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STUDY QUESTIONWhat is the effect of the ketone β-hydroxybutyrate (βOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability?SUMMARY ANSWERIn vitro βOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner.WHAT IS KNOWN ALREADYA maternal KD in humans elevates gamete and offspring βOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to βOHB at supraphysiological concentrations (8–80 mM) perturbs preimplantation mouse embryo development.STUDY DESIGN, SIZE, DURATIONA mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with βOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured βOHB-exposed embryos was assessed.PARTICIPANTS/MATERIALS, SETTING, METHODSMouse embryos were cultured in vitro with or without βOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of βOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, βOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 β-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed.MAIN RESULTS AND THE ROLE OF CHANCEAll βOHB concentrations tested slowed embryo development (P LIMITATIONS, REASONS FOR CAUTIONThis study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro βOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of βOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown.WIDER IMPLICATIONS OF THE FINDINGSThese findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to βOHB within a physiological range. Maternal diets which increase βOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of βOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

Summary Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. Therefore, gonadal sex-specific transcription factors are required to prevent transdifferentiation of gonadal somatic cells to the other sexual fate. Mouse genetic experiments have shown that oestrogen receptor signalling and the transcription factor FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying this protective mechanism is unknown. Here, we show that one post-translational modification (i.e. SUMOylation catalysed by TRIM28) is sufficient to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of TRIM28 SUMO-E3 ligase activity, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type different from gonadal embryonic progenitors. TRIM28 binds to chromatin close to the critical transcription factor FOXL2 to maintain the female pathway through SUMOylation of specific chromatin regions. Therefore, FOXL2 signalling might maintain the adult ovary cell fate via TRIM28-dependent SUMOylation. Improper SUMOylation of chromatin regions in granulosa cells might lead to female reproductive disorders and infertility, the incidence of which is currently increasing.

Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLC) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially co-express Wnt4 and Sox9. SLCs become sexually dimorphic around E12.5, progressively acquire a Sertoli- or granulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Finally, we found that WNT4 is a crucial regulator of the SLC lineage and is required for the formation of the rete testis.TeaserDescription of an uncharacterized multipotent gonadal cell lineage involved in testis and ovary development

Heterozygous mutations in the human SOX9 gene cause the skeletal malformation syndrome campomelic dysplasia which in 75% of 46, XY individuals is associated with male-to-female sex reversal. Although studies in homozygous Sox9 knockout mouse models confirmed that SOX9 is critical for testis development, mice heterozygous for the Sox9-null allele were reported to develop normal testes. This led to the belief that the SOX9 dosage requirement for testis differentiation is different between humans, which often require both alleles, and mice, in which one allele is sufficient. However, in prior studies, gonadal phenotypes in heterozygous Sox9 XY mice were assessed only by either gross morphology, histological staining or analyzed on a mixed genetic background. In this study, we conditionally inactivated Sox9 in somatic cells of developing gonads using the Nr5a1-Cre mouse line on a pure C57BL/6 genetic background. Section and whole-mount immunofluorescence for testicular and ovarian markers showed that XY Sox9 heterozygous gonads developed as ovotestes. Quantitative droplet digital PCR confirmed a 50% reduction of Sox9 mRNA as well as partial sex reversal shown by an upregulation of ovarian genes. Our data show that haploinsufficiency of Sox9 can perturb testis development in mice, suggesting that mice may provide a more accurate model of human disorders/differences of sex development than previously thought.

Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of Nedd4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis. Significance: Ubiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored. Refereed/Peer-reviewed

Sex in mammals is determined genetically with the acquisition of either the XX (female) or XY (male) chromosomes at the time of fertilization. The chromosomal sex will determine if, during embryogenesis, ovaries or testes form from the bipotential gonadal anlage, the genital ridges. Hormones produced by testes and ovaries will then drive most, if not all, secondary sexual characteristics, resulting in the female or male phenotype. Each of these sequential steps is promoted by specific genes and mutations in these genes can lead to disorders of sex development (DSDs).

Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising β-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/β-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/β-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.

