Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.

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.

MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function.

The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, we show that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.

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.

A scoping review of the information provided by fertility smartphone applications.

The growth of smartphone application use across areas of female reproductive health has led to increased interest into their functions and benefits. This scoping review aims to determine the nature and extent of the peer-reviewed literature presented on fertility-based apps, to identify the reliability of the information within the apps, and to determine the ability of this information to educate users. A systematic search of six databases was conducted in April 2020, returning a total of 21,158 records. After duplicate removal, title and abstract screening exclusionary steps, 27 records were reviewed and charted. Records covered a variety of reproductive health themes including contraception, sexual health, and family planning, and used a range of methodologies. The accuracy of fertility information within the apps reported in these studies was variable, but overall there was a lack of depth in the coverage of content in apps. It was common for studies in this review to base fertile window algorithms on stringent cycle length and variability requirements, limiting the applicability of information delivered to users. Furthermore, studies from app affiliates often lacked collaborations with researchers, minimising the potential for fertility knowledge improvements integrated across the suite of female reproductive health apps.

Biocompatible Nanomaterials as an Emerging Technology in Reproductive Health; a Focus on the Male.

A growing body of research has confirmed that nanoparticle (NP) systems can enhance delivery of therapeutic and imaging agents as well as prevent potentially damaging systemic exposure to these agents by modifying the kinetics of their release. With a wide choice of NP materials possessing different properties and surface modification options with unique targeting agents, bespoke nanosystems have been developed for applications varying from cancer therapeutics and genetic modification to cell imaging. Although there remain many challenges for the clinical application of nanoparticles, including toxicity within the reproductive system, some of these may be overcome with the recent development of biodegradable nanoparticles that offer increased biocompatibility. In recognition of this potential, this review seeks to present recent NP research with a focus on the exciting possibilities posed by the application of biocompatible nanomaterials within the fields of male reproductive medicine, health, and research.

The Impact of Aging on Macroautophagy in the Pre-ovulatory Mouse Oocyte.

Accompanying the precipitous age-related decline in human female fertility is an increase in the proportion of poor-quality oocytes within the ovary. The macroautophagy pathway, an essential protein degradation mechanism responsible for maintaining cell health, has not yet been thoroughly investigated in this phenomenon. The aim of this study was to characterize the macroautophagy pathway in an established mouse model of oocyte aging using in-depth image analysis-based methods and to determine mechanisms that account for the observed changes. Three autophagy pathway markers were selected for assessment of gene and protein expression in this model: Beclin 1; an initiator of autophagosome formation, Microtubule-associated protein 1 light chain 3B; a constituent of the autophagosome membrane, and lysosomal-associated membrane protein 1; a constituent of the lysosome membrane. Through quantitative image analysis of immunolabeled oocytes, this study revealed impairment of the macroautophagy pathway in the aged oocyte with an attenuation of both autophagosome and lysosome number. Additionally, an accumulation of amphisomes greater than 10 µm2 in area were observed in aging oocytes, and this accumulation was mimicked in oocytes treated with lysosomal inhibitor chloroquine. Overall, these findings implicate lysosomal dysfunction as a prominent mechanism by which these age-related changes may occur and highlight the importance of macroautophagy in maintaining mouse pre-ovulatory oocyte quality. This provides a basis for further investigation of dysfunctional autophagy in poor oocyte quality and for the development of therapeutic or preventative strategies to aid in the maintenance of pre-ovulatory oocyte health.

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.

The association between reproductive health smartphone applications and fertility knowledge of Australian women.

