Ovarian reserve (OR) and fertility are critical in women's healthcare. Clinical methods for encoding OR and fertility rely on the combination of tests, which cannot serve as a multi-functional platform with limited information from specific biofluids. Herein, metabolic fingerprinting of follicular fluid (MFFF) from follicles is performed, using particle-assisted laser desorption/ionization mass spectrometry (PALDI-MS) to encode OR and fertility. PALDI-MS allows efficient MFFF, showing fast speed (≈30 s), high sensitivity (≈60 fmol), and desirable reproducibility (coefficients of variation <15%). Further, machine learning of MFFF is applied to diagnose diminished OR (area under the curve of 0.929) and identify high-quality oocytes/embryos (p < 0.05) by a single PALDI-MS test. Meanwhile, metabolic biomarkers from MFFF are identified, which also determine oocyte/embryo quality (p < 0.05) from the sampling follicles toward fertility prediction in clinics. This approach offers a powerful platform in women's healthcare, not limited to OR and fertility.

There are a few studies indicating that small molecular compounds affect the proliferation, differentiation, apoptosis, and autophagy of female germline stem cells (FGSCs). However, whether small molecular compound 28 (C28) affect development of FGSCs remains unknown. In this study, we found that C28 reduced the viability and proliferation of FGSCs, respectively. Additionally, western blotting showed that the expression of autophagy marker light chain 3 beta II (LC3B-II) was significantly increased and expression of sequestosome-1 (SQSTM1) was significantly reduced in C28-treated groups. Immunofluorescence showed that, in C28-treated groups, the number of LC3B-II-positive puncta was increased significantly. These results indicated that C28 induced autophagy of FGSCs in vitro. Furthermore, data from Chromatin Immunoprecipitation Sequencing for H3K27ac showed that autophagy-related biological processes such as regulation of mitochondrial membrane potential, Golgi vesicle transport, and cellular response to reactive oxygen species were different after C28-treated. In addition, RNA-Seq showed that the expression of genes (Trib3, DDIT3, and ATF4) related to endoplasmic reticulum (ER) stress was enhanced by C28. These results suggest that the changes of H3K27ac and ER stress might be associated with C28-induced FGSC autophagy.

N6-methyladenosine (m6A) methylation modification is the most prevalent and abundant internal modification of eukaryotic mRNAs. Increasing evidence has shown that mRNA m6A plays important roles in the development of stem cells. However, to the best of our knowledge, no reports about the roles of mRNA m6A in mouse female germline stem cells (mFGSCs) have been published. In this study, we compared the genome-wide profiles of mRNA m6A methylation and DNA methylation between FGSCs and sandosinbred mice (SIM) embryo-derived thioguanine and ouabain-resistant (STO) cells. qRT-PCR revealed that the expression levels of mRNA m6A-related genes (Mettl3, Alkbh5, Ythdf1, Ythdf2, Ythdc1, and Ythdc2) in FGSCs were significantly higher than those in STO cells. m6A RNA immunoprecipitation sequencing (MeRIP-seq) data further showed that the unique m6A-methylated mRNAs in FGSCs and STO cells were related to cell population proliferation and somatic development, respectively. Additionally, knockdown of Ythdf1 inhibited FGSC self-renewal. Comparison of methylated DNA immunoprecipitation sequencing (MeDIP-seq) results between FGSCs and STO cells identified that DNA methylation contributed to FGSC proliferation by suppressing the somatic program. These results suggested that m6A regulated FGSC self-renewal possibly through m6A binding protein YTHDF1, and DNA methylation repressed somatic programs in FGSCs to maintain FGSC characteristics.

Autophagy is required for oogenesis and plays a critical role in response to aging caused by oxidative stress. However, there have been no reports on regulation of cytoprotective autophagy in female germline stem cells (FGSCs) in response to aging caused by oxidative stress.

