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.

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.

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.

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.

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.