BACKGROUND: Previous studies have identified that women living in developed countries have insufficient knowledge of factors which may be contributing to the increasingly high global infertility rates such as maternal age and assisted reproductive technologies. There is a large market of reproductive health smartphone applications, yet little is known about the advantages these apps may confer to users in regards to reproductive health knowledge. METHODS: An anonymous, online survey of women living in Australia aged 18 and above was open March-June 2018, until ≥200 responses were acquired for statistical power. Respondents answered questions regarding knowledge about general fertility and related factors (age, cyclic fertility, smoking, obesity, miscarriage rate, and success of assisted reproductive technologies). Fertility knowledge was compared in respondents who did or did not use apps relating to female reproductive health. Additionally the functions preferred in reproductive health apps was described by app using respondents. Sociodemographic information was also collected, and relevant data within the dataset was subject to multivariable modelling for the outcome of the fertility knowledge questions. RESULTS: Of the 673 respondents that completed the survey, 43.09% reported using mobile phone applications relating to female reproductive health. On average, respondents answered only three of the six fertility knowledge questions correctly. App using respondents were more likely to score better on one question, related to fertility during the menstrual cycle (p < 0.001). App users most commonly reported using the menstrual tracking function in apps (82.4%), which may account for the increased knowledge of cyclic fertility. CONCLUSIONS: This data provides preliminary evidence toward the usefulness of smartphone applications as a medium for providing information about fertility to women. A limited understanding of one's own fertility was demonstrated despite being essential for the decision-making of women throughout their reproductive years.

Dynamin 2-dependent endocytosis is essential for mouse oocyte development and fertility.

During folliculogenesis, oocytes are dependent on metabolic and molecular support from surrounding somatic cells. Here, we examined the role of the dynamin (DNM) family of mechanoenzymes in mediating endocytotic uptake into growing follicular oocytes. We found DNM1 and DNM2 to be highly expressed in growing follicular oocytes as well as in mature germinal vesicle (GV) and metaphase II (MII) stage oocytes. Moreover, oocyte-specific conditional knockout (cKO) of DNM2 (DNM2Δ) led to complete sterility, with follicles arresting at the preantral stage of development. In addition, DNM2Δ ovaries were characterized by disrupted follicular growth as well as oocyte and follicle apoptosis. Further, the loss of DNM activity, either through DNM2 cKO or through pharmacological inhibition (Dyngo 6a) led to the impairment of endocytotic pathways in preantral oocytes as well as in mature GV and MII oocytes, respectively. Loss of DNM activity resulted in the redistribution of endosomes and the misslocalization of clathrin and actin, suggesting dysfunctional endocytosis. Notably, there was no observable effect on the fertility of DNM1Δ females. Our study has provided new insight into the complex and dynamic nature of oocyte growth during folliculogenesis, suggesting a role for DNM2 in mediating the endocytotic events that are essential for oocyte development.

Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development†.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality.

Male Infertility: Shining a Light on Lipids and Lipid-Modulating Enzymes in the Male Germline.

Despite the prevalence of male factor infertility, most cases are defined as idiopathic, thus limiting treatment options and driving increased rates of recourse to assisted reproductive technologies (ARTs). Regrettably, our current armory of ARTs does not constitute therapeutic treatments for male infertility, thus highlighting an urgent need for novel intervention strategies. In our attempts to fill this void, we have come to appreciate that the production of pathological levels of oxygen radicals within the male germline are a defining etiology of many idiopathic infertility cases. Indeed, an imbalance of reactive oxygen species can precipitate a cascade of deleterious sequelae, beginning with the peroxidation of membrane lipids and culminating in cellular dysfunction and death. Here, we shine light on the importance of lipid homeostasis, and the impact of lipid stress in the demise of the male germ cell. We also seek to highlight the utility of emerging lipidomic technologies to enhance our understanding of the diverse roles that lipids play in sperm function, and to identify biomarkers capable of tracking infertility in patient cohorts. Such information should improve our fundamental understanding of the mechanistic causes of male infertility and find application in the development of efficacious treatment options.

Autophagy in Female Fertility: A Role in Oxidative Stress and Aging.

