homologue of the human syndrome campomelic dysplasia, which is caused by mutations in SOX9. Our results suggest that whole-genome radiation hybrid technology will be a useful adjunct to mapping the genomes of nonhuman mammalian species. PMID: 8661048 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 157: Hum Genet. 1996 Feb;97(2):186-93. Translocation breakpoints in three patients with campomelic dysplasia and autosomal sex reversal map more than 130 kb from SOX9. Wirth J, Wagner T, Meyer J, Pfeiffer RA, Tietze HU, Schempp W, Scherer G. Institut fur Humangenetik und Anthropologie der Universitat, Freiburg, Germany. Campomelic dysplasia (CMPD1) and autosomal XY sex reversal (SRA1) are caused by mutations in the SRY-related gene SOX9 on 17q. Unexpectedly, the 17q breakpoints in four CMPD1 translocation cases previously analyzed by us and others map 50 kb or more from SOX9. Here, we present clinical, cytogenetic, and molecular data from a new CMPD1/SRA1 patient with t(6;17)(q14;q24). Fluorescence in situ hybridization has shown that the 17q breakpoint in this case maps to the same region as the breakpoints in the other translocation cases, at least 130 kb from SOX9. Likewise, the breakpoints in two of the previously described cases also map more than 130 kb and, as shown by pulsed field gel electrophoresis analysis, at most 400 kb or 690 kb from SOX9. By using a SOX9 coding sequence polymorphism, expression of both SOX9 alleles has been demonstrated by the reverse transcriptase polymerase chain reaction in lymphoblastoid cells from one of the translocation cases. Publication Types: Case Reports PMID: 8566951 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 158: Cytogenet Cell Genet. 1996;72(2-3):246-9. SOX20, a new member of the SOX gene family, is located on chromosome 17p13. Meyer J, Wirth J, Held M, Schempp W, Scherer G. Institute of Human Genetics, University of Freiburg, Germany. SOX genes share a high sequence identity with the HMG box present in the testis determining gene SRY. We have identified a HMG box-like sequence motif on six contiguous cosmids, which cross-hybridize to a SOX9 cDNA probe. A data base search revealed a high similarity of the deduced amino acid sequence to the human SOX12 and the murine Sox16 HMG domains. The cosmids were assigned to chromosome 17p13 by FISH analysis. PMID: 8978787 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 159: World J Urol. 1996;14(5):278-94. Molecular determinants of sexual differentiation. Wiener JS, Marcelli M, Lamb DJ. Department of Urology, Baylor College of Medicine, Houston, Texas, USA. The processes of sexual differentiation have been greatly clarified by molecular biologic discoveries over the past five years. Gonadal differentiation into a testis or ovaries is controlled by a multitude of genes beginning with SRY which is believed to represent the testis determining factor. Other genes involved include SF-1, WT-1, DAX-1, and SOX9. The fully developed testis produces Mullerian inhibiting substance and testosterone to create the male phenotype; the female phenotype develops in their absence. This hormonally-driven process also requires additional factors and appropriate receptors. Errors in this pathway may be manifested clinically as intersex disorders, and the study of these disorders has helped to further elucidate the molecular mechanisms of sexual differentiation. Publication Types: Review PMID: 8912468 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 160: Heredity. 1995 Dec;75 ( Pt 6):599-611. The genetic basis of murine and human sex determination: a review. McElreavey K, Barbaux S, Ion A, Fellous M. Immunogenetique Humaine, Institut Pasteur, Paris, France. Determination of mammalian sex depends on the presence or absence of a functional testis. Testes are determined by the activity of the testis determining factor encoded by the sex determining gene, Y (SRY) located on the Y chromosome. Considerable evidence suggests that the SRY gene is the only gene on the Y chromosome that is both necessary and sufficient to initiate testis determination. Other steps in the mammalian sex determining pathway are unknown, although recent advances have shown that mutations in X chromosome and autosomal loci are also associated with sex reversal, suggesting the presence of at least one other sex determining gene. Duplications of sequences on the short arm of the human X chromosome, including the DAX-1 (DSS-AHC critical region on the X chromosome, gene 1) gene, are occasionally associated with XY male-to-female sex reversal. In addition, mutations in the SRY-related gene SOX9 (SRY-related box) are associated with a failure of human testicular determination. Furthermore, the occurrence of inherited sex reversed conditions in both mice and men indicate the presence of at least one other sex determining gene. Breeding the Y chromosome from certain Mus musculus domesticus strains into the laboratory mouse