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Ovotestis development in B6-XY(POS) mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XY(POS) ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development.

Gene function during mouse development is often studied through the production and analysis of transgenic and knockout models. However, these techniques are time- and resource-consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with microinjection and magnetically induced transfection ("magnetofection") of gene expression constructs. As proof-of-principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain-of-function analysis, and shRNA constructs for loss-of-function analysis. Ectopic expression of Sry induced female-to-male sex-reversal, whereas knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Furthermore, ectopic expression of Tmem184a, a gene of unknown function, in female genital ridges, resulted in failure of gonocytes to enter meiosis. This technique will likely be applicable to the study of gene function in a broader range of developing organs and tissues.

The present decade is witnessing a paradigm shift in our understanding of gene regulation. RNA, once relegated to an intermediary between DNA and protein, has emerged as a key contributor in the coordination of complex developmental pathways. For sexually reproducing organisms, propagation of the species is accomplished via an elaborate sexual phenotype. In mammals this consists of a highly complex cell lineage that has the capacity for intricate self-differentiation whilst maintaining the potential to generate all cell types upon fertilization. In addition, mammals possess a diverse range of somatic reproductive tissues and organs that often undergo dynamic morphological changes in response to a variety of external and internal cues. Although the protein component required to mediate these processes continues to be vigorously investigated, it is becoming increasingly apparent that an understanding of the non-coding RNA (ncRNA) component is required to develop a comprehensive picture of mammalian sexual development.

In mammals, the Y-linked sex-determining gene Srycell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9),leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However,how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.

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.

The Y chromosome gene SRY is the initiator of male sexual differentiation in mammals, but the molecular and cellular mechanisms operating downstream of SRY remain undefined. A deeper understanding of these issues relies on the ability to visualize SRY protein endogenously under a number of experimental conditions. Here we compare the specificity and effectiveness of several available antibodies to mouse SRY. Two antibodies cross-reacted with other SOX proteins in immunofluorescence analyses of transfected cells, and one of these two was unable to detect SRY on Western blots. A third antibody was both avid and specific, and was able to detect endogenous SRY in developing Sertoli cells in mouse genital ridges. Our findings underline the need to distinguish between useful and spurious reagents for biochemical and immunolocalization studies involving mouse SRY protein.

Until recently, sex determination in mammals has often been described as a male determination process, with male differentiation being the active and dominant pathway, and only in its absence is the passive female pathway followed. This picture has been challenged recently with the discovery that the gene encoding R-spondin1 is mutated in human patients with female-to-male sex reversal. These findings might place R-spondin1 in the exceptional position of being the female-determining gene in mammals. In this review, possible roles of R-spondin1 during sex determination as well as questions arising from this study will be discussed.

Arguably the most defining moment in our lives is fertilization, the point at which we inherit either an X or a Y chromosome from our father. The profoundly different journeys of male and female life are thus decided by a genetic coin toss. These differences begin to unfold during fetal development, when the Y-chromosomal Sry (“sex-determining region Y”) gene is activated in males and acts as a switch that diverts the fate of the undifferentiated gonadal primordia, the genital ridges, towards testis development. This sex-determining event sets in train a cascade of morphological changes, gene regulation, and molecular interactions that directs the differentiation of male characteristics. If this does not occur, alternative molecular cascades and cellular events drive the genital ridges toward ovary development. Once testis or ovary differentiation has occurred, our sexual fate is further sealed through the action of sex-specific gonadal hormones. We review here the molecular and cellular events (differentiation, migration, proliferation, and communication) that distinguish testis and ovary during fetal development, and the changes in gene regulation that underpin these two alternate pathways. The growing body of knowledge relating to testis development, and the beginnings of a picture of ovary development, together illustrate the complex mechanisms by which these organ systems develop, inform the etiology, diagnosis, and management of disorders of sexual development, and help define what it is to be male or female.