Significance: The precipitous age-related decline in female fertility is intimately associated with a reduction in both the quantity and quality of the germline (oocytes). Although complex etiologies undoubtedly contribute to the deterioration of oocyte quality, increasing attention has focused on the pervasive impact of oxidative stress. Indeed, the prolonged lifespan of the meiotically arrested oocyte places this cell at heightened risk of oxidative lesions, which commonly manifest in dysregulation of protein homeostasis (proteostasis). Although oocytes are able to mitigate this threat via the mobilization of a sophisticated network of surveillance, repair, and proteolytic pathways, these defenses are themselves prone to age-related defects, reducing their capacity to eliminate oxidatively damaged proteins. Recent Advances: Here, we give consideration to the quality control mechanisms identified within the ovary that afford protection to the female germline. Our primary focus is to review recent advances in our understanding of the autophagy pathway and its contribution to promoting oocyte longevity and modulating pathophysiological responses to oxidative stress. In addition, we explore the therapeutic potential of emerging strategies to fortify autophagic activity. Critical Issues: The complex interplay of oxidative stress and autophagy has yet to be fully elucidated within the context of the aging oocyte and surrounding ovarian environment. Future Directions: Emerging evidence provides a strong impetus to resolve the causal link between autophagy and oxidative stress-driven pathologies in the aging oocyte. Such research may ultimately inform novel therapeutic strategies to combat the age-related loss of female fertility via fortification of intrinsic autophagic activity.

Mechanistic Insight into the Regulation of Lipoxygenase-Driven Lipid Peroxidation Events in Human Spermatozoa and Their Impact on Male Fertility.

A prevalent cause of sperm dysfunction in male infertility patients is the overproduction of reactive oxygen species, an attendant increase in lipid peroxidation and the production of cytotoxic reactive carbonyl species such as 4-hydroxynonenal. Our previous studies have implicated arachidonate 15-lipoxygenase (ALOX15) in the production of 4-hydroxynonenal in developing germ cells. Here, we have aimed to develop a further mechanistic understanding of the lipoxygenase-lipid peroxidation pathway in human spermatozoa. Through pharmacological inhibition studies, we identified a protective role for phospholipase enzymes in the liberation of peroxidised polyunsaturated fatty acids from the human sperm membrane. Our results also revealed that arachidonic acid, linoleic acid and docosahexanoic acid are key polyunsaturated fatty acid substrates for ALOX15. Upon examination of ALOX15 in the spermatozoa of infertile patients compared to their normozoospermic counterparts, we observed significantly elevated levels of ALOX15 protein abundance in the infertile population and an increase in 4-hydroxynonenal adducts. Collectively, these data confirm the involvement of ALOX15 in the oxidative stress cascade of human spermatozoa and support the notion that increased ALOX15 abundance in sperm cells may accentuate membrane lipid peroxidation and cellular dysfunction, ultimately contributing to male infertility.

Advances in human primordial follicle activation and premature ovarian insufficiency.

In women, the non-growing population of follicles that comprise the ovarian reserve is determined at birth and serves as the reservoir for future fertility. This reserve of dormant, primordial follicles and the mechanisms controlling their selective activation which constitute the committing step into folliculogenesis are essential for determining fertility outcomes in women. Much of the available data on the mechanisms responsible for primordial follicle activation focuses on a selection of key molecular pathways, studied primarily in animal models, with findings often not synonymous in humans. The excessive induction of primordial follicle activation may cause the development of premature ovarian insufficiency (POI), a condition characterised by menopause before age 40 years. POI affects 1-2% of all women and is accompanied by additional health risks. Therefore, it is critical to further our understanding of primordial follicle activation in order to diagnose, treat and prevent premature infertility. Research in primordial follicle activation has focused on connecting new molecules to already established key signalling pathways, such as phosphatidylinositol 3-Kinase (PI3K) and mammalian target of rapamycin (mTOR). Additionally, other aspects of the ovarian environment, such as the function of the extracellular matrix, in contributing to primordial follicle activation have gained traction. Clinical applications are examining replication of this extracellular environment through the construction of biological matrices mimicking the 3D ovary, to support follicular growth through to ovulation. This review outlines the importance of the events leading to the establishment of the ovarian reserve and highlights the fundamental factors known to influence primordial follicle activation in humans presenting new horizons for female infertility treatment.

The small non-coding RNA profile of mouse oocytes is modified during aging.

