Cell proliferation is necessary for the determination of male fate in the gonad

Cell proliferation has been shown to have multiple functions in development and pattern formation, including roles in growth, morphogenesis, and gene expression. Previously, we determined that the earliest known morphological event downstream of the male sex determining gene, Sry, is the induction of proliferation. In this study, we used proliferation inhibitors to block cell division during early gonad development, at stages before the XY gonad has committed to the testis pathway. Using the expression of sex-specific genes and the formation of testis morphology as markers of testis determination, we found that proliferation within a specific 8-h window was critical for the establishment of the male pathway and the formation of the testis. Inhibition of proliferation before or after this critical period led to smaller gonads, but did not block testis formation. The critical period of proliferation coincides with the initiation of Sry expression and is essential for the differentiation of Sertoli cells, suggesting that proliferation is a vital component of the initiation of the male pathway by Sry. We believe these studies suggest that proliferation is involved not only in the elaboration of organ pattern, but also in the choice between patterns (male and female) in the bipotential gonad.     

Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal

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.     

Sry-negative XX sex reversal in the American cocker spaniel dog

The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY-negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y-specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley-Liss, Inc.     

siRNA介导Sry基因沉默对小鼠胚胎性别决定基因表达的影响

为研究Sry基因的调控网络, 采用siRNA表达载体介导的RNAi技术, 特异性地抑制睾丸决定因子Sry在小鼠胚胎中的表达, 并观察Sry基因沉默后对在两性性腺分化中起重要作用的Wt1, Sf1, Dax1, Gata4, Sox9及Amh基因表达的影响。利用本课题组先前构建的siRNA重组表达载体(pSilencer4.1/Sry217及pSilencer4.1/Sry565), 通过尾静脉注射法导入妊娠9.5天(9.5 dpc)的母鼠体内, 在11.5 dpc时取胚胎, 对性别鉴定为雄性的胚胎以RT-PCR法和Western-blot检测Sry基因的表达抑制效果, 并同时用定量PCR法检测Wt1等上述性别决定相关基因表达变化情况。结果表明, 注射干扰质粒后48 h Sry基因的mRNA和蛋白表达水平均降低, 其中siRNA表达质粒pSilencer 4.1/Sry 565的抑制效果显著, 可达到80%的抑制率。Sry基因沉默后, Wt1基因表达量显著升高; Sf1, Dax1, Gata4, Sox9基因表达水平没有明显变化; Amh基因无表达。试验结果表明, Sry基因表达抑制会导致Wt1基因表达升高; 另外, Sry基因激活Sox9基因的表达可能需要其他的辅助因子协同作用。     

In vitro Cre/loxP system in cells from developing gonads: investigation of the Sry promoter

There have been few studies on the regulatory elements of the Sry gene, mainly because no Sry-expressing cell lines have yet been established. This paper describes a useful tool for investigating the regulation and upstream region of Sry by means of the in vitro Cre/loxP system. Using plasmids containing the 9.9 kb mouse genomic Sry previously shown to induce testis development in XX transgenic mice, we constructed a Sry/Cre fusion gene plasmid in which Cre expression is controlled by the 5' and 3' untranslated regions of mouse Sry. To distinguish between male and female gonads of 11.5 days post-coitus (d.p.c.) fetuses, double transgenic fetuses carrying both the CAG (cytomegalovirus enhancer and beta-actin promoter)/loxP/lacZ transgene on the autosome and the green fluorescent protein transgene ubiquitously expressed on the Y chromosome were produced by crossing between two transgenic mouse lines. When Sry/Cre plasmids were transfected into the cells that had been prepared from the gonads, brains and livers of double transgenic fetuses, only a small number of X-gal-stained cells were detected among the primary cultured cells from male and female gonads, and none were detected among the cells from the other tissues. The X-gal-positive cells were negative for alkaline phosphatase, indicating that these cells were somatic cells expressing Sry. The Sry/Cre plasmids with a 0.4 kb upstream region of Sry yielded a large number of X-gal-positive cells in the cells from gonads, including various tissues of 11.5 d.p.c. fetuses, indicating the loss of the tissue-specific expression of Sry. The Sry/Cre with a 1.4 kb upstream region maintained tissue-specific activity of Sry. The results indicate that the present in vitro Cre/loxP system using transgenic mice is a simple and useful system for investigating the regulatory element of sex determination-related genes, including Sry.     

Direct regulation of adult brain function by the male-specific factor SRY

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

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.     

Evidence that the testis determination pathway interacts with a non-dosage compensated, X-linked gene.

In a number of mammals, including mouse and man, it has been shown that at equivalent gestational ages, males are developmentally more advanced than females, even before the gonads form. In mice, although some strains of Y chromosome exert a minor accelerating effect in pre-implantation development, it is a post-implantation effect of the difference in X chromosome constitution that is the major cause of the male/female developmental difference. Thus XX females are retarded in their development by about 1.5 h relative to X(M)O females or XY males; however, they are more advanced than X(P)O females by about 4 h. It has been suggested that this early developmental difference between XX and XY embryos may "weight the dice" in favour of ovarian and testicular development, respectively, although expression of Sry will normally overcome any such bias. Here we test this proposal by comparing the relative frequencies of female, hermaphrodite and male development in X(P)O, XX and X(M)O mice that carry an incompletely penetrant Sry transgene. The results show that testicular tissue develops more frequently in XX,Sry transgenics than in either of the two types of XO transgenics. Thus the incidence of testicular development is affected by X dosage rather than by the developmental hierarchy. This implies there is a non-dosage compensated gene (or genes) on the X chromosome, which interacts with the testis-determining pathway. Since the pseudoautosomal region (PAR) is known to escape X-inactivation, penetrance of the Sry transgene was also assessed in X(M)Y(*X) mice that have two doses of the PAR but have a single dose of all genes proximal to the distal X marker Amel. These mice showed similar levels of testicular development to X(M)O mice with the transgene; thus the non-dosage compensated X gene maps outside the PAR.     

Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination

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.     

Potency of testicular somatic environment to support spermatogenesis in XX/Sry transgenic male mice

The sex-determining region of Chr Y (Sry) gene is sufficient to induce testis formation and the subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males, such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth because of germ cell-autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here, we demonstrate that the testicular somatic environment of XX/Sry males is defective in supporting the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, of entering meiosis and of differentiating to the round-spermatid stage. XY-donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, resulting in severe deficiency of elongated spermatid stages. By contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY-donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed the missing expression of several Y-linked genes and the alterations in the expression profile of genes associated with spermiogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, highlighting the idea that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice.     

Testis determination: soft talk and kinky sex.

The activity of the Y-linked Sry gene during a critical period of gonadal differentiation is the normal trigger for testis determination and subsequent male development in mammals. This gene encodes a DNA-binding protein of the HMG-box class. It has been shown to induce a dramatic kink in target DNA-binding sites, which allows for much speculation on how the gene functions to regulate testis-specific gene expression. It is also clear that cell interactions are vital to its mode of action, and generally in the process of gonadal differentiation.     

SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development

In mammals, male sex is determined by the Y-chromosomal gene Sry (sex-determining region of Y chromosome). The expression of Sry and subsequently Sox9 (SRY box containing gene 9) in precursors of the supporting cell lineage results in the differentiation of these cells into Sertoli cells. Sertoli cells in turn orchestrate the development of all other male-specific cell types. To ensure that Sertoli cells differentiate in sufficient numbers to induce normal testis development, the early testis produces prostaglandin D(2) (PGD(2)), which recruits cells of the supporting cell lineage to a Sertoli cell fate. Here we show that the gene encoding prostaglandin D synthase (Pgds), the enzyme that produces PGD(2), is expressed in Sertoli cells immediately after the onset of Sox9 expression. Promoter analysis in silico and in vitro identified a paired SOX/SRY binding site. Interestingly, only SOX9, and not SRY, was able to bind as a dimer to this site and transactivate the Pgds promoter. In line with this, a transgenic mouse model showed that Pgds expression is not affected by ectopic Sry expression. Finally, chromatin immunoprecipitation proved that SOX9 but not SRY binds to the Pgds promoter in vivo.     

Cloning and mutational analysis of SRY.

A candidate for the male sex-determining gene has recently been isolated. This sex-determining gene (SRY) has been found to be mutated in some individuals with failed testis development, and, in mouse transgenesis, the SRY murine homologue (Sry) causes female-to-male sex reversal. The cloning of SRY should facilitate the characterisation of other genes in the testis-determining pathway and provide information on the mechanism of mammalian developmental decisions.     

Sex-determining mechanisms in animals

Biological mechanisms leading to the development of males and females are extremely varied. In the XX/XY system, the male has an unequal pair of chromosomes, while in the ZZ/ZW system, the unequal pair is in the female. Sex can also be determined by the temperature of incubation. Recent research has focused on the identification of sex-determining genes, culminating in the demonstration that the Sry gene on the Y chromosome of mice can induce male development in genetically female XX mouse embryos. Nevertheless, the occurrence of phenotypes in apparent contrast to the genotype suggests that the genetic male/female switch is not simple, and there may be common features linking different sex-determining mechanisms. There is increasing evidence that such a link may be provided by the induction of growth differences, and that the primary sex difference may result from the distinction between fast versus slow growth.     

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.     

Matrix metalloproteinases regulate mesonephric cell migration in developing XY gonads which correlates with the inhibition of tissue inhibitor of metalloproteinase-3 by Sry

In the mouse, the sex determining gene Sry, on the Y chromosome, controls testis differentiation during embryogenesis. Following Sry expression, indifferent XY gonads increase their size relative to XX gonads and form cord-like structures with the adjacent mesonephros, providing XY gonad somatic cells. This mesonephric cell migration is known to depend on Sry, but the molecular mechanism of mesonephric cell migration remains unknown. In this study, it was shown that cells expressing Sry induced proliferation of mesonephric cells migrating into male gonads, and inhibited expression of the tissue inhibitor of metalloproteinases (TIMP)-3 gene, which is the endogenous inhibitor of matrix metalloproteinases (MMP). In addition, the mesonephric cell migration was blocked by a chemically synthesized inhibitor of MMP in a gonad/mesonephros organ co-culture system with enhanced green fluorescent protein transgenic embryos. The findings indicate that MMP may play a critical role in mesonephric cell migration, and the function of MMP may be regulated by a Sry-TIMP-3 cascade. These findings are an important clue for the elucidation of testicular formation in developing gonads.