The forkhead box (FOX), FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown here, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation. Immunostaining and Western blot analyses confirmed FOXO1 and phosphatase and tensin homolog (PTEN) depletion, maintenance of globin transcription factor (GATA) 4 and nuclear localization of FOXL2 and phosphorylated small mothers against decapentaplegic (SMAD) 2/3 in the tumor cells, recapitulating results we observed in human adult GCTs. Microarray and quantitative PCR analyses of mouse GCTs further confirmed expression of specific genes (Foxl2, Gata4, and Wnt4) controlling granulosa cell fate specification and proliferation, whereas others (Emx2, Nr0b1, Rspo1, and Wt1) were suppressed. Key genes (Amh, Bmp2, and Fshr) controlling follicle growth, apoptosis, and differentiation were also suppressed. Inhbb and Grem1 were selectively elevated, whereas reduction of Inha provided additional evidence that activin signaling and small mothers against decapentaplegic (SMAD) 2/3 phosphorylation impact GCT formation. Unexpectedly, markers of Sertoli/epithelial cells (SRY [sex determining region Y]-box 9/keratin 8) and alternatively activated macrophages (chitinase 3-like 3) were elevated in discrete subpopulations within the mouse GCTs, indicating that Foxo1/3/Pten depletion not only leads to GCTs but also to altered granulosa cell fate decisions and immune responses. Thus, analyses of the Foxo1/3/Pten mouse GCTs and human adult GCTs provide strong evidence that impaired functions of the FOXO1/3/PTEN pathways lead to dramatic changes in the molecular program within granulosa cells, chronic activin signaling in the presence of FOXL2 and GATA4, and tumor formation.

ROBO2 plays a key role in regulating ureteric bud (UB) formation in the embryo, with mutations in humans and mice leading to supernumerary kidneys. Previous studies have established that the number and position of UB outgrowths is determined by the domain of metanephric mesenchymal Gdnf expression, which is expanded anteriorly in Robo2 mouse mutants. To clarify how this phenotype arises, we used high-resolution 3D imaging to reveal an increase in the number of nephrogenic cord cells, leading to extension of the metanephric mesenchyme field in Robo2-null mouse embryos. Ex vivo experiments suggested a dependence of this effect on proliferative signals from the Wolffian duct. Loss of Robo2 resulted in a failure of the normal separation of the mesenchyme from the Wolffian duct/ureteric epithelium, suggesting that aberrant juxtaposition of these two compartments in Robo2-null mice exposes the mesenchyme to abnormally high levels of proliferative stimuli. Our data suggest a new model in which SLIT-ROBO signalling acts not by attenuating Gdnf expression or activity, but instead by limiting epithelial/mesenchymal interactions in the nascent metanephros and restricting the extent of the nephrogenic field. These insights illuminate the aetiology of multiplex kidney formation in human individuals with ROBO2 mutations.

A complex network of gene regulation and interaction drives male sex determination and differentiation. While many important protein-coding genes that are necessary for proper male development have been identified, many disorders in human sex development are still unexplained at the molecular level. This suggests that key factors and regulatory mechanisms are still unknown. In recent years, extensive data have shown that different classes of non-coding RNAs (ncRNAs) play a role in almost all developmental and physiological pathways. Here we review what is known about their role in male sex determination and differentiation not only in mammals, but also other species. While for some processes a key role for ncRNA has been identified, we are still far from having a complete picture.

Primary sex determination refers to the developmental processes by which the bipotential gonads develop as either testes or ovaries. Many of the genes involved in testis or ovary determination have been identified either by genetic studies of individuals suffering from disorders of sex development (DSD) or from gene knockout experiments in mice. In this review, we summarize our current knowledge of the major genetic and signaling pathways underlying testis and ovarian development in mammals and discuss how the advent of powerful sequencing and gene editing technologies will affect the future of this important field of sex determination and DSD research.

