Analyzing the cellular and molecular atlas of ovarian mesenchymal cells provides a strategy against female reproductive aging.

Ovarian mesenchymal cells (oMCs) constitute a distinct microenvironment that supports folliculogenesis under physiological conditions. Supplementation of exogenous non-ovarian mesenchymal-related cells has been reported to be an efficient approach to improve ovarian functions. However, the development and cellular and molecular characteristics of endogenous oMCs remain largely unexplored. In this study, we surveyed the single-cell transcriptomic landscape to dissect the cellular and molecular changes associated with the aging of oMCs in mice. Our results showed that the oMCs were composed of five ovarian differentiated MC (odMC) populations and one ovarian mesenchymal progenitor (oMP) cell population. These cells could differentiate into various odMCs via an oMP-derived route to construct the ovarian stroma structures. Comparative analysis revealed that ovarian aging was associated with decreased quantity of oMP cells and reduced quality of odMCs. Based on the findings of bioinformatics analysis, we designed different strategies involving supplementation with young oMCs to examine their effects on female fertility and health. Our functional investigations revealed that oMCs supplementation prior to ovarian senescence was the optimal method to improve female fertility and extend the reproductive lifespan of aged females in the long-term.

Lineage tracing of mutant granulosa cells reveals in vivo protective mechanisms that prevent granulosa cell tumorigenesis.

Ovarian granulosa cell tumors (GCTs) originate from granulosa cells (GCs) and represent the most common sex cord-stromal tumor in humans. However, the developmental regulations and molecular mechanisms underlying their etiology are largely unknown. In the current study, we combined a multi-fluorescent reporter mouse model with a conditional knockout mouse model, in which the tumor suppressor genes Pten and p27 were deleted in GCs, to perform cell lineage tracing of mutant GCs. We found that only 30% of ovaries with substantial mutant GCs developed into GCTs that derived from a single mutant GC. In-depth molecular analysis of the process of tumorigenesis demonstrated that up-regulation of immune evasion genes Cd24a and Cd47 led, in part, to the transition of mutant GCs to GCTs. Therefore, treatment with the Cd47 inhibitor RRX-001 was tested and found to efficiently suppress the growth of GCTs in vivo. Together, our study has revealed an immune evasion mechanism via CD24/CD47 upregulation to GCT formation, shedding light on the future potential clinical therapies for GCTs.

Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells.

Ovarian follicles are the fundamental structures that support oocyte development, and communications between oocytes and follicle somatic cells are crucial for oogenesis. However, it is unknown that whether exposure to microgravity influences cellular communications and ovarian follicle development, which might be harmful for female fertility. By 3D culturing of ovarian follicles under simulated microgravity (SMG) conditions in a rotating cell culture system, we found that SMG treatment did not affect the survival or general growth of follicles but decreased the quality of cultured follicles released oocytes. Ultrastructure detections by high-resolution imaging showed that the development of cellular communicating structures, including granulosa cell transzonal projections and oocyte microvilli, were markedly disrupted. These abnormalities caused chaotic polarity of granulosa cells (GCs) and a decrease in oocyte-secreted factors, such as Growth Differentiation Factor 9 (GDF9), which led to decreased quality of oocytes in these follicles. Therefore, the quality of oocytes was dramatically improved by the supplementations of GDF9 and NADPH-oxidase inhibitor apocynin. Together, our results suggest that exposure to simulated microgravity impairs the ultrastructure of ovarian follicles. Such impairment may affect female fertility in space environment.

Quantitative proteomic analysis uncovers protein-expression profiles during gonadotropin-dependent folliculogenesis in mice†.

Ovarian follicle is the basic functional unit of female reproduction, and is composed of oocyte and surrounding granulosa cells. In mammals, folliculogenesis strictly rely on gonadotropin regulations to determine the ovulation and the quality of eggs. However, the dynamic changes of protein-expressing profiles in follicles at different developmental stages remain largely unknown. By performing mass-spectrometry-based quantitative proteomic analysis of mouse follicles, we provide a proteomic database (~3000 proteins) that covers three key stages of gonadotropin-dependent folliculogenesis. By combining bioinformatics analysis with in situ expression validation, we showed that our proteomic data well reflected physiological changes during folliculogenesis, which provided potential to predict unknown regulators of folliculogenesis. Additionally, by using the oocyte structural protein zona pellucida protein 2 as the internal control, we showed the possibility of our database to predict the expression dynamics of oocyte-expressing proteins during folliculogenesis. Taken together, we provide a high-coverage proteomic database to study protein-expression dynamics during gonadotropin-dependent folliculogenesis in mammals.

In vivo promotion of primordial follicle activation by stem cell factor treatment in mice with premature ovarian insufficiency and advanced age.

