Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.     

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.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.     

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.     

Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development.

Sex-determining mechanisms are highly variable between phyla. Only one example has been found in which structurally and functionally related genes control sex determination in different phyla: the sexual regulators mab-3 of Caenorhabditis elegans and doublesex of Drosophila both encode proteins containing the DM domain, a novel DNA-binding motif. These two genes control similar aspects of sexual development, and the male isoform of DSX can substitute for MAB-3 in vivo, suggesting that the two proteins are functionally related. DM domain proteins may also play a role in sexual development of vertebrates. A human gene encoding a DM domain protein, DMRT1, is expressed only in the testis in adults and maps to distal 9p24.3, a short interval that is required for testis development. Earlier in development we find that murine Dmrt1 mRNA is expressed exclusively in the genital ridge of early XX and XY embryos. Thus Dmrt1 and Sry are the only regulatory genes known to be expressed exclusively in the mammalian genital ridge prior to sexual differentiation. Expression becomes XY-specific and restricted to the seminiferous tubules of the testis as gonadogenesis proceeds, and both Sertoli cells and germ cells express Dmrt1. Dmrt1 may also play a role in avian sexual development. In birds the heterogametic sex is female (ZW), and the homogametic sex is male (ZZ). Dmrt1 is Z-linked in the chicken. We find that chicken Dmrt1 is expressed in the genital ridge and Wolffian duct prior to sexual differentiation and is expressed at higher levels in ZZ than in ZW embryos. Based on sequence, map position, and expression patterns, we suggest that Dmrt1 is likely to play a role in vertebrate sexual development and therefore that DM domain genes may play a role in sexual development in a wide range of phyla.     

Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressing cells in Lepidochelys olivacea

Bipotential gonads represent the structural framework from which alternative molecular sex determination networks have evolved. Maintenance of Sox9 expression in Sertoli cells is required for the structural and functional integrity of male gonads in mammals and probably in most amniote vertebrates. However, spatial and temporal patterns of Sox9 expression have diversified along evolution. Species with temperature sex determination are an interesting predictive model since one of two alternative developmental outcomes, either ovary or testis occurs under controlled laboratory conditions. In the sea turtle Lepidochelys olivacea, Sox9 is expressed in the medullary cords of bipotential gonads when incubated at both female- or male-promoting temperature (FT or MT). Sox9 is then turned off in presumptive ovaries, while it remains turned on in testes. In the current study, Sox9 was used as a marker of the medullary cell lineage to investigate if the medullary cords originate from mesothelial cells at the genital ridges where Sox9 is upregulated, or, if they derive from a cell population specified at an earlier developmental stage, which maintains Sox9 expression. Using immunofluorescence and in situ hybridization, embryos were analyzed prior to, during and after gonadal sex determination. A T-shaped domain (T-Dom) formed by cytokeratin (CK), N-cadherin (Ncad) and SOX9-expressing cells was found at the upper part of the hindgut dorsal mesentery. The arms of the T-Dom were extended to both sides towards the ventromedial mesonephric ridge before the thickening of the genital ridges, indicating that they contained gonadal epithelial cell precursors. Thereafter, expression of Sox9 was maintained in medullary cords while it was downregulated at the surface epithelium of bipotential gonads in both FT and MT. This result contrasts with observations in mammals and birds, in which Sox9 upregulation starts at a later stage in the inner cells underlying the Sox9-negative surface epithelium, suggesting that the establishment of a self-regulatory Sox9 loop required for Sertoli cell determination has evolved. The T-shaped domain at the upper part of the hindgut dorsal mesentery found in the current study may represent the earliest precursor of the genital ridges, previously unnoticed in amniote vertebrates.     

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.     

A cystatin-related gene, testatin/cresp, shows male-specific expression in germ and somatic cells from the initial stage of murine gonadal sex-differentiation

Sex-differentiation in mammals initiates at mid-gestation when the differentiation of Sertoli cells is triggered by the expression of the testis-determining gene, Sry. However, little is known about the succeeding germ-soma interaction that directs the sex-differentiation of germ cells. We carried out subtraction and differential screening between male and female gonads at 13.5 dpc (days post coitum). A novel cystatin-related gene was identified and named cresp (cystatin-related expressed in Sertoli and spermatogonia), and has recently been reported independently under the name testatin (T?h?nen et al., 1998). The presumed amino acid sequence of testatin/cresp showed considerable homology to the cystatin family, but it lacked a few critical amino acid residues for the cysteine-protease inhibitory activity. A 0.7 kb RNA was detected by northern blotting specifically in the fetal and adult testes from 11.5 dpc and expression increased between 11.5 dpc and 12.5 dpc. Using RT-PCR analysis, the testatin/cresp mRNA was first detectable at 9.5 dpc in both male and female embryos but it was maintained only in the male. In females, the expression became weaker at 11.5 dpc and was undetectable after 12.0 dpc. In situ hybridization and immunohistochemical analyses, as well as single cell RT-PCR analysis, showed that the testatin/cresp mRNA was localized specifically in both the (pro)spermatogonia and Sertoli cells in the testis from 12.5 dpc to adult. Thus, expression of the testatin/cresp gene is upregulated in male gonads but downregulated in females immediately after the initiation of sex-differentiation, suggesting roles in the early developmental cascade of testis such as the germ-soma interaction.     

Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis

During embryogenesis, primordial germ cells (PGCs) have the potential to enter either spermatogenesis or oogenesis. In a female genital ridge, or in a non-gonadal environment, PGCs develop as meiotic oocytes. However, male gonadal somatic cells inhibit PGCs from entering meiosis and direct them to a spermatogenic fate. We have examined the ability of PGCs from male and female embryos to respond to the masculinising environment of the male genital ridge, defining a temporal window during which PGCs retain a bipotential fate. To help understand how PGCs respond to the male gonadal environment, we have identified molecular differences between male PGCs that are committed to spermatogenesis and bipotential female PGCs. Our results suggest that one way in which PGCs respond to this masculinising environment is to synthesise prostaglandin D(2). We show that this signalling molecule can partially masculinise female embryonic gonads in culture, probably by inducing female supporting cells to differentiate into Sertoli cells. In the developing testis, prostaglandin D(2) may act as a paracrine factor to induce Sertoli cell differentiation. Thus part of the response of PGCs to the male gonadal environment is to generate a masculinising feedback loop to ensure male differentiation of the surrounding gonadal somatic cells.