Establishing and maintaining fertility: the importance of cell cycle arrest.

Development of the ovary or testis is required to establish reproductive competence. Gonad development relies on key cell fate decisions that occur early in embryonic development and are actively maintained. During gonad development, both germ cells and somatic cells proliferate extensively, a process facilitated by cell cycle regulation. This review focuses on the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) in mouse gonad development. We particularly highlight recent single-cell RNA sequencing studies that show the heterogeneity of cyclin-dependent kinase inhibitors. This diversity highlights new roles for cell cycle inhibitors in controlling and maintaining female fertility.

Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation†.

The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signaling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system, which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR. Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1, and ZFX, was investigated with in-situ immunolocalization in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signaling pathway's role in primordial follicle activation.

Investigation into the presence and functional significance of proinsulin C-peptide in the female germline†.

Diabetes is associated with poor oocyte quality and the dysregulation of ovarian function and is thus a leading contributor to the increasing prevalence of female reproductive pathologies. Accordingly, it is well-established that insulin fulfills a key role in the regulation of several facets of female reproduction. What remains less certain is whether proinsulin C-peptide, which has recently been implicated in cellular signaling cascades, holds a functional role in the female germline. In the present study, we examined the expression of insulin, C-peptide, and its purported receptor; GPR146, within the mouse ovary and oocyte. Our data establish the presence of abundant C-peptide within follicular fluid and raise the prospect that this bioactive peptide is internalized by oocytes in a G-protein coupled receptor-dependent manner. Further, our data reveal that internalized C-peptide undergoes pronounced subcellular relocalization from the ooplasm to the pronuclei postfertilization. The application of immunoprecipitation analysis and mass spectrometry identified breast cancer type 2 susceptibility protein (BRCA2), the meiotic resumption/DNA repair protein, as a primary binding partner for C-peptide within the oocyte. Collectively, these findings establish a novel accumulation profile for C-peptide in the female germline and provide the first evidence for an interaction between C-peptide and BRCA2. This interaction is particularly intriguing when considering the propensity for oocytes from diabetic women to experience aberrant meiotic resumption and perturbation of traditional DNA repair processes. This therefore provides a clear imperative for further investigation of the implications of dysregulated C-peptide production in these individuals.

Janus kinase JAK1 maintains the ovarian reserve of primordial follicles in the mouse ovary.

STUDY QUESTION: Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? SUMMARY ANSWER: JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. WHAT IS KNOWN ALREADY: A series of integrated, intrinsic signalling pathways (including PI3K/AKT, mTOR and KITL) are responsible for regulating the ovarian reserve of non-growing primordial follicles and ultimately female fertility. The JAK-STAT signal transduction pathway is highly conserved with established roles in cell division and differentiation. Key pathway members (specifically JAK1, STAT3 and SOCS4) have been previously implicated in early follicle development. STUDY DESIGN, SIZE, DURATION: A laboratory animal study was undertaken using the C57Bl/6 inbred mouse strain as a model for human ovarian follicle development. To determine which Jak genes were most abundantly expressed during primordial follicle activation, mRNA expression was analysed across a developmental time-course, with ovaries collected from female mice at post-natal days 1 (PND1), 4 (PND4), 8 (PND8), as well as at 6 weeks (6WK) and 7 months (7MTH) (n ≥ 4). Functional analysis of JAK1 was performed on PND2 mouse ovaries subjected to in vitro explant culture treated with 12.5 µM Ruxolitinib (JAK inhibitor) or vehicle control (DMSO) for 48 h prior to histological assessment (n ≥ 4). PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression and localization of the JAK family during ovarian follicle development in the C57Bl/6 inbred mouse strain were evaluated using quantitative PCR, immunoblotting and immunolocalisation. Functional studies were undertaken using the JAK inhibitor Ruxolitinib to investigate the underpinning cellular mechanisms via biochemical in vitro inhibition and histological assessment of intact neonate ovaries. All experiments were replicated at least three times using tissue from different mice unless otherwise stated. MAIN RESULTS AND THE ROLE OF CHANCE: Jak1 is the predominant Jak mRNA expressed in the C57Bl/6 mouse ovary across all developmental time-points assessed (P ≤ 0.05). Forty-eight hour inhibition of JAK1 with Ruxolitinib of PND2 ovaries in vitro demonstrated concomitant acceleration of primordial follicle activation and apoptosis (P ≤ 0.001) and upregulation of downstream JAK-STAT pathway members STAT3 and suppressors of cytokine signalling 4 (SOCS4). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Results are shown in one species, the C57Bl/6 mouse strain as an established model of human ovary development. Ruxolitinib also inhibits JAK2, with decreased efficacy. However, Jak2 mRNA had limited expression in the mouse ovary, particularly at the neonatal stages of follicle development, thus any effect of Ruxolitinib on primordial follicle activation was unlikely to be mediated via this isoform. WIDER IMPLICATIONS OF THE FINDINGS: This study supports a key role for JAK1 in the maintenance and activation of primordial follicles, with potential for targeting the JAK-STAT pathway as a method of regulating the ovarian reserve and female fertility. STUDY FUNDING AND COMPETING INTEREST(S): This project has been funded by the Australian National Health and Medical Research Council (G1600095) and The Hunter Medical Research Institute Bob and Terry Kennedy Children's Research Project Grant in Pregnancy & Reproduction (G1501433). All authors declare no conflict of interests.

