Sexually dimorphic gene expression in the developing mouse gonad

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Sry induces cell proliferation in the mouse gonad

Sry is the only gene on the Y chromosome that is required for testis formation in mammals. One of the earliest morphological changes that occurs as a result of Sry expression is a size increase of the rudimentary XY gonad relative to the XX gonad. Using 5'-bromo-2'-deoxyuridine (BrdU) incorporation to label dividing cells, we found that the size increase corresponds with a dramatic increase in somatic cell proliferation in XY gonads, which is not detected in XX gonads. This male-specific proliferation was observed initially in the cells of the coelomic epithelium and occurred in two distinct stages. During the first stage, proliferation in the XY gonad was observed largely in SF1-positive cells and contributed to the Sertoli cell population. During the second stage, proliferation was observed in SF1-negative cells at and below the coelomic epithelium and did not give rise to Sertoli cells. Both stages of proliferation were dependent on Sry and independent of any other genetic differences between male and female gonads, such as X chromosome dosage or other genes on the Y chromosome. The increase in cell proliferation began less than 24 hours after the onset of Sry expression, before the establishment of male-specific gene expression patterns, and before the appearance of any other known male-specific morphological changes in the XY gonad. Therefore, an increase in cell proliferation in the male coelomic epithelium is the earliest identified effect of Sry expression.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad.

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.     

金钱鱼下丘脑转录组分析及其对雌激素在体注射的响应

为探究XX雌性和XY雄性金钱鱼(Scatophagus argus)下丘脑中的差异表达基因及雌激素(17β-Estradiol,E2)在体注射对XY雄鱼下丘脑中基因表达的影响,开展了转录组测序分析,包括测序数据质控、基因功能注释,差异基因筛选、鉴定和差异基因功能富集分析等,并利用实时定量PCR(Real-time quantitative PCR, qPCR)检测了金钱鱼下丘脑中相关基因的表达。金钱鱼下丘脑转录组共测得Clean reads 275833710,测序质量30(Q30)大于95%, GC含量值大于48%。其中, Ctrl-XX-H vs. Ctrl-XY-H组,共筛选和鉴定了91个差异表达基因,包括36个上调基因和55个下调基因。在Ctrl-XY-H vs. E2-XY-H组,筛选和鉴定了28个差异表达基因,包括11个上调基因和17个下调基因。GO和KEGG富集分析发现,差异表达基因主要显著富集在细胞、单生物过程,膜、膜组分,结合等生物学功能及泛醌和其他萜类-醌生物合成及类固醇激素生物合成通路等。转录组和qPCR结果表明, XX和XY金钱鱼下丘脑中prl、ccna1和hs...     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.     

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3–4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.     

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G₁/G₀ arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours. Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G₁/G₀ arrest. Lastly, in XX germ cells we observed a down‐regulation of genes involved in both G₁‐ and G₂‐phases of the cell cycle consistent with their entry into meiosis. Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.     

Characterization of Mesonephric Cells That Migrate into the XY Gonad during Testis Differentiation

In mouse fetal gonads, sex differentiation begins at 10.5-11.5 days postcoitum (dpc). With XY gonads of 12.5 dpc, cord-like structures are visible and stromal cells migrate from adjacent mesonephros, unlike in XX gonads. However, the migrated mesonephric cells, except for the endothelial cells, have not been specifically identified because they have not expressed differentiation markers over the course of organ coculture in previous experiments. In this study, we have for the first time succeeded in isolating only the mesonephric cells that migrate into the XY gonad from the mesonephros with alive and then cultured these cells in vitro through the use of an organ coculture system using EGFP-transgenic mice and a FACS Vantage. The migrated and isolated cells were used for morphological and molecular characterization. The migrated mesonephric cells contained three cell forms; a sharp cell form, a round cell form, and a cluster-forming cell. The sharp cells have the characters of peritubular myoid cells. The round cells and cluster-forming cells have the potential to differentiate into Leydig cells, as some of them are 3beta-HSD-positive. In in vitro culture of migrated mesonephric cells, the cluster-forming cells proliferated well and then differentiated into round cells, suggesting that the cluster-forming cells may be stem or precursor cells for the round cells. Thus, our findings provide important information related to the migration and differentiation of migrated mesonephric cells in the XY gonad.     

Hormonal induction and stability of monosex populations in the medaka (Oryzias latipes): expression of sex-specific marker genes

The model teleost medaka (Oryzias latipes, d-rR.YHNI strain) was used to produce offspring of a defined sex (monosex populations) by crossing experimentally produced YY and XX males to normal females. These monosex populations had the predicted chromosomal constitution as shown by a sex chromosome-specific DNA sequence. However, in XX populations the spontaneous development of males without previous exposure to androgens was observed. Differences in the percentage of male offspring from individual XX breeding pairs indicate a possible variation of unknown genetic factors to be responsible for the development of XX males. The expression of two gonadal genes that are involved in sex differentiation, Dmrt1b(Y) and Fig1a (factor in the germ line alpha), was analyzed in monosex populations. Dmrt1b(Y) expression correlated strictly with the genotype but not the sexual phenotype. When XY juvenile fish were exposed to 17 alpha-ethynylestradiol at concentrations that induce sex reversal, Dmrt1b(Y) expression was not repressed. However, Dmrt1b(Y) was expressed in XY or YY gonads regardless of the sex and could not be detected in XX individuals. In contrast, the expression of Fig1a correlated with the phenotypic sex: Fig1a was expressed in male juvenile fish exposed to 17 alpha-ethynylestradiol and repressed in fish exposed to 17 alpha-methyltestosterone. The Dmrt1b(Y) expression appears to reflect an early and important event in sex determination and lends support to the suggested key regulatory role of the Dmrt1b(Y) gene in sex determination. This process is apparently hormone insensitive, and the expression of further downstream acting genes can be regulated (directly or indirectly) by sex steroids.     

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein.     

Analysis of sex differences in EGC imprinting

Sex-specific differences are apparent in the methylation patterns of H19 and Igf2 imprinted genes in embryonic germ cells (EGCs) derived from 11.5 or 12.5 days post coitum (dpc) primordial germ cells (PGCs). Here we studied whether these differences are associated either with the sex chromosome constitution of the EGCs or with the sex of the genital ridge (testis versus ovary) from which the PGCs were isolated. For this purpose we derived pluripotent EGC lines from sex-reversed embryos, either XY embryos deleted for Sry (XY(Tdym1)) or XX embryos carrying an Sry transgene. Southern blotting of the EGC DNA was used to analyze the differentially methylated regions of Igf2 and H19. The analysis revealed that both genes were more methylated in EGCs with an XY sex chromosome constitution than in those with an XX sex chromosome constitution, irrespective of the phenotypic sex of the genital ridge from which the EGCs had been derived. We conclude that the sex-specific methylation is intrinsic and cell-autonomous, and is not due to any influence of the genital ridge somatic cells upon the PGCs.