Characterization of Oogonial Stem Cells in Adult Mouse Ovaries with Age and Comparison to In Silico Data on Human Ovarian Aging.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

Inhibition of EED activity enhances cell survival of female germline stem cell and improves the oocytes production during oogenesis in vitro.

Ovarian organoids, based on female germline stem cells (FGSCs), are nowadays widely applied for reproductive medicine screening and exploring the potential mechanisms during mammalian oogenesis. However, there are still key issues that urgently need to be resolved in ovarian organoid technology, one of which is to establish a culture system that effectively expands FGSCs in vitro, as well as maintaining the unipotentcy of FGSCs to differentiate into oocytes. Here, FGSCs were EED226 treated and processed for examination of proliferation and differentiation in vitro. According to the results, EED226 specifically increased FGSC survival by decreasing the enrichment of H3K27me3 on Oct4 promoter and exon, as well as enhancing OCT4 expression and inhibiting P53 and P63 expression. Notably, we also found that FGSCs with EED226 treatment differentiated into more oocytes during oogenesis in vitro, and the resultant oocytes maintained a low level of P63 versus control at early stage development. These results demonstrated that inhibition of EED activity appeared to promote the survival of FGSCs and markedly inhibited their apoptosis during in vitro differentiation. As a result of our study, we propose an effective culture strategy to culture FGSCs and obtain oocytes in vitro, which provides a new vision for oogenesis in vitro.

Identification of oogonial stem cells in chicken ovary.

OBJECTIVES: Oogonial stem cells (OSCs) are germ cells that can sustain neo-oogenesis to replenish the pool of primary follicles in adult ovaries. In lower vertebrates, fresh oocytes are produced by numerous OSCs through mitosis and meiosis during each reproduction cycle, but the OSCs in adult mammals are rare. The birds have retained many conserved features and developed unique features of ovarian physiology during evolution, and the presence of OSCs within avian species remain unknown. MATERIALS AND METHODS: In this study, we investigated the existence and function of OSCs in adult chickens. The chicken OSCs were isolated and expanded in culture. We then used cell transplantation system to evaluate their potential for migration and differentiation in vivo. RESULTS: DDX4/SSEA1-positive OSCs were identified in both the cortex and medulla of the adult chicken ovary. These putative OSCs undergo meiosis in the reproductively active ovary. Furthermore, the isolated OSCs were expanded in vitro for months and found to express germline markers similar to those of primordial germ cells. When transplanted into the bloodstream of recipient embryos, these OSCs efficiently migrated into developing gonads, initiated meiosis, and then derived oocytes in postnatal ovaries. CONCLUSIONS: This study has confirmed the presence of functional OSCs in birds for the first time. The identification of chicken OSCs has great potential for improving egg laying and preserving endangered species.

Isolation of female germline stem cells from neonatal piglet ovarian tissue and differentiation into oocyte-like cells.

It has been generally accepted that the number of oocyte pool in mammalian ovaries is limited and irreversibly consumed throughout the adulthood until menopause, which has been challenged by the existence of female germline stem cells (FGSCs) and their differentiation potentials into oocytes through mitosis. However, there have been a few reports about the existence of porcine FGSCs (pFGSCs) in the neonatal piglet ovarian tissues. In this study, the pFGSCs were isolated from the one day post partum (1 dpp) piglet ovaries by a differential anchoring velocity method combined with the magnetic cell sorting (MACS) using VASA antibody. The gene expression levels and in vitro differentiation potentials of pFGSCs were subsequently analyzed. The results showed that Oct4, C-kit, Vasa, Stella, Ifitm3 and Dazl were expressed in the pFGSCs. A small portion of pFGSCs (2.81 ± 0.76%) spontaneously differentiated into oocyte-like cells (OLCs) with a mean diameter of 50 µm and gene expressions of Vasa, Ifitm3, Blimp1, Gdf9, Zp3, Dazl and Stella. Compared with that of the spontaneous differentiation system, the differentiation rates of pFGSCs into OLCs were significantly increased after the co-supplementations of porcine follicular fluid (PFF) and retinoic acid (RA). Taken together, these above results revealed the direct evidences for the existence of pFGSCs in 1 dpp piglet ovaries and the in vitro differentiation potential of pFGSCs into OLCs, benefiting future research related to the in vitro establishment of livestock FGSCs and the in vitro differentiation of pFGSCs.

Expansion Microscopy-based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells.

Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.

Mitotic exchange in female germline stem cells is the major source of Sex Ratio chromosome recombination in Drosophila pseudoobscura.