strain C57BL/6J results in XY male-to-female sex reversal. This suggests both allelic variation of the Sry gene and the presence of autosomal sex determining genes. In humans, familial cases of SRY-negative XX males occur. Analysis of the transmission of the trait indicates the segregation of an autosomal or X-linked recessive mutation. The mutation may be in a gene whose wild-type function is to inhibit male sex determination. SRY may trigger male sex determination by repressing or functionally antagonizing the product of this gene. Publication Types: Review Review, Tutorial PMID: 8575930 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 161: Philos Trans R Soc Lond B Biol Sci. 1995 Nov 29;350(1333):271-7; discussion 277-8. The role of SOX9 in autosomal sex reversal and campomelic dysplasia. Schafer AJ, Dominguez-Steglich MA, Guioli S, Kwok C, Weller PA, Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN, et al. Department of Genetics, University of Cambridge, U.K. In eutherian mammals, the Y-chromosome gene SRY is required for induction of testis development. Although the Y chromosome is sex determining, loci located elsewhere in the genome participate in the complex cascade of genetic interactions required to form a testis. Male to female sex reversal (46,XY females) occurs at a high frequency in individuals afflicted with the skeletal malformation syndrome campomelic dysplasia. Chromosomal translocations in individuals with both syndromes had localized an autosomal sex reversal locus (SRA1) and a campomelic dysplasia locus (CMPD1) to the long arm of human chromosome 17. The molecular cloning of a translocation breakpoint in a sex reversed campomelic dysplasia patient revealed its proximity to SOX9, a gene which is related to SRY. Analysis of SO X9 in patients without chromosomal rearrangements demonstrated single allele mutations in sex reversed campomelic individuals, linking this gene with both bone formation and control of testis development. Identification of SO X9 as SRA1/CMPD1 and the role of SO X9 mutations in sex reversal and campomelic dysplasia are discussed. PMID: 8570691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 162: Nat Genet. 1995 Jan;9(1):15-20. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, Gangadharan U, Greenfield A, Koopman P. Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia. Mutations in the human SRY-related gene, SOX9, located on chromosome 17, have recently been associated with the sex reversal and skeletal dysmorphology syndrome, campomelic dysplasia. In order to clarify the role of this gene in skeletal development, we have studied the expression of mouse Sox9 during embryogenesis. Sox9 is expressed predominantly in mesenchymal condensations throughout the embryo before and during the deposition of cartilage, consistent with a primary role in skeletal formation. Interspecific backcross mapping has localized mouse Sox9 to distal chromosome 11. The expression pattern and chromosomal location of Sox9 suggest that it may be the gene defective in the mouse skeletal mutant Tail-short, a potential animal model for campomelic dysplasia. PMID: 7704017 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 163: Cell. 1994 Dec 16;79(6):1111-20. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E, et al. Institute of Human Genetics, University of Freiburg, Federal Republic of Germany. A human autosomal XY sex reversal locus, SRA1, associated with the skeletal malformation syndrome campomelic dysplasia (CMPD1), has been placed at distal 17q. The SOX9 gene, a positional candidate from the chromosomal location and expression pattern reported for mouse Sox9, was isolated and characterized. SOX9 encodes a putative transcription factor structurally related to the testis-determining factor SRY and is expressed in many adult tissues, and in fetal testis and skeletal tissue. Inactivating mutations on one SOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal. The 17q breakpoints in three CMPD1 translocation cases map 50 kb or more from SOX9. Publication Types: Case Reports PMID: 8001137 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 164: Nature. 1994 Dec 8;372(6506):525-30. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Foster JW, Dominguez-Steglich MA, Guioli S, Kowk G, Weller PA, Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN, et al. Department of Genetics, University of Cambridge, UK. Induction of testis development in mammals requires the presence of the Y-chromosome gene SRY. This gene must exert its effect by interacting with other genes in the sex-determination pathway. Cloning of a translocation chromosome breakpoint from a sex-reversed patient with campomelic dysplasia, followed by mutation analysis of an adjacent gene, indicates that SOX9, an SRY-related gene, is involved in both bone formation and control of testis development. PMID: 7990924 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------