Dormant primordial follicles (PFs) are the most abundant reproductive resource in mammalian ovaries. With advances in the mechanism of study of the regulation of PF activation, PFs have been used to improve fertility in clinical practice. As a central controlling element of follicle activation signaling, the pre-granulosa cell-secreted stem cell factor (SCF; also known as KIT ligand, KITL), which initiates the growth of dormant oocytes, is an ideal natural activator that stimulates follicle activation. However, no systematic study has been conducted to identify the activating effect of SCF in vivo and in vitro. In this study, by combining an in vitro whole ovary culture system and several mouse models, we provide a series of experimental evidence that SCF is an efficient activator for improving PF activation in mouse ovaries. Our in vitro study showed that SCF increased phosphatidylinositol 3-kinase (PI3K) signaling and PF activation ratio in neonatal ovaries. In vivo ovarian non-invasive topical administrations of SCF to the ovaries efficiently improved follicle activation and development, oocyte retrieval ratio and fertility in inducible premature ovarian insufficiency mouse models and aged mice. Our study suggests that SCF is an efficient growth factor that can be applied to improve PF activation.

Polycomb subunit Pcgf2 mediates ovulation and fertility through transcriptional regulation progesterone receptor.

Ovarian follicles are the fundamental structure to support oocyte development, which provides mature oocytes for offspring. This process requires granulosa cells (GCs) to respond to the midcycle surge of hormones, leading to GC proliferation and differentiation by a series of genes' transcriptional expression changes. Epigenetic mediator, Polycomb Repressive Complex 1 (PRC1) has been reported to function in fetal ovarian development. However, its functional relevance to folliculogenesis and ovulation remains unknown. In this study, we demonstrated that GC-selective depletion of PCGF2, a key component of PRC1, led to the loss of follicles, ovulation defects, and a lengthened estrus cycle, resulting in subfertility in female mice. The expression of PCGF2 is in the GCs of growing follicles and increases after human chorionic gonadotropin (hCG) stimulation. PCGF2 bound to the promoter of the key ovulation gene progesterone receptor (Pgr) and upregulated the expression of Pgr by targeting the epigenetic modification of H2AK119ub1 after hCG surge. Consistently, the expression of downstream genes of Pgr also sharply decreased, which resulted in the follicular rupture failed and oocyte entrapped in corpus luteum in GC-specific Pcgf2 knockout mice. Together, our study identified that PCGF2 is essential for folliculogenesis and ovulation via modulating hormone receptor expression.

Asynchronous embryonic germ cell development leads to a heterogeneity of postnatal ovarian follicle activation and may influence the timing of puberty onset in mice.

BACKGROUND: Ovarian follicles, which are the basic units of female reproduction, are composed of oocytes and surrounding somatic (pre) granulosa cells (GCs). A recent study revealed that signaling in somatic preGCs controlled the activation (initial recruitment) of follicles in the adult ovaries, but it is also known that there are two waves of follicle with age-related heterogeneity in their developmental dynamics in mammals. Although this heterogeneity was proposed to be crucial for female reproduction, our understanding of how it arises and its significance is still elusive. RESULTS: In the current study, by deleting the key secreted factor KIT ligand from preGCs and analyzing the follicle cell developmental dynamics, we revealed distinct patterns of activation and growth associated with the two waves of follicles in mouse ovary. Our results confirmed that activation of adult wave follicles is initiated by somatic preGCs and dependent on the KIT ligand. By contrast, activation of first wave follicles, which are awakened from germ cells before follicle formation, can occur in the absence of preGC-secreted KIT ligand in postnatal ovaries and appears to be oocyte-initiated. We also found that the asynchronous activity of phosphatidylinositol 3 kinases (PI3K) signaling and meiotic process in embryonic germ cells lead to the follicle heterogeneity in postnatal ovaries. In addition, we supplied evidence that the time sequence of embryonic germ cell development and its related first wave follicle growth are correlated to the time of puberty onset in females. CONCLUSION: Taken together, our study provides evidence that asynchronous development of embryonic oocytes leads to the heterogeneity of postnatal ovarian follicle activation and development, and affects the timing of onset of puberty in females.

Imaging and tracing the pattern of adult ovarian angiogenesis implies a strategy against female reproductive aging.

Robust angiogenesis is continuously active in ovaries to remodel the ovary-body connections in mammals, but understanding of this unique process remains elusive. Here, we performed high-resolution, three-dimensional ovarian vascular imaging and traced the pattern of ovarian angiogenesis and vascular development in the long term. We found that angiogenesis was mainly active on ovarian follicles and corpus luteum and that robust angiogenesis constructs independent but temporary vascular networks for each follicle. Based on the pattern of ovarian angiogenesis, we designed an angiogenesis-blocking strategy by axitinib administration to young females, and we found that the temporary suppression of angiogenesis paused ovarian development and kept the ovarian reserve in the long term, leading to postponed ovarian senescence and an extension of the female reproductive life span. Together, by uncovering the detailed model of physiological ovarian angiogenesis, our experiments suggest a potential approach to delay female reproductive aging through the manipulation of angiogenesis.