Sex Ratio chromosomes in Drosophila pseudoobscura are selfish X chromosome variants associated with 3 nonoverlapping inversions. In the male germline, Sex Ratio chromosomes distort the segregation of X and Y chromosomes (99:1), thereby skewing progeny sex ratio. In the female germline, segregation of Sex Ratio chromosomes is mendelian (50:50), but nonoverlapping inversions strongly suppress recombination establishing a 26-Mb haplotype (constituting ∼20% of the haploid genome). Rare crossover events located between nonoverlapping inversions can disrupt this haplotype, and recombinants have sometimes been found in natural populations. We recently reported on the first lab-generated Sex Ratio recombinants occurring at a rate of 0.0012 crossovers per female meiosis. An improved experimental design presented here reveals that these recombination events were at least 4 times more frequent than previously estimated. Furthermore, recombination events were strongly clustered, indicating that the majority arose from mitotic exchange in female germline stem cells and not from meiotic crossing-over in primary oocytes. Finally, asymmetric recovery of complementary recombinants was consistent with unequal exchange causing the recombination-induced viability defects. Incorporating these experimental results into population models for Sex Ratio chromosome evolution provided a substantially better fit to natural population frequencies and allowed maintenance of the highly differentiated 26-Mb Sex Ratio haplotype without invoking strong epistatic selection. This study provides the first estimate of spontaneous mitotic exchange for naturally occurring chromosomes in Drosophila female germline stem cells, reveals a much higher Sex Ratio chromosome recombination rate, and develops a mathematical model that accurately predicts the rarity of recombinant Sex Ratio chromosomes in natural populations.

Novel intrinsic factor Yun maintains female germline stem cell fate through Thickveins.

Germline stem cells (GSCs) are critical for the reproduction of an organism. The self-renewal and differentiation of GSCs must be tightly controlled to avoid uncontrolled stem cell proliferation or premature stem cell differentiation. However, how the self-renewal and differentiation of GSCs are properly controlled is not fully understood. Here, we find that the novel intrinsic factor Yun is required for female GSC maintenance in Drosophila. GSCs undergo precocious differentiation due to de-repression of differentiation factor Bam by defective BMP/Dpp signaling in the absence of yun. Mechanistically, Yun associates with and stabilizes Thickveins (Tkv), the type I receptor of Dpp/BMP signaling. Finally, ectopic expression of a constitutively active Tkv (TkvQD) completely suppresses GSC loss caused by yun depletion. Collectively, these data demonstrate that Yun functions through Tkv to maintain GSC fate. Our results provide new insight into the regulatory mechanisms of how stem cell maintenance is properly controlled.

Female germline stem cells: aging and anti-aging.

The delay of ovarian aging and the fertility preservation of cancer patients are the eternal themes in the field of reproductive medicine. Acting as the pacemaker of female physiological aging, ovary is also considered as the principle player of cancer, cardiovascular diseases, cerebrovascular diseases, neurodegenerative diseases and etc. However, its aging mechanism and preventive measures are still unclear. Some researchers attempt to activate endogenous ovarian female germline stem cells (FGSCs) to restore ovarian function, as the most promising approach. FGSCs are stem cells in the adult ovaries that can be infinitely self-renewing and have the potential of committed differention. This review aims to elucidate FGSCs aging mechanism from multiple perspectives such as niches, immune disorder, chronic inflammation and oxidative stress. Therefore, the rebuilding nichs of FGSCs, regulation of immune dysfunction, anti-inflammation and oxidative stress remission are expected to restore or replenish FGSCs, ultimately to delay ovarian aging.

[Metformin improves polycystic ovary syndrome and activates female germline stem cells in mice].

Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4-5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2 and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E2 levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.

Daidzein Activates Akt Pathway to Promote the Proliferation of Female Germline Stem Cells through Upregulating Clec11a.

Female germline stem cells (FGSCs) have been successfully isolated and characterized from postnatal mammalian and human ovarian tissues. However, the effects and mechanisms of action of natural small-molecule compounds on FGSCs are largely unknown. Here, we found that daidzein promoted the viability and proliferation of FGSCs. To elucidate the mechanism underlying this, we performed RNA-Sequence in daidzein-treated FGSCs and controls. The results showed that there were 153 upregulated and 156 downregulated genes in daidzein treatment. We confirmed the expression of some genes related to cell proliferation in the sequencing results by RT-PCR, such as Type C lectin domain family 11 member a (Clec11a), Mucin1 (Muc1), Glutathione peroxidase 3 (Gpx3), and Tet methylcytosine dioxygenase 1 (Tet1). The high expression of Clec11a at the protein level after daidzein treatment was also confirmed by western blotting. Furthermore, recombinant mouse Clec11a (rmClec11a) protein was shown to promote the viability and proliferation of FGSCs. However, knockdown of Clec11a inhibited the viability and proliferation of FGSCs, which could not be rescued by the administration of daidzein. These results indicate that daidzein promoted the viability and proliferation of FGSCs through Clec11a. In addition, both daidzein and rmClec11a activated the Akt signaling pathway in FGSCs. However, Clec11a knockdown inhibited this pathway, which could not be rescued by daidzein administration. Taken together, our findings revealed that daidzein activates the Akt signaling pathway to promote cell viability and proliferation through upregulating Clec11a. This study should deepen our understanding of the developmental mechanism of FGSCs and female infertility.