International audience; Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan., Author summary Fertility is a concern in women’s health, especially when the reported average age of mothers is rising (2016, CDC DB232). Of concern in aging mothers is ovarian follicle health, which requires active communication between its cell components that include a single egg and its surrounding granulosa support cells. The process used to establish cell-cell communication within follicles is unknown but begins during fetal development. We discovered that two related genes, Irx3 and Irx5, direct network construction. If Irx3 and Irx5 are eliminated in mice, oocyte-granulosa cell contact is lost, and all follicles die within short order. Analysis of additional Irx3 and Irx5 mutant mice highlighted that each factor is important for establishing follicle integrity. During fetal development, they work together in the same cell to establish the identity of future granulosa support cells. Then, as the follicles form, they continue to work cooperatively, but in different cells. While Irx5 expression remains in granulosa cells, Irx3 expression transitions to the oocyte and together, they promote cell patterns and interacting zones to synchronize the follicle as an interactive unit. Although the work of Irx3 and Irx5 is finished in the perinatal ovary, their impact lasts a lifetime as it establishes long-term follicle integrity and therefore, fertility.

Sex determination, although crucially important for the survival of a species, is arguably the least conserved mechanism in evolution and is instead one of the most variable developmental pathways. There are two main mechanisms of sex determination: genetic (GSD) and environmental sex determination (ESD). Amongst vertebrates, many reptiles such as crocodiles and turtles determine sex by ESD, whereas birds and mammals, including human display GSD. GSD can be further subdivided into those for which the males are heterogametic, that is, an XX-XY system, and those with heterogametic females, that is, ZZ-ZW system. Here, we review genetic mechanisms of sex determination.

Exploring stem cells in the mammalian ovary has unleashed a Pandora's box of new insights and questions. Recent evidence supports the existence of stem cells of a number of the different cell types within the ovary. The evidence for a stem cell model producing mural granulosa cells and cumulus cells is strong, despite a limited number of reports. The recent identification of a precursor granulosa cell, the gonadal ridge epithelial-like cell, is exciting and novel. The identification of female germline (oogonial) stem cells is still very new and is currently limited to just a few species. Their origins and physiological roles, if any, are unknown, and their potential to produce oocytes and contribute to follicle formation in vivo lacks robust evidence. The precursor of thecal cells remains elusive, and more compelling data are needed. Similarly, claims of very small embryonic-like cells are also preliminary. Surface epithelial cells originating from gonadal ridge epithelial-like cells and from the mesonephric epithelium at the hilum of the ovary have also been proposed. Another important issue is the role of the stroma in guiding the formation of the ovary, ovigerous cords, follicles, and surface epithelium. Immune cells may also play key roles in developmental patterning, given their critical roles in corpora lutea formation and regression. Thus, while the cellular biology of the ovary is extremely important for its major endocrine and fertility roles, there is much still to be discovered. This review draws together the current evidence and perspectives on this topic.

The development of any organ system requires a complex interplay of cellular signals to initiate the differentiation and development of the heterogeneous cell and tissue types required to carry out the organs' functions. In this way, an extracellular stimulus is transmitted to an intracellular target through an array of interacting protein intermediaries, ultimately enabling the target cell to elicit a response. Surprisingly, only a small number of signaling pathways are implicated throughout embryogenesis and are used over and over again. Gonadogenesis is a unique process in that 2 morphologically distinct organs, the testes and ovaries, arise from a common precursor, the bipotential genital ridge. Accordingly, most of the signaling pathways observed throughout embryogenesis also have been shown to be important for mammalian sex determination and gonad development. Here, we review the mechanisms of signal transduction within these pathways and the importance of these pathways throughout mammalian gonad development, mainly concentrating on data obtained in mouse but including other species where appropriate.

Male sex determination in mammals relies on sex determining region Y-mediated upregulation of sex determining region-box 9 (SOX9) expression in XY gonads, whereas Wnt family member (WNT)/R-spondin 1 signaling and forkhead box L2 (FOXL2) drive female sex determination in XX gonads. Fibroblast growth factor (FGF) 9 signaling ensures sustained SOX9 expression through repression of one of the ovarian pathways (WNT signaling), whereas the significance of FGF-mediated repression of the FOXL2 pathway has not been studied. Previously, we demonstrated that FGFR2 is the receptor for FGF9 in the XY gonad. Whether a specific isoform (FGFR2b or FGFR2c) is required was puzzling. Here, we show that FGFR2c is required for male sex determination. Initially, in developing mouse embryos at 12.5 to 13.5 days postcoitum (dpc), XY Fgfr2c-/- gonads appear as ovotestes, with SOX9 and FOXL2 expression predominantly localized to the posterior and anterior gonadal poles, respectively. However, by 15.5 dpc, XY Fgfr2c-/- gonads show complete male-to-female sex reversal, evident by the lack of SOX9 and ectopic expression of FOXL2 throughout the gonads. Furthermore, ablation of the Foxl2 gene leads to partial or complete rescue of gonadal sex reversal in XY Fgfr2c-/- mice. Together with previous findings, our data suggest that testis determination involves FGFR2c-mediated repression of both the WNT4- and FOXL2-driven ovarian-determining pathways.