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 D2 (PGD2) and that PGD2 can induce Sox9 transcription in cultured XX gonads. Our data indicate a mechanism whereby Sry uses both a cell-autonomous mechanism and a PGD2-mediated signaling mechanism to stimulate expression of Sox9 and induce the differentiation of Sertoli cells in vivo.

XX sex reversal syndromes not involving Sry provide an opportunity to identify and study genes important for sexual development. The polled intersex syndrome (PIS) in goats, which shares some features with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) in humans, exemplifies such syndromes. BPES is caused by defects in the forkhead transcription factor gene FOXL2, while PIS is caused by a large deletion of goat chromosome 1q43 that affects transcription of the genes Pisrt1 and Foxl2. Pisrt1 is a non-translated gene that has a sexually dimorphic expression pattern in goats. Here, we describe the structure and expression of the mouse Pisrt1 locus, to investigate its likely role in ovarian development more broadly in mammals. This gene showed some sequence similarity, and was found in a similar genomic context, to its goat and human orthologues. Expression analyses indicated that Pisrt1 is transcribed, and its mRNA polyadenylated and exported to the cytoplasm, but no significant open reading frames were found in a 1.5 kb mouse genomic region corresponding to goat Pisrt1. Pisrt1 transcripts were expressed very broadly among tissues of the developing mouse embryo, and at similar levels in male and female gonads at each stage examined, as determined by in situ hybridisation and RT-PCR. This profile of expression suggests that Pisrt1 is unlikely to contribute to sex-specific events during gonadal development in mice and that divergent pathways of ovarian development operate among different mammalian species.

Sox genes encode transcription factors belonging to the HMG (High Mobility Group) superfamily. They are conserved across species and involved in a number of developmental processes. In vitro studies have shown at least one Sox gene to be capable of inducing oncogenic transformation of fibroblast cells. In addition, overexpression and/or amplification of Sox genes are associated with a large number of tumour types in vivo. We review here the available evidence linking Sox gene expression and cancer, and show that this link is supported by extensive EST database analysis. This work provides a basis for further studies aimed at investigating the possible role of Sox genes in the oncogenic process.

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.

Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression.

International audience; The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development., Author Summary Disorders of gonadal development represent a clinically and genetically heterogeneous class of DSD caused by defects in gonadal development and/or a failure of testis/ovarian differentiation. Unfortunately, in many cases the genetic aetiology of DSD is unknown, indicating that our knowledge of the factors mediating sex determination is limited. Using exome sequencing on a case of autosomal recessive syndromic 46,XY DSD with testicular dysgenesis and chondrodysplasia, we found a homozygous missense mutation (G287V) within the coding sequence of the O-acetyl-transferase HHAT gene. The HHAT gene encodes an enzyme required for the attachment of palmitoyl residues that are critical for multimerization and long range signaling potency of hedgehog secreted proteins. We found that HHAT is widely expressed in human organs during fetal development, including testes and ovaries around the time of sex determination. In vitro assays show that G287V mutation impairs HHAT palmitoyl-transferase activity and mice lacking functional Hhat exhibit testicular dysgenesis as well as other skeletal, neuronal and growth defects that recapitulate most aspects of the syndromic 46,XY DSD patient. These data provide the first clinical evidence of the essential role played by lipid modification of Hedgehog proteins in human testicular organogenesis and embryonic development.

Pre-natal and early post-natal ovarian development has become a field of increasing importance over recent years. The full effects of perturbations of ovarian development on adult fertility, through environmental changes or genetic anomalies, are only now being truly appreciated. Mitigation of these perturbations requires an understanding of the processes involved in the development of the ovary. Herein, we review some recent findings from mice, sheep, and cattle on the key events involved in ovarian development. We discuss the key process of germ cell migration, ovigerous cord formation, meiosis, and follicle formation and activation. We also review the key contributions of mesonephric cells to ovarian development and propose roles for these cells. Finally, we discuss polycystic ovary syndrome, premature ovarian failure, and pre-natal undernutrition; three key areas in which perturbations to ovarian development appear to have major effects on post-natal fertility.