Three-dimensional genome structure shapes the recombination landscape of chromatin features during female germline stem cell development.

BACKGROUND: During meiosis of mammalian cells, chromatin undergoes drastic reorganization. However, the dynamics of the three-dimensional (3D) chromatin structure during the development of female germline stem cells (FGSCs) are poorly understood. METHODS: The high-throughput chromosome conformation capture technique was used to probe the 3D structure of chromatin in mouse germ cells at each stage of FGSC development. RESULTS: The global 3D genome was dramatically reorganized during FGSC development. In topologically associating domains, the chromatin structure was weakened in germinal vesicle stage oocytes and still present in meiosis I stage oocytes but had vanished in meiosis II oocytes. This switch between topologically associating domains was related to the biological process of FGSC development. Moreover, we constructed a landscape of chromosome X organization, which showed that the X chromosome occupied a smaller proportion of the active (A) compartment than the autosome during FGSC development. By comparing the high-order chromatin structure between female and male germline development, we found that 3D genome organization was remodelled by two different potential mechanisms during gamete development, in which interchromosomal interactions, compartments, and topologically associating domain were decreased during FGSC development but reorganized and recovered during spermatogenesis. Finally, we identified conserved chromatin structures between FGSC development and early embryonic development. CONCLUSIONS: These results provide a valuable resource to characterize chromatin organization and for further studies of FGSC development.

Cistanche deserticola polysaccharides extracted from Cistanche deserticola Y.C. Ma promote the differentiation of mouse female germline stem cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal medicine Cistanche deserticola Y.C. Ma has been recorded and treatment for infertility and impotence since ancient times, which is widely distributed in northwest China, and is mainly composed of phenylethanol glycosides, iridoids, lignans, polysaccharides, alkaloids, etc. C. deserticola polysaccharides (CDPs) is one of its main active ingredients, studies of its effect on germline stem cells are limited so far. AIM OF THE STUDY: The aim of this study was to clarify that CDPs promoted the differentiation of FGSCs in vitro, and to initially clarify its possible cell signaling pathways. MATERIAL AND METHODS: The cells were randomly divided into two groups. Normal FGSCs culture medium and the optimal concentration of CDPs (0.5 µg/mL) were added for culture, which was the selected treatment concentration that could promote cell differentiation on the basis of maintaining cell viability. After treatment for different time periods (12 h, 24 h, 36 h, 48 h), the cell proliferation and differentiation were evaluated by CCK-8, real-time PCR (qPCR), cell immunofluorescence and Western blot. Subsequently, RNA-Seq and data analysis were used to preliminarily analyze and verify the different genes and possible signal pathways. RESULTS: Under the treatment of CDPs, cell viability was relatively better, and the expression of meiotic markers stimulated by retinoic acid gene 8 protein (Stra8) and synaptonemal complex protein 3 (Sycp3) significantly increased. In addition, their cell morphology was more similar to oocytes. Comparison of gene expression in FGSCs identified key differential expression genes (DEGs) by RNA-Seq that consisted of 549 upregulated and 465 downregulated genes. The DEGs enriched in the functional categories of germline cell development and relevant signaling pathways, which jointly regulate self-renewal and differentiation of FGSCs. The transforming growth factor ß (TGF-ß) signaling pathway and bone morphogenetic protein (BMP) signaling pathway might be activated to synergistically influence cell differentiation during the CDPs treatment of FGSCs. CONCLUSION: These findings indicated that CDPs could promote the differentiation of FGSCs in vitro and could be regulated by different DEGs and signal transduction. Preliminary mechanism studies have shown that CDPs can exert their biological activities by regulating the TGF-ß and BMP signaling pathways.

Retinoic acid induced meiosis initiation in female germline stem cells by remodelling three-dimensional chromatin structure.