Axel Honneth proposes a recognition theoretical approach to justice, where justice is a matter of the recognition of individuals in the three spheres of ethical life he identifies. The value of recognition is based on the value of ‘self-realization’. Given the intimate relation between self-realization on the one hand and autonomy and authenticity on the other, autonomy and authenticity also play an important normative role in Honneth’s recognition theoretical approach to justice. In this paper I will examine Honneth’s conceptions of autonomy and authenticity. I will argue that these conceptions manage to meet some of the descriptive challenges faced by recognition theories, but fail to provide recognition with the required normative underpinnings. In order to gain or retain normative force, Honneth needs to provide an account of ‘true self’ or objective human well-being, which in turn would fail to meet the descriptive challenge and move dangerously close to traditional theory.

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.

Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination., ほ乳類の性決定遺伝子Sryの発現制御メカニズムの解明に成功 : 人間の性分化疾患の原因解明に期待. 京都大学プレスリリース. 2013-09-06.

A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

The Reproductive system, Andrew Pask.- Non-coding RNAs: an introduction. Jennifer X Yang, Raphael H Rastetter, and Dagmar Wilhelm.- How many non-coding RNAs does it take to compensate for male/female genetic imbalance?, Jean-Francois Ouimette and Claire Rougelle.- The piRNA pathway guards the germline genome against transposable elements, Katalin Fejes Toth, Dubravka Pezic, Evelyn Stuwe, Alexandre Webster,- Non-coding RNA in ovarian development and disease, J Browning Fitzgerald, Jitu George, and Lane K Christenson.- Non-coding RNA in spermatogenesis and epididymal maturation.- Janet E Holt, Simone J Stanger, Brett Nixon, and Eileen A McLaughlin.- Non-coding RNAs in mammary gland development and disease.- Gurveen K Sandhu, Michael J Milevskiy, Wesley Wilson, Annette M Shewan, and Melissa A Brown.- Non-coding RNAs in prostate cancer: From discovery to clinical applications, Yvonne Ceder.- Non-coding RNAs n uterine development, function and disease.- Warren B Nothnick.

For many years the main role of RNA, it addition to the housekeeping functions of for example tRNAs and rRNAs, was believed to be a messenger between the genes encoded on the DNA and the functional units of the cell, the proteins. This changed drastically with the identification of the first small non-coding RNA, termed microRNA, some 20 years ago. This discovery opened the field of regulatory RNAs with no or little protein-coding potential. Since then many new classes of regulatory non-coding RNAs, including endogenous small interfering RNAs (endo-siRNAs), PIWI-associated RNAs (piRNAs), and long non-coding RNAs, have been identified and we have made amazing progress in elucidating their expression, biogenesis, mechanisms and mode of action, and function in many, if not all, biological processes. In this chapter we provide an introduction about the current knowledge of the main classes of non-coding RNAs, what is know about their biogenesis and mechanism of function.

For many years the main role of RNA, it addition to the housekeeping functions of for example tRNAs and rRNAs, was believed to be a messenger between the genes encoded on the DNA and the functional units of the cell, the proteins. This changed drastically with the identification of the first small non-coding RNA, termed microRNA, some 20 years ago. This discovery opened the field of regulatory RNAs with no or little protein-coding potential. Since then many new classes of regulatory non-coding RNAs, including endogenous small interfering RNAs (endo-siRNAs), PIWI-associated RNAs (piRNAs), and long non-coding RNAs, have been identified and we have made amazing progress in elucidating their expression, biogenesis, mechanisms and mode of action, and function in many, if not all, biological processes. In this chapter we provide an introduction about the current knowledge of the main classes of non-coding RNAs, what is know about their biogenesis and mechanism of function.