Several studies have recently revealed the regulatory mechanisms underlying female germline stem cell (FGSC) differentiation, proliferation, and apoptosis, but other biological processes such as autophagy and its mechanism in FGSCs are largely unclear. The use of small chemical compounds may be a good approach to further investigate the process and mechanism of autophagy in FGSC development. In this study, we used ZCL-082, a derivative of benzoxaboroles, to treat FGSCs. Using a cell counting kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, we found that ZCL-082 could significantly reduce the viability, proliferation, and number of FGSCs in vitro. Moreover, western blotting revealed that the expression of light chain 3 beta 2 (LC3B-II) in FGSCs was significantly increased after treatment with ZCL-082 for 3 and 6 h. Meanwhile, the expression of sequestosome-1 (SQSTM1) was significantly decreased. These results suggested that ZCL-082 can induce autophagy of FGSCs in vitro. Regarding the molecular mechanism, ZCL-082 could significantly reduce the expression of growth arrest-specific 5 (GAS5) long non-coding RNA, which could directly bind to microRNA-21a (miR-21a) and negatively regulate each other in FGSCs. Knockdown of GAS5 induced the autophagy of FGSCs, while GAS5 overexpression inhibited the autophagy of FGSCs in vitro and rescued FGSC autophagy induced by ZCL-082. Additionally, overexpression of miR-21a significantly enhanced LC3B-II protein expression while significantly reducing the expression of programmed cell death protein 4 (PDCD4) and SQSTM1 protein in FGSCs compared with control cells. The inhibition of miR-21a significantly reduced the basal or ZCL-082-induced upregulated expression of LC3B-II, and it significantly enhanced the expression of PDCD4 while downregulating the basal or ZCL-082-induced expression of SQSTM1 in FGSCs. Furthermore, the overexpression of GAS5 enhanced the protein expression of PDCD4, but knockdown of GAS5 reduced the protein expression of PDCD4. Taken together, these results suggested that ZCL-082 induced autophagy through GAS5 functioning as a competing endogenous RNA (ceRNA) sponge for miR-21a in FGSCs. It also suggested that the GAS5/miR-21a axis may be a potential therapeutic target for premature ovarian failure in the clinic.

During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs).

Female germline stem cells (FGSCs) are proposed to be a key factor for ameliorating female infertility. Previously we have shown that neonatal and adult FGSCs could be isolated and purified from mouse ovarian tissues. The long noncoding (lnc) RNA growth arrest-specific 5 sequence (GAS5) transcribed from mammalian genomes plays important regulatory roles in various developmental processes. However, there is no study on the relationship between GAS5 and FGSC development in vitro. In this study, we showed that GAS5 was highly expressed in the neonatal mouse ovary and was located in both FGSCs and oocytes. GAS5 facilitated FGSC proliferation and promoted their survival in vitro. Moreover, GAS5 also inhibited apoptosis of cultured FGSCs. These findings indicate that GAS5 is a crucial regulator of FGSC development. This might serve as a foundation for a strategy of lncRNA-directed diagnosis or treatment of female infertility.

Metformin as a first-line clinical anti-diabetic agent prolongs the lifespan of model animals and promotes cell proliferation. However, the molecular mechanisms underlying the proliferative phenotype, especially in epigenetics, have rarely been reported. The aim of this study was to investigate the physiological effects of metformin on female germline stem cells (FGSCs) in vivo and in vitro, uncover β-hydroxybutyrylation epigenetic modification roles of metformin and identify the mechanism of histone H2B Lys5 β-hydroxybutyrylation (H2BK5bhb) in Gata-binding protein 2 (Gata2)-mediated proliferation promotion of FGSCs.

Ubiquitin-specific-processing protease 7 (Usp7) is a key deubiquitinase controlling epigenetic modification and regulating the self-renewal, proliferation, and differentiation of stem cells. However, the functions and mechanisms of action of Usp7 on female germline stem cells (FGSCs) are unknown. Here, we demonstrated that Usp7 regulated FGSC self-renewal via DNA methylation. The results of Cell Counting Kit-8 and 5-ethynyl-20-deoxyuridine assays showed that the viability and proliferation of FGSCs were negatively regulated by Usp7. Moreover, Usp7 downregulated the expression of self-renewal genes, such as Oct4, Etv5, Foxo1, and Akt, but upregulated the expression of differentiation-related genes including Stra8 and Sycp3. Mechanistically, RNA-seq results showed that Usp7 negatively regulated FGSC self-renewal but positively modulated differentiation in FGSCs. Meanwhile, both overexpression and knockdown of Usp7 resulted in significant changes in DNA methylation and histone modification in FGSCs. Additionally, RNA-seq and MeDIP-seq analyses showed that Usp7 regulates the self-renewal and differentiation of FGSCs mainly through DNA methylation rather than histone modification, which was also confirmed by a rescue assay. Our study not only offers a novel method to research FGSC self-renewal and differentiation in view of epigenetic modifications, but also provides a deep understanding of FGSC development. Graphical Abstract.