The African pygmy mouse Mus minutoides is characterized by the presence of a high proportion of fertile XY females in natural populations. This species displays 2 morphologically different X chromosomes: the ancestral X and a shorter one designated as X*, feminizing the X*Y individuals. This strongly suggests that in the presence of an X* chromosome, the male differentiation program is not activated despite a functional Y chromosome. In this study, we compared the histology of the adult ovaries of the 3 female genotypes (XX, XX* and X*Y) and investigated the expression of some of the main genes involved in male and female differentiation. We found that X*Y gonads display a typical ovarian structure without any testicular organization. Moreover, the ovarian somatic marker FOXL2 is detected in X*Y follicle cells and exhibits the same pattern as in XX and XX* ovaries, whereas SOX9 and DMRT1 are absent at all stages of follicular differentiation. However, surprisingly, X*Y ovaries display a higher level of Sry transcripts compared to testes. Our findings confirm the complete sex reversal in X*Y individuals with no apparent sign of masculinization, providing an attractive model to unravel new gene interactions involved in the mammalian sex determination system.

From a developmental biology perspective, gonadogenesis is of particular interest because it provides a unique example of how distinct organs, the testis and ovary, can arise from a common bipotential primordium. Gonadogenesis is also highly relevant from a clinical perspective, as congenital disorders of sex development (DSDs) are not uncommon, occurring in approximately 1 in 4500 live births. In recent years, there has been significant progress in our understanding of the genes and pathways that control important aspects of gonadogenesis including the initial decision to develop as a testis or ovary (sex determination), elaboration and cross-repression of the testis and ovary developmental pathways, and maintenance of the gonadal phenotype after birth. In this chapter, we provide an overview of gonadal morphogenesis and cell lineage specification, focusing primarily on these processes in mice and humans. We also provide an update on the genetic mechanisms that control murine gonadogenesis and maintenance and, where possible, discuss new insights into the pathological mechanisms of DSDs associated with mutation of orthologous genes in mice and humans.

The interferon regulatory factor 1 (IRF-1) acts as a transcriptional inducer of the interferon beta (IFN-beta) gene and interferon-stimulated genes. Here we report that IRF-1-mediated IFN-beta induction depends on NFkappaB activity. IRF-1 by itself initiates NFkappaB activation by inducing a reduction in cellular MAD3/IkappaBalpha, an inhibitor of NFkappaB. After nuclear translocation, NFkappaB synergizes with IRF-1 on the cis-elements positive regulatory domain (PRD)II and PRDI/III to induce transcription of the IFN-beta gene. In contrast with IFN-beta transcription induced by dsRNA or virus, c-Jun/ATF-2 binding to PRDIV is not involved. Recombinant MAD3/IkappaBalpha is phosphorylated in vitro by extracts from IRF-1-expressing cells. IRF-1-dependent MAD3/IkappaBalpha degradation is not detectable in cells expressing a dominant negative mutant of the protein kinase PKR, suggesting that PKR mediates MAD3/IkappaBalpha degradation.

We report here that stress stimuli such as gamma-irradiation or the anticancer drug doxorubicin activate expression of the death-inducing ligands (DILs) CD95-L, TNF-alpha and TRAIL. Apoptosis induced by gamma-irradiation or doxorubicin engages a FADD- and caspase-dependent apoptosis pathway which is inhibited by dominant negative FADD or the caspase inhibitor zVAD. zVAD did not prevent activity of JNK/SAPKs in response to doxorubicin suggesting that JNK/SAPK activity is independent of death receptor triggering during cellular stress-induced apoptosis. In addition, JNK/SAPKs remained activated by doxorubicin in resistant cell lines in which cleavage of caspases and apoptosis was not observed. These data uncouple JNK/SAPK activation and apoptosis signaling and indicate that cellular stress-induced apoptosis involves signaling via DILs which is paralleled by activation of JNK/SAPKs. Activation of these kinases may contribute e.g., to the expression of molecules involved in apoptosis but is not sufficient for induction of the apoptosis program following cellular stress.

MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.

Sexual reproduction is fundamental to the survival and propagation of metazoan species. In mice, as in most mammals, sex is determined at the time of fertilization when a Y- or X-chromosome-bearing sperm fuses with the X-chromosome-containing egg, resulting in an XY or XX embryo. The Y chromosome contains the male-determining factor Sry, which drives the differentiation of the bipotential gonads into testes. Without Sry, the gonads develop into ovaries. Testes and ovaries then regulate the development of all other secondary sexual characteristics through the secretion of steroid hormones. In this article, we discuss the cell and molecular biology that underlies sex determination in the mouse.

The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both the temporal and spatial development of the lymphatic vessel network in mouse testes and ovaries. We find that lymphangiogenesis in the testis is initiated during late gestation, but in contrast to other organs, lymphatic vessels remain confined to the testis cap and, unlike blood vessels, do not infiltrate the entire organ. Conversely, lymphatic vessels invade the ovarian tissue, beginning postnatally, and sprouting from preexisting lymphatic vessels at the extraovarian rete. The ovary develops a rich network of lymphatic vessels, extending from the medulla into the surrounding cortex adjacent to developing follicles. This study reveals distinct patterns of lymphangiogenesis in the testes and ovaries and will serve as the basis for the identification of the divergent molecular pathways that control morphogenesis and the function of the lymphatic vasculature in these two organs.

MOTIVATION: Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS: To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (>80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION: Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT: m.dinger@garvan.org.au or j.mattick@garvan.org.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The major regulators of the c-jun promoter are ATF-2 and c-Jun. They act as pre-bound heterodimers on two 'AP-1-like' sites, and are preferentially addressed by different types of extracellular signals. The transactivating potential of ATF-2 is stimulated to a higher extent than that of c-Jun by a broad group of agents causing DNA damage and other types of cellular stress, such as short-wavelength UV, or the alkylating compounds N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) or methylmethanesulphonate (MMS). In contrast, treatment with the phorbol ester TPA preferentially enhances c-Jun-dependent transactivation but does not affect ATF-2. Accordingly, UV and MMS but not TPA induce c-jun transcription in F9 cells, which express ATF-2, but not c-Jun. Stimulation of ATF-2-dependent transactivation by genotoxic agents requires the presence of threonines 69 and 71 located in the N-terminal transactivation domain. These sites are the target of p54 and p46 stress-activated protein kinases (SAPKs) which bind to, and phosphorylate ATF-2 in vitro. However, p46 and p54 kinase activity is not increased by phorbol ester, which strongly suggests that the protein kinase phosphorylating c-Jun in response to TPA is distinct from SAPKs and does not act on ATF-2. Our data demonstrate that distinct signal transduction pathways converge at c-Jun/ATF-2, whereby each subunit is individually addressed by a specific class of protein kinases. This allows fine tuned modulation of c-jun expression by a large spectrum of extracellular signals.

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.

Human DAX1 duplications cause dosage-sensitive sex reversal (DSS) whereby chromosomally XY individuals can develop as females due to gonadal dysgenesis. However, the mechanism of DSS-adrenal hypoplasia congenita on X, gene 1 (DAX1) action in the fetal testis is unknown. We show that in fetal testes from XY Dax1-overexpressing transgenic mice, the expression of the key testis-promoting gene sex-determining region on Y (SRY)-box-9 (Sox9) is reduced. Moreover, in XY Sox9 heterozygotes, in which testis development is usually normal, Dax1 overexpression results in ovotestes, suggesting a DAX1-SOX9 antagonism. The ovarian portion of the XY ovotestes was characterized by expression of the granulosa cell marker, Forkhead box-L2, with complete loss of the Sertoli cell markers, SOX9 and anti-Müllerian hormone, and the Leydig cell marker CYP17A1. However, the expression of SRY and steroidogenic factor-1 (SF1), two key transcriptional regulators of Sox9, was retained in the ovarian portion of the XY ovotestes. Using reporter mice, Dax1 overexpression reduced activation of TES, the testis enhancer of Sox9, indicating that DAX1 might repress Sox9 expression via TES. In cultured cells, increasing levels of DAX1 antagonized SF1-, SF1/SRY-, and SF1/SOX9-mediated activation of TES, due to reduced binding of SF1 to TES, providing a likely mechanism for DSS.