Making human eggs in a dish: are we close?

In the space of 50 years, we have seen incredible achievements in human reproductive medicine. With these leaps forward, it is no wonder that there is a major interest in women's reproductive health research, including extension of reproductive lifespan. Substantial effort is currently being made to address this challenge, including from the commercial sector. In vitro gametogenesis (IVG) in mice is a spectacular breakthrough and has the potential to offer hope to women with intractable infertility. However, with such lofty goals, some reflection may be called for: mastering all of the techniques required for complete and safe IVG in women is likely to be extraordinarily difficult.

A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

Genetic control of typical and atypical sex development.

Sex development relies on the sex-specific action of gene networks to differentiate the bipotential gonads of the growing fetus into testis or ovaries, followed by the differentiation of internal and external genitalia depending on the presence or absence of hormones. Differences in sex development (DSD) arise from congenital alterations during any of these processes, and are classified depending on sex chromosomal constitution as sex chromosome DSD, 46,XY DSD or 46,XX DSD. Understanding the genetics and embryology of typical and atypical sex development is essential for diagnosing, treating and managing DSD. Advances have been made in understanding the genetic causes of DSD over the past 10 years, especially for 46,XY DSD. Additional information is required to better understand ovarian and female development and to identify further genetic causes of 46,XX DSD, besides congenital adrenal hyperplasia. Ongoing research is focused on the discovery of further genes related to typical and atypical sex development and, therefore, on improving diagnosis of DSD.

Somatic FGFR2 is Required for Germ Cell Maintenance in the Mouse Ovary.

During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.

A New Understanding, Guided by Single-Cell Sequencing, of the Establishment and Maintenance of the Ovarian Reserve in Mammals.

BACKGROUND: Oocytes are a finite and non-renewable resource that are maintained in primordial follicle structures. The ovarian reserve is the totality of primordial follicles, present from birth, within the ovary and its establishment, size, and maintenance dictates the duration of the female reproductive lifespan. Understanding the cellular and molecular dynamics relevant to the establishment and maintenance of the reserve provides the first steps necessary for modulating both individual human and animal reproductive health as well as population dynamics. SUMMARY: This review details the key stages of establishment and maintenance of the ovarian reserve, encompassing germ cell nest formation, germ cell nest breakdown, and primordial follicle formation and activation. Furthermore, we spotlight several formative single-cell sequencing studies that have significantly advanced our knowledge of novel molecular regulators of the ovarian reserve, which may improve our ability to modulate female reproductive lifespans. KEY MESSAGES: The application of single-cell sequencing to studies of ovarian development in mammals, especially when leveraging genetic and environmental models, offers significant insights into fertility and its regulation. Moreover, comparative studies looking at key stages in the development of the ovarian reserve across species has the potential to impact not just human fertility, but also conservation biology, invasive species management, and agriculture.

Dataset of differentially expressed genes in mouse P12 testes in response to the loss of ATRX in Sertoli cells.

This dataset represents genes that are dysregulated in the postnatal day 12 (P12) mouse testis when ATRX is specifically inactivated in Sertoli cells (ScAtrxKO mice). The differentially expressed genes included in the dataset may play important roles in the testicular phenotypes observed in the ScAtrxKO mice, which were first reported in our previous work [1]. In fetal ScAtrxKO mice, Sertoli cells undergo apoptosis due to cell cycle defects, resulting in smaller testes with reduced tubule volume [1]. Adult ScAtrxKO mice show a wide range of spermatogenesis defects probably due to a failure of the dysfunctional ATRX protein to interact with the androgen receptor (AR) [1]. ATRX, a chromatin remodeling protein, is widely expressed in the human testis including Sertoli cells [2,3]. In XY individuals, the loss of ATRX leads to ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome associated with a wide range of genital abnormalities such as hypospadias, ambiguous genitalia, and small testes with reduced tubule volume [4], [5], [6], [7], [8]. Our dataset contributes towards understanding the mechanism underlying ATRX regulation of testis development and spermatogenesis.