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 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.     

Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes

Testicular type Sox9 is the most upstream conserved gene in the sex determining cascade among vertebrate. However, in medaka, only one Sox9 gene was identified as expressed in the ovary; no other Sox9 gene was reported expressed in the testis. We explored the medaka genome and cloned a novel testicular type Sox9 cDNA. Phylogenetic analysis revealed that both our isolated Sox9 and the already reportedly cloned medaka Sox9 belongs zebrafish Sox9a branch. Therefore, we named our gene Sox9a2. Unexpectedly, Sox9a2 mRNA was expressed in somatic cells surrounding germ cells at similar high levels in both sexes during early gonadal sex differentiation. However, at the initial stage of testicular tubules development, the expression of Sox9a2 was maintained only in XY gonads, and was remarkably reduced in XX gonads. These results suggest that Sox9a2 is not involved in early sex determination and differentiation, but is involved in the later development of testicular tubules in medaka.     

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.     

Sex differentiation in mammals and tempo of growth: probabilities vs. switches

In the conventional model of sex differentiation in placental mammals, a switch is envisaged to steer the indifferent gonad into the path of either testicular or ovarian development. The immediate cause of the switch is thought to be the presence or absence of Sertoli cells, which in turn is controlled by the presence or absence of the testis-determining factor on the Y chromosome (TDF in humans, Tdy in mice). Quantitative investigations indicate, however, that the rate of growth of XY gonads is faster than that of XX gonads before the formation of Sertoli cells, and furthermore, that XY embryos develop faster than XX embryos long before the formation of gonadal ridges. Since the genetic constitution of the sex chromosomes appears to manifest itself from the earliest embryonic stages onwards, the concept of indifferent gonads being switched into alternate pathways becomes inappropriate. A model is proposed in which gonadal differentiation depends on developmental thresholds: the formation of Sertoli cells needs to occur by a particular stage in time in a sufficiently developed gonad, failing which the gonad will enter the ovarian pathway. While TDF is the principal factor enhancing the rate of gonadal growth, other factors which influence development rates can modulate the probability of a gonad becoming either a testis or an ovary.     

High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder

The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.     

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.     

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.     

Homozygous inactivation of Sox9 causes complete XY sex reversal in mice

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. Because Sox9(-/-) embryos die around Embryonic Day 11.5 (E11.5) at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can be analyzed only 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 downregulation 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.     

Estrogen rescues masculinization of genetically female medaka by exposure to cortisol or high temperature

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by exposure to high water temperature (HT) during gonadal sex differentiation, possibly by elevation of cortisol, the major glucocorticoid produced by the interrenal cells in teleosts. Yet, it remains unclear how the elevation of cortisol levels by HT causes female‐to‐male sex reversal. This paper reports that exposure to cortisol or HT after hatching inhibited both the proliferation of female‐type germ cells and the expression of ovarian‐type aromatase (cyp19a1), which encodes a steroidogenic enzyme responsible for the conversion of androgens to estrogens, and induced the expression of gonadal soma‐derived growth factor (gsdf) in XX gonads during gonadal sex differentiation. In contrast, exposure to either cortisol or HT in combination with 17β‐estradiol (E2) did not produce these effects. Moreover, E2 completely rescued cortisol‐ and HT‐induced masculinization of XX medaka. These results strongly suggest that cortisol and HT cause female‐to‐male sex reversal in medaka by suppression of cyp19a1 expression, with a resultant inhibition of estrogen biosynthesis. This mechanism may be common among animals with temperature‐dependent sex determination. Mol. Reprod. Dev. 79: 719–726, 2012. © 2012 Wiley Periodicals, Inc.     

Testis-specific expression of a novel mouse defensin-like gene,Tdl

In order to isolate genes regulating sex differentiation of embryonic gonads, we have attempted to obtain genes specifically expressed in male embryonic gonads by means of subtractive hybridization screening, and we have cloned a novel mouse gene, Tdl. It potentially encodes a protein showing sequence homology to anti-microbial protein, beta-defensins. Tdl reveals structural features such as the six cysteine residues with invariant spacing, which are found in beta-defensins, but the overall amino acid similarity of Tdl to other members of the beta-defensin family is low. In addition, the Tdl gene shows genomic organization similar to that of other beta-defensin genes. We have found that Tdl is specifically expressed in Sertoli cell-lineage in seminiferous cords in embryonic testes, but not in embryonic ovaries after 12.5dpc when the sexual differentiation of gonads is initiated. Tdl is specifically expressed in gonads among adult tissues, and its expression persisted in Sertoli cells.