Mice lacking the function of the polycomb group protein CBX2 (chromobox homolog 2; also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout (KO) mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes. However, how CBX2 regulates development of these tissues remains largely unknown. In the present study, we used microarray, RT-PCR, and immunohistochemical analyses to show that the expression of Sry, Sox9, Lhx9, Ad4BP/SF-1, Dax-1, Gata4, Arx, and Dmrt1, genes encoding transcription factors essential for gonadal development, is affected in Cbx2 KO gonads. Male-to-female sex reversal in Cbx2 KO mice was rescued by crossing them with transgenic mice displaying forced expression of Sry or Sox9. However, testes remained hypoplastic in these mice, indicating that the size and the sex of the gonad are determined by different sets of genes. Our study implicates Cbx2 in testis differentiation through regulating Sry gene expression.

In mammals, Sry (sex-determining region Y gene) is the master regulator of male sex determination. It induces a cascade of gene expression that regulates the differentiation of the bipotential genital ridges into testicular tissue. In the absence of Sry, or if SRY function is impaired, the genital ridges develop into ovaries. Subsequently, hormones produced by the testes and ovaries direct the differentiation of all secondary sexual characteristics (for review, see Wilhelm and Koopman [1]).

AGO proteins are universal effectors of eukaryotic small RNA-directed regulatory pathways. In this study, we used a comparative genomics approach to explore the AGO sub-family in the teleost clade. We identified five Ago homologues in teleost genomes, one more than encoded in other vertebrate clades. The additional teleost homologue was preserved most likely due to the differential retention of regulatory elements following the fish-specific genome duplication event that occurred approximately 350 million years ago. Analysis of all five Ago genomic loci in teleosts revealed that orthologues contain specific, conserved sequence elements in non-coding regions indicating that the teleost Ago paralogues are differentially regulated. This was supported by qRT-PCR analysis that showed differential expression of the zebrafish homologues across development and between adult tissues indicating stage and tissue-specific function of individual AGO proteins. Multiple sequence alignments showed not only that all teleost homologues possess critical residues for AGO function, but also that teleost homologues contain multiple orthologue-specific features, indicative of structural diversification. Notably, these are retained throughout the vertebrate lineage arguing these may be important for orthologue-specific functions.

The 3′ untranslated regions (3′UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3′UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3′UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3′UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3′UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3′UTRs, as well as caution in the use of 3′UTR sequence probes to analyze gene expression.

Mammalian sex determination is a dynamic process involving balanced gene expression leading to the development of either a testis or an ovary. Candidate sex-determining genes have been identified through microarray-based studies of gonadal gene expression; however, few methods exist for validation. This study describes a new technique for transfecting gonads using nucleofection. Fifteen micrograms of expression plasmid DNA was transfected into E11.5 gonads, cultured for 3 days and gene expression analyzed. Following optimization, we consistently achieved cell transfection efficiencies of 11% of cells using pMax-GFP plasmid. To test the applicability of nucleofection to studies of gene function, a testis-determining gene was transfected into gonads and its ability to sex-reverse was examined. When Sry was transfected into female (XX) gonads, upregulation of its target gene Sox9 was observed, as well as a downregulation of the ovarian gene Foxl2. Conversely, when shSox9 was introduced into male (XY) gonads, reduction of Sox9 and its target gene, Amh was observed, with a concomitant upregulation of Foxl2. Nucleofection-based gene delivery can recapitulate in vivo events of gonadal development that demonstrates ‘proof-of-principle’ of the method as a screening tool to evaluate the cellular function of potential sex-determining and gonadal differentiation genes.