It has long been believed that most female mammalian species lose the ability to generate oocytes in postnatal ovaries. Recent evidence has demonstrated the isolation and culture of female germline stem cells (FGSCs) from adult mice and humans. However, the process and mechanisms of FGSC differentiation in vivo following transplantation have not yet been studied. Here, we isolated and characterized FGSCs from a single EGFP-transgenic mouse, and traced the development and behavior of transplanted FGSCs (F-TFs) in vivo. Comparisons of folliculogenesis between recipients with FGSC transplantation and wild-type (WT) mice were performed by single follicle RNA-sequencing (RNA-seq). Results showed that FGSCs exhibited a homing ability and began to differentiate into early-stage oocytes only when they reached the edge of the ovarian cortex. The F-TFs restored function of premature ovarian failure (gdf9iCre; PtenloxP/loxP genotype) and generated offspring. Furthermore, results demonstrated that the developmental mechanisms of follicles derived from F-TFs were similar to that of WT follicles. Weighted gene co-expression network analysis identified two potential sub-networks and core genes that played a critical role in follicular development. These findings provide a theoretical basis and lay a technology platform for specific or personalized medical treatment of ovarian failure or other ovarian diseases.

In recent years, advancements in genome-wide analyses of the mammalian transcriptome have revealed that long noncoding RNAs (lncRNAs) is pervasively transcribed in the genome and an increasing number of studies have demonstrated lncRNAs as a new class of regulatory molecules are involved in mammalian development (Carninci et al. (2005); Fatica and Bozzoni (2014)), but very few studies have been conducted on the potential roles of lncRNAs in mammalian testis development. To get insights into the expression patterns of lncRNA during mouse testis development, we investigated the lncRNAs expression profiles of neonatal and adult mouse testes using microarray platform and related results have been published (Sun et al., PLoS One 8 (2013) e75750.). Here, we describe in detail the experimental system, methods and validation for the generation of the microarray data associated with our recent publication (Sun et al., PLoS One 8 (2013) e75750.). Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE43442.

Germline stem cells play an essential role in establishing the fertility of an organism. Although extensively characterized, the regulatory mechanisms that govern the fundamental properties of mammalian female germline stem cells remain poorly understood.

In vitro production of oocytes capable of producing offspring has exciting potential applications in reproductive medicine. Here, we generated and characterized an ovarian organoid model derived from female germline stem cells using a three-dimensional culture system. We show that this model generated normal offspring and detected drug toxicity. The ovarian organoids could produce oocytes and exhibited endocrine functions. Single-cell analysis of ovarian organoids identified six ovarian cell lineages, such as germ, granulosa and theca cells, and produced gene-expression signatures for each cell type. Investigation of the expression patterns of genes related to meiosis and gene ontogeny analysis for germ cell clusters showed that a germ cell population was maintained in the ovarian organoids. Moreover, flow cytometric analysis confirmed that the population of germ cells could be maintained on the organoids and showed that ascorbic acid treatment had a beneficial effect of germ cell population maintenance on the organoids. Furthermore, we demonstrated the successful production of offspring from oocytes derived from ovarian organoids. Finally, we showed the ovarian organoids had the potential to drug toxicological detection. For example, we found that salinomycin impaired the formation of ovarian organoids and germ cell population maintenance by inducing apoptosis. These results indicate that the female germline stem cell-derived ovarian organoids represent a valuable model system for generating oocytes that can yield offspring, and provide a novel model for drug screening and toxicological detection.

The existence of mammalian female germline stem cells (FGSCs) indicates that mammalian ovaries possess germline stem cells analogous to testis, and continue to produce gametes postnatally, which provides new insights into female fertility. In this study, we compared the morphological and molecular characteristics between FGSCs and spermatogonial stem cells (SSCs) by analysis of morphology, immunofluorescence, alkaline phosphatase activity assay, reverse transcription polymerase chain reaction (RT-PCR) and microarray hybridization. The results demonstrated that the morphology and growth patterns of FGSCs are similar to those of SSCs. Microarray analysis of global gene expression profiles of FGSCs and SSCs showed similar signatures in the transcriptome level. A list of 853 co-highly expressed genes (CEG) in female and male germline stem cells may be responsible for the morphological and molecular similarity. We constructed a continuous network of the CEG based on I2D protein-protein interaction database by breadth first search. From the network, we could observe the interactions of the CEG may be responsible for maintaining the properties of germline stem cells. This study was the first attempt to compare morphological and molecular characteristics between FGSCs and SSCs. These findings would provide some clues for further research on mammalian FGSCs.