Follistatin is a secreted glycoprotein required for female sex determination and early ovarian development, but the precise mechanisms regulating follistatin (Fst) gene expression are not known. Here, we investigate the roles of bone morphogenetic protein 2 (BMP2) and forkhead-domain transcription factor L2 (FOXL2) in the regulation of Fst expression in the developing mouse ovary. Bmp2 and Fst showed similar temporal profiles of mRNA expression, whereas FOXL2 protein and Fst mRNA were coexpressed in the same ovarian cells. In a cell culture model, both FOXL2 and BMP2 up-regulated Fst expression. In ex vivo mouse fetal gonad culture, exogenous BMP2 increased Fst expression, but this effect was counteracted by the BMP antagonist Noggin. Moreover, in Foxl2-null mice, Fst expression was reduced throughout fetal ovarian development, and Bmp2 expression was also reduced. Our data support a model in which FOXL2 and BMP2 cooperate to ensure correct expression of Fst in the developing ovary. Further, Wnt4-knockout mice showed reduced expression of Fst limited to early ovarian development, suggesting a role for WNT4 in the initiation, but not the maintenance, of Fst expression.

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 toads, both males and females develop a unique gonadal structure called the Bidder's organ (BO), which resembles ovarian tissue and is attached to the anterior part of the gonad. It is not clear whether the BO is a vestigial organ, or has an endocrine function. In this study, we investigated the expression of the gonadal development genes Dmrt1, Sox9, Sf1, Dax1, and p450arom in the developing BO as compared with the gonads of male and female cane toads. We demonstrate that Sf1, Dax1, and p450arom, key genes involved in vertebrate steroidogenesis, are transcriptionally active in the BO during developmental milestones associated with sexual development and maturation. Furthermore, the pattern of transcriptional activity in the BO is completely independent of the corresponding gonads in both sexes, despite its ovary-like morphology. These results suggest that the BO likely has a steroidogenic role in the development of the cane toad, distinct from that of the gonads.

During mouse germ cell development, the first sign of sex differentiation occurs when XY germ cells enter G1/G0 arrest from 12.5 days postcoitum (dpc). Retinoblastoma 1 (RB1), a potent cell cycle regulator, was investigated in XY germ cell arrest by studying germ cell proliferation in Rb1−/− mutant mouse embryos. Because mice homozygous for the Rb1 deletion die in utero around 14.5 dpc, we used ex vivo culture techniques to allow analysis of developing gonads to 16.5 dpc. In Rb1−/− gonads, we observed normal somatic cell development, assessed by immunofluorescence for markers HSD3B1 and anti-Mullerian hormone. However, at 14.5 dpc, when wild-type XY germ cells had arrested, we could detect actively proliferating germ cells using the proliferation markers MKI67, pHH3, and bromodeoxyuridine incorporation. The increased proliferation was reflected with a trend of increased germ cell number and expression of germ cell markers Ddx4 and Pou5f1 in the Rb1−/− testes. By 16.5 dpc, this phenotype was resolved such that the entire germ cell population had entered G1/G0 arrest, although the total germ cell number remained elevated. At each stage analyzed, we saw no increase in expression of RB1 family members Rbl1 and Rbl2 in the Rb1−/− testes, but we saw a significant increase of cyclin-dependent kinase (CDK) inhibitor Cdkn1b and Cdkn2b expression. We conclude that Rb1 is required for correct germ cell entry into G1/G0 arrest in the wild-type gonad at 14.5 dpc, but in its absence, upregulation of other cell cycle suppressors, including Cdkn1b and Cdkn2b, can induce delayed germ cell arrest.