Germ cell sex differentiation in the mouse embryo is denoted by meiosis entry in females and mitotic arrest in males. Because p38 mitogen-activated protein kinase (MAPK) signaling initiates mitotic arrest in other differentiating cell types, we investigated its potential role in XY germ cell differentiation in mice. We report that p38 MAPK is phosphorylated and therefore activated only in XY germ cells around the time of sex differentiation. Quantitative RT-PCR analysis showed that 14 known targets of p38 MAPK signaling are expressed in the embryonic gonads at this time and that five of these targets (Mapkapk5, Max, Myc, Hbp1, and Cebpa) have expression profiles similar to that of activated p38 MAPK. Inhibition of p38 MAPK signaling in XY germ cells ex vivo reduced expression of the pluripotency marker POU5F1 and increased the expression of Stra8 and SYCP3, premeiosis and meiosis markers, respectively, to levels approaching those observed in XX germ cells. These data suggest that p38 MAPK signaling antagonizes entry into meiosis in XY germ cells, instead directing them toward mitotic quiescence and a spermatogenic fate.

In mammals, biological differences between males and females, which influence many aspects of their physical, social, and psychological environments, are solely determined genetically. In the presence of a Y chromosome, the gonadal primordium will differentiate into a testis, whereas in the absence of the Y chromosome an ovary will develop. Testis and ovary subsequently direct the differentiation of all secondary sex characteristics down the male and female pathway, respectively. The male-determining factor on the Y chromosome, SRY, was identified some 20 years ago. Since then, significant progress has been made toward understanding the molecular and cellular pathways that result in the formation of a testis. Here, we review what is known about testis differentiation in mice and humans, with reference to other species where appropriate.

In mammals, biological differences between males and females, which influence many aspects of their physical, social, and psychological environments, are solely determined genetically. In the presence of a Y chromosome, the gonadal primordium will differentiate into a testis, whereas in the absence of the Y chromosome an ovary will develop. Testis and ovary subsequently direct the differentiation of all secondary sex characteristics down the male and female pathway, respectively. The male-determining factor on the Y chromosome, SRY, was identified some 20 years ago. Since then, significant progress has been made toward understanding the molecular and cellular pathways that result in the formation of a testis. Here, we review what is known about testis differentiation in mice and humans, with reference to other species where appropriate.

Male development in mammals is normally initiated by the Y-linked gene Sry, which activates expression of Sox9, leading to a cascade of gene activity required for testis formation. Although defects in this genetic cascade lead to human disorders of sex development (DSD), only a dozen DSD genes have been identified, and causes of 46,XX DSD (XX maleness) other than SRY translocation are almost completely unknown. Here, we show that transgenic expression of Sox10, a close relative of Sox9, in gonads of XX mice resulted in development of testes and male physiology. The degree of sex reversal correlated with levels of Sox10 expression in different transgenic lines. Sox10 was expressed at low levels in primordial gonads of both sexes during normal mouse development, becoming male-specific during testis differentiation. SOX10 protein was able to activate transcriptional targets of SOX9, explaining at a mechanistic level its ability to direct male development. Because over-expression of SOX10 alone is able to mimic the XX DSD phenotypes associated with duplication of human chromosome 22q13, and given that human SOX10 maps to 22q13.1, our results functionally implicate SOX10 in the etiology of these DSDs.