Oocytes are reliant on messenger RNA (mRNA) stores to support their survival and integrity during a protracted period of transcriptional dormancy as they await ovulation. Oocytes are, however, known to experience an age-associated alteration in mRNA transcript abundance, a phenomenon that contributes to reduced developmental potential. Here we have investigated whether the expression profile of small non-protein-coding RNAs (sRNAs) is similarly altered in aged mouse oocytes. The application of high throughput sequencing revealed substantial changes to the global sRNA profile of germinal vesicle stage oocytes from young (4-6 weeks) and aged mice (14-16 months). Among these, 160 endogenous small-interfering RNAs (endo-siRNAs) and 10 microRNAs (miRNAs) were determined to differentially accumulate within young and aged oocytes. Further, we revealed decreased expression of two members of the kinesin protein family, Kifc1 and Kifc5b, in aged oocytes; family members selectively targeted for expression regulation by endo-siRNAs of elevated abundance. The implications of reduced Kifc1 and Kifc5b expression were explored using complementary siRNA-mediated knockdown and pharmacological inhibition strategies, both of which led to increased rates of aneuploidy in otherwise healthy young oocytes. Collectively, our data raise the prospect that altered sRNA abundance, specifically endo-siRNA abundance, could influence the quality of the aged oocyte.

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.

Oxidative damage in naturally aged mouse oocytes is exacerbated by dysregulation of proteasomal activity.

An increase in oxidative protein damage is a leading contributor to the age-associated decline in oocyte quality. By removing such damaged proteins, the proteasome plays an essential role in maintaining the fidelity of oocyte meiosis. In this study, we established that decreased proteasome activity in naturally aged, germinal vesicle (GV) mouse oocytes positively correlates with increased protein modification by the lipid aldehyde 4-hydroxynonenal (4-HNE). Furthermore, attenuation of proteasome activity in GV oocytes of young animals was accompanied by an increase in 4-HNE-modified proteins, including α-tubulin, thereby contributing to a reduction in tubulin polymerization, microtubule stability, and integrity of oocyte meiosis. A decrease in proteasome activity was also recapitulated in the GV oocytes of young animals following exposure to oxidative insults in the form of either hydrogen peroxide (H2O2) or 4-HNE. We also observed that upon oxidative insult, 4-HNE exhibits elevated adduction to multiple proteasomal subunits. Notably, the inclusion of the antioxidant penicillamine, to limit propagation of oxidative stress cascades, led to a complete recovery of proteasome activity and enhanced clearance of 4-HNE-adducted α-tubulin during a 6-h post-treatment recovery period. This strategy also proved effective in reducing the incidence of oxidative stress-induced aneuploidy following in vitro oocyte maturation, but was ineffective for naturally aged oocytes. Taken together, our results implicate proteasome dysfunction as an important factor in the accumulation of oxidatively induced protein damage in the female germline. This discovery holds promise for the design of therapeutic interventions to address the age-dependent decline in oocyte quality.

Vitamin D and folate: A reciprocal environmental association based on seasonality and genetic disposition.

OBJECTIVES: The purpose of this study was (1) to elucidate any reciprocal seasonal relationship that might exist between red cell folate (RCF) and serum vitamin D3 Levels; (2) to explore whether folate-related gene variants that influence/alter DNA-thymidylate and methyl group biosynthesis modify any associations detected in objective 1; and (3) to consider whether these processes might influence reproductive success consistent with the "folate-vitamin D-UV hypothesis of skin pigmentation" evolutionary model. METHODS: A large (n = 649) Australian cross-sectional study population was examined. Polymerase chain reaction (PCR)/Restriction fragment length polymorphism (RFLP) analysis was used to genotype C677T-MTHFR, C1420T-SHMT, T401C-MTHFD and 2R > 3R-TS. RCF was measured by chemiluminescent immunoassay and vitamin D2 and D3 by HPLC. RESULTS: RCF and photosynthesized vitamin D3 , but not RCF and dietary vitamin D2 , exhibit a significant reciprocal association in spring and summer. Three folate genes (C677T-MTHFR, C1420T-SHMT, and 2R > 3R-TS) strengthen this effect in spring, and another (T401C-MTHFD) in summer. Effects are seasonal, and do not occur over the whole year. CONCLUSIONS: Findings are consistent with what might be required for the "folate-vitamin D-UV hypothesis of skin pigmentation" model. It suggests genetic influence in provision of one-carbon units by 5,10-methylene-H4 folate, may be an important factor in what appears to be a clear seasonal relationship between vitamin D3 and folate status.

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.