Mammalian gonadal development is crucial for fertility. Sexual differentiation, meiosis and gametogenesis are critical events in the process of gonadal development. Abnormalities in any of these events may cause infertility. However, owing to the complexity of these developmental events, the underlying molecular mechanisms are not fully understood and require further research.

Circular RNAs (circRNAs) are a class of noncoding RNAs that can regulate gene expression at the post-transcriptional level. The contribution of circRNAs in the regulation of granulosa cells (GCs) functions is not yet clear. The aim of this study was to analyze circRNA expression in adult and neonate ovaries, uncover the biological roles of circ_0002861 (circEGFR) and identify the mechanism by which it modulates follicular development.

This study aimed to clarify the regulation and mechanism of meiotic initiation in FGSC development.

Multiglycosides of Tripterygium wilfordii Hook f (GTW), a Chinese herb-derived medicine used as a remedy for rheumatoid arthritis, are considered to be a reversible anti-fertility drug affecting the mammalian spermatids. However, the mechanism behind this effect is still unknown. To study the possible mechanism behind the impact of GTW on spermatogenesis, we administered 4 groups of 4-week-old male mice with different doses of GTW. We found a dose-dependent decrease in the number of germ cells after 40 days of GTW treatment, and an increase in apoptotic cells from the low-dose to the high-dose group. During this same period the dimethylated level of histone H3 lysine 9 (H3K9me2) in GTW-treated testes germ cells declined. Additionally, spermatogonial stem cells (SSCs) from 6-day-old mice were isolated to evaluate the possible effect of GTW or triptolide on development of SSCs. We found a significantly higher incidence of apoptosis and lower dimethylation level of H3K9me2 in the SSCs of GTW or triptolide treatment than in controls. Thus, these data suggest that the GTW-induced apoptosis might be responsible for the fertility impairment in mice. This damage could be traced back to the early stages of spermatogenesis. GTW also affected the epigenetic modification of H3K9 in spermatogenesis. Molecular dynamics simulation suggested that triptolide and dimethylated or trimethylated H3K9 might have similar interaction mechanisms with EED (embryonic ectoderm development). These candidate activation mechanisms provide the first glimpse into the pathway of GTW-induced gonad toxicity, which is crucial for further research and clinical application.

N6-methyladenosine (m6A) modification controls cell fate determination. Here, we show that liquid-liquid phase separation (LLPS) of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a pivotal m6A "reader" protein, promotes the transdifferentiation of spermatogonial stem cells (SSCs) into neural stem cell-like cells by activating the IκB-nuclear factor κB (NF-κB)-CCND1 axis. The inhibition of IκBα/β mRNA translation mediated by YTHDF1 LLPS is the key to the activation of the IκB-NF-κB-CCND1 axis. Disrupting either YTHDF1 LLPS or NF-κB activation inhibits transdifferentiation efficiency. Moreover, overexpression of the YTH domain of YTHDF1 inhibits the activation of the IκB-NF-κB-CCND1 axis by promoting IκBα/β mRNA translation. Overexpression of the tau-YTH fusion protein reactivates IκB-NF-κB-CCND1 axis by inhibiting the translation of IκBα/β mRNAs, and tau LLPS is observed, which can restore transdifferentiation efficiency. Our findings demonstrate that the protein-RNA LLPS plays essential roles in cell fate transition and provide insights into translational medicine and the therapy of neurological diseases.

Spermatogonial stem cells (SSCs) are essential for spermatogenesis and male fertility. MicroRNAs (miRs) are key regulators of gene expression involved in self-renewal, differentiation, and apoptosis. However, the function and mechanisms of individual miR in regulating self-renewal and differentiation of SSCs remain unclear. Here, we report for the first time that miR-322 regulates self-renewal of SSCs. Functional assays revealed that miR-322 was essential for SSC self-renewal. Mechanistically, miR-322 promoted SSC self-renewal by targeting RASSF8 (ras association domain family 8). Moreover, the WNT/β-catenin signaling pathway was involved in the miR-322-mediated regulation. Furthermore, miR-322 overexpression increased GFRα1, ETV5 and PLZF expression but decreased STRA8, C-KIT and BCL6 expression. Our study provides not only a novel insight into molecular mechanisms regulating SSC self-renewal but also a basis for the diagnosis, treatment, and prevention of male infertility.