OBJECTIVES: This study aimed to clarify the regulation and mechanism of meiotic initiation in FGSC development. MATERIALS AND METHODS: FGSCs were induced to differentiate into meiosis in differentiation medium. RNA sequencing was performed to analysis the difference of transcription level. High-through chromosome conformation capture sequencing (Hi-C) was performed to analysis changes of three-dimensional chromatin structure. Chromosome conformation capture further confirmed a spatial chromatin loop. ChIP-qPCR and dual luciferase reporter were used to test the interaction between Stimulated by retinoic acid gene 8 (STRA8) protein and Trip13 promoter. RESULTS: Compared with FGSCs, the average diameter of STRA8-positive germ cells increased from 13 µm to 16.8 µm. Furthermore, there were 4788 differentially expressed genes between the two cell stages; Meiosis and chromatin structure-associated terms were significantly enriched. Additionally, Hi-C results showed that FGSCs underwent A/B compartment switching (switch rate was 29.81%), the number of topologically associating domains (TADs) increasing, the average size of TADs decreasing, and chromatin loop changes at genome region of Trip13 from undifferentiated stage to meiosis-initiation stage. Furthermore, we validated that Trip13 promoter contacted distal enhancer to form spatial chromatin loop and STRA8 could bind Trip13 promoter to promote gene expression. CONCLUSION: FGSCs underwent chromatin structure remodelling from undifferentiated stage to meiosis-initiation stage, which facilitated STRA8 binding to Trip13 promoter and promoting its expression.

Stella Regulates the Development of Female Germline Stem Cells by Modulating Chromatin Structure and DNA Methylation.

Female germline stem cells (FGSCs) have the ability to self-renew and differentiate into oocytes. Stella, encoded by a maternal effect gene, plays an important role in oogenesis and early embryonic development. However, its function in FGSCs remains unclear. In this study, we showed that CRISPR/Cas9-mediated knockout of Stella promoted FGSC proliferation and reduced the level of genome-wide DNA methylation of FGSCs. Conversely, Stella overexpression led to the opposite results, and enhanced FGSC differentiation. We also performed an integrative analysis of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), high-throughput genome-wide chromosome conformation capture (Hi-C), and use of our published epigenetic data. Results indicated that the binding sites of STELLA and active histones H3K4me3 and H3K27ac were enriched near the TAD boundaries. Hi-C analysis showed that Stella overexpression attenuated the interaction within TADs, and interestingly enhanced the TAD boundary strength in STELLA-associated regions. Taking these findings together, our study not only reveals the role of Stella in regulating DNA methylation and chromatin structure, but also provides a better understanding of FGSC development.

Workflow Optimization for Identification of Female Germline or Oogonial Stem Cells in Human Ovarian Cortex Using Single-Cell RNA Sequence Analysis.

In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post-natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non-human primates and humans, a recent study rooted in single-cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non-overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies.

Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery.

Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. Here, we explain the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. We find that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.

Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells.

The three-dimensional configuration of the genome ensures cell type-specific gene expression profiles by placing genes and regulatory elements in close spatial proximity. Here, we used in situ high-throughput chromosome conformation (in situ Hi-C), RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to characterize the high-order chromatin structure signature of female germline stem cells (FGSCs) and identify its regulating key factor based on the data-driven of multiple omics data. By comparison with pluripotent stem cells (PSCs), adult stem cells (ASCs), and somatic cells at three major levels of chromatin architecture, A/B compartments, topologically associating domains, and chromatin loops, the chromatin architecture of FGSCs was most similar to that of other ASCs and largely different from that of PSCs and somatic cells. After integrative analysis of the three-dimensional chromatin structure, active compartment-associating loops (aCALs) were identified as a signature of high-order chromatin organization in FGSCs, which revealed that CCCTC-binding factor was a major factor to maintain the properties of FGSCs through regulation of aCALs. We found FGSCs belong to ASCs at chromatin structure level and characterized aCALs as the high-order chromatin structure signature of FGSCs. Furthermore, CTCF was identified to play a key role in regulating aCALS to maintain the biological functions of FGSCs. These data provide a valuable resource for future studies of the features of chromatin organization in mammalian stem cells and further understanding of the fundamental characteristics of FGSCs.

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages.

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves-tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 µm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments.

Serum- and Feeder-Free Culture of Juvenile Monkey Female Germline Stem Cells and Testosterone Regulation of their Self-Renewal.

Female germline stem cells (FGSCs) have been found in mouse, rat, pig, sheep and human ovaries. However, there is no information on the isolation or long-term culture of FGSCs from non-human primates. Here, we identified the presence of FGSCs in the ovaries of juvenile (3-4-year-old) cynomolgus monkeys using DDX4 and Ki67 double immunofluorescence. Then, a long-term serum- and cell feeder-free culture system for these FGSCs was used to establish a cell line, and its biological characteristics were analyzed. We found that testosterone promoted self-renewal of the cells. This study confirmed for the first time the presence of FGSCs in the ovary of non-human primates. This culture system and cell line will be of great significance for research in medicine and reproductive biology.

Metformin improves polycystic ovary syndrome and activates female germline stem cells in mice / 生理学报

Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4-5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2 and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E2 levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.