Sex determination is regulated by a molecular antagonism between testis- and ovary-determining pathways in the supporting cell lineage of the gonadal primordia. Genes important for maintaining this lineage play critical roles in early gonadal development, but their influence on testis and ovary differentiation is unclear due to the severity of loss-of-function phenotypes. The transcription factor SF1 (Nr5a1/Ad4BP) is one such factor, required for establishing the supporting cell lineage, and for propagating the male pathway. In the gonad, Sf1 expression is enhanced by the transcriptional co-factor Cited2. We have used the reduced levels of Sf1 expression in Cited2(-/-) mice as a hypomorphic model to gain insight into the sex-specific roles of SF1 function in gonadal development. In XY mutant mice, we found that testis development was delayed in Cited2(-/-) gonads, and that testis structure was permanently disrupted. In XX Cited2(-/-) gonads, ectopic cell migration was observed which correlated with a transient upregulation of Fgf9, and a delay in Wnt4 then Foxl2 expression. These data suggest a novel role for SF1 in promoting ovarian development in addition to its roles in testis differentiation.

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.

Developmental defects caused by targeted gene inactivation in mice are commonly subject to strain-specific modifiers that modulate the severity of the phenotype. Although several genetic modifier loci have been mapped in mice, the gene(s) residing at these loci are mostly unidentified, and the molecular mechanisms of modifier action remain poorly understood. Mutations in Sox18 cause a variable phenotype in the human congenital syndrome hypotrichosis-lymphedema-telangiectasia, and the phenotype of Sox18-null mice varies from essentially normal to completely devoid of lymphatic vasculature and lethal, depending on the strain of the mice,suggesting a crucial role for strain-specific modifiers in this system. Here we show that two closely related Group F Sox factors, SOX7 and SOX17, are able to functionally substitute for SOX18 in vitro and in vivo. SOX7 and SOX17 are not normally expressed during lymphatic development, excluding a conventional redundancy mechanism. Instead, these genes are activated specifically in the absence of SOX18 function, and only in certain strains. Our studies identify Sox7 and Sox17 as modifiers of the Sox18 mutant phenotype, and reveal their mechanism of action as a novel mode of strain-specific compensatory upregulation.

Human patients with Frasier syndrome express reduced levels of the +KTS isoforms of the developmental regulator WT1 and exhibit complete XY gonadal dysgenesis and male-to-female sex reversal. Mice with a targeted mutation that blocks production of these isoforms show a reduction in Sry mRNA in the gonad, but the molecular and cellular basis of this reduction has not been established. Using immunofluorescence analysis, we found a significantly lower level of SRY protein per cell in XY Wt1(+KTS)-null mouse gonads. We also found a reduced number of SRY-expressing cells, correlating with a decrease in cell proliferation at and near the coelomic epithelium at 11.5 dpc. No reduction in somatic cell numbers was seen in XX Wt1(+KTS)-null gonads, indicating that the effect of WT1 on cell proliferation is mediated by Sry. Sertoli cell differentiation was blocked in XY Wt1(+KTS)-null mouse gonads, as indicated by the loss of SOX9 and Fgf9 expression, but the addition of recombinant FGF9 to ex vivo gonad cultures rescued the mutant phenotype, as indicated by the induction of the Sertoli-cell specific marker anti-Müllerian hormone. Our data suggest that WT1(+KTS) is involved in the cell-autonomous regulation of Sry expression, which in turn influences cell proliferation and Sertoli cell differentiation via FGF9. Thus, sex reversal in Wt1(+KTS)-null mice and Frasier syndrome patients results from a failure of Sertoli cells both to fully differentiate and to reach sufficient numbers to direct testis development.

In most mammals, the expression of SRY (sex-determining region on the Y chromosome) initiates the development of testes, and thus determines the sex of the individual. However, despite the pivotal role of SRY, its mechanism of action remains elusive. One important missing piece of the puzzle is the identification of genes regulated by SRY. In this study we used chromatin immunoprecipitation to identify direct SRY target genes. Anti-mouse SRY antibody precipitated a region 7.5 kb upstream of the transcriptional start site of cerebellin 4 precursor (Cbln4), which encodes a secreted protein. Cbln4 is expressed in Sertoli cells in the developing gonad, with a profile mimicking that of the testis-determining gene SRY-box containing gene 9 (Sox9). In transgenic XY mouse embryos with reduced Sox9 expression, Cbln4 expression also was reduced, whereas overexpression of Sox9 in XX mice caused an upregulation of Cbln4 expression. Finally, ectopic upregulation of SRY in vivo resulted in ectopic expression of Cbln4. Our findings suggest that both SRY and SOX9 contribute to the male-specific upregulation of Cbln4 in the developing testis, and they identified a direct in vivo target gene of SRY.