The boygirl (byg) mouse mutant reveals that MAP3K4-mediated signaling is necessary for normal SRY expression and testis specification in the developing mouse gonad., Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans., Author Summary In mammals, whether an individual develops as a male or female depends on its sex chromosome constitution: those with a Y chromosome become males because of the development of the embryonic gonad into a testis. The Y-linked sex determining gene SRY regulates this process by initiating a pathway of gene and protein expression, including the expression of critical autosomal genes such as SOX9. We identified a mouse mutant that causes embryonic gonadal sex reversal: the development of ovaries in an XY embryo. This mutant, which we called boygirl (byg), was shown to contain an early stop codon that disrupts the autosomal gene encoding MAP3K4, a component of the mitogen-activated protein kinase (MAPK) signaling pathway. Analysis of embryonic XY gonads suggests that sex reversal is caused by delayed and reduced expression of the sex-determining gene SRY. Our data indicate, for the first time, a requirement for MAPK signaling in the developing XY gonad in order to facilitate normal expression of SRY and the downstream testis-determining genes and also suggest that reduced dosage of MAP3K4 may be the cause of a previously described autosomal sex-reversing mutation in the mouse. We predict that loss of MAP3K4 or other MAPK components may underlie disorders of sexual development (DSD) in humans as well.

The development of an embryo as male or female depends on differentiation of the gonads as either testes or ovaries. A number of genes are known to be important for gonadal differentiation, but our understanding of the regulatory networks underpinning sex determination remains fragmentary. To advance our understanding of sexual development beyond the transcriptome level, we performed the first global survey of the mouse gonad proteome at the time of sex determination by using two-dimensional nanoflow LC-MS/MS. The resulting data set contains a total of 1037 gene products (154 non-redundant and 883 redundant proteins) identified from 620 peptides. Functional classification and biological network construction suggested that the identified proteins primarily serve in RNA post-transcriptional modification and trafficking, protein synthesis and folding, and post-translational modification. The data set contains potential novel regulators of gonad development and sex determination not revealed previously by transcriptomics and proteomics studies and more than 60 proteins with potential links to human disorders of sexual development.

Testis cords are specialised tubes essential for generation and export of sperm, yet the mechanisms directing their formation, and the regulation of their position, size, shape and number, remain unclear. Here, we use a novel fluorescence-based three-dimensional modelling approach to show that cords initially form as a network of irregular cell clusters that are subsequently remodelled to form regular parallel loops, joined by a flattened plexus at the mesonephric side. Variation in cord number and structure demonstrates that cord specification is not stereotypic, although cord alignment and diameter becomes relatively consistent, implicating compensatory growth mechanisms. Branched, fused and internalised cords were commonly observed. We conclude that the tubule-like structure of testis cords arise through a novel form of morphogenesis consisting of coalescence, partitioning, and remodelling. The methods we describe are applicable to investigating defects in testis cord development in mouse models, and more broadly, studying morphogenesis of other tissues.

Background information. SRY (sex-determining region Y), the master regulator of male development in mammals, has been studied extensively for more than 17 years, but how the SRY protein triggers the chain of events leading to testis development remains unclear. SRY probably requires a partner protein to elicit its molecular function. KRAB-O, a novel protein containing a KRAB (Kruppel-associated box) domain only, was suggested recently as a candidate SRY partner. In order to investigate the possible role of KRAB-O in sex determination, we studied its expression and conducted functional assays of the SRY—KRAB interaction. Results. More than 100 KRAB genes were found to be expressed in mouse developing gonads, including 19 transcripts encoded by the KRAB-O cluster that were found to be expressed in somatic cells at 11.5 dpc (days post-coitum). Loss-of-function analysis in Sry-expressing cultured cells, using shRNA (small hairpin RNA) constructs directed against KRAB-O and its homologous genes, resulted in a reduced ability to up-regulate Sox9 [SRY-related HMG (high-mobility group)-box 9]; however, KRAB-knockdown mice exhibited normal testis development. Conclusions. Reduced Sox9 expression in KRAB-knockdown cells supports a role for KRAB-O and perhaps other KRAB genes in mediating SRY function. Overlapping expression and potential redundancy between members of the large KRAB-O gene cluster may mask any loss-of-function in vivo, presenting clear challenges for further functional analysis.