Gap junctions are essential for murine primordial follicle assembly immediately before birth

The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking.

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Single-cell transcriptome landscape of ovarian cells during primordial follicle assembly in mice

PLoS Biology ◽

10.1371/journal.pbio.3001025 ◽

2020 ◽

Vol 18 (12) ◽

pp. e3001025

Author(s):

Jun-Jie Wang ◽

Wei Ge ◽

Qiu-Yue Zhai ◽

Jing-Cai Liu ◽

Xiao-Wen Sun ◽

...

Keyword(s):

Single Cell ◽

Germ Cells ◽

Granulosa Cells ◽

Network Inference ◽

Primordial Follicle ◽

Cell Types ◽

Bioinformatics Analyses ◽

Primordial Follicles ◽

Ovarian Cells ◽

New Genes

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.

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miR-378-3p maintains the size of mouse primordial follicle pool by regulating cell autophagy and apoptosis

Cell Death and Disease ◽

10.1038/s41419-020-02965-1 ◽

2020 ◽

Vol 11 (9) ◽

Author(s):

Xiaowen Sun ◽

Francesca Gioia Klinger ◽

Jing Liu ◽

Massimo De Felici ◽

Wei Shen ◽

...

Keyword(s):

Post Partum ◽

Expression System ◽

Primordial Follicle ◽

Underlying Mechanism ◽

Newborn Mice ◽

Primordial Follicles ◽

Reproductive Life ◽

Reproductive Life Span

Abstract Primordial follicle pool provides all available oocytes throughout the whole reproductive life span. Abnormal regulation in primordial follicle assembly leads to abnormal size of primordial follicle pool, even causes infertility. Here, miR-378-3p was proved to regulate mouse primordial follicle assembly both in vivo and in vitro. The expression of miR-378-3p significantly increased in mice ovaries from 17.5 dpc (days post coitum) up to 3 dpp (day post partum) compared with the expression of 16.5 dpc ovaries, which suggested that miR-378-3p was involved in primordial follicle assembly. To uncover the underlying mechanism, newborn mice ovaries were cultured in vitro in the presence of rapamycin and 3-methyladenine, which showed that the expression of miR-378-3p changed together with the percentage of primordial follicle. Moreover, during the normal process of primordial follicle assembly between 17.6 dpc and 3 dpp, autophagy is activated, while, apoptosis is inhibited. The in vivo results showed that newborn mice starved for 1.5 days showing the increased miR-378-3p, activated autophagy and inhibited apoptosis in the ovaries, had more percentage of primordial follicles. Over-expression of miR-378-3p using miR-378-3p agomir caused increased percentage of primordial follicle, increased level of autophagy, and decreased level of apoptosis. Knockdown of miR-378-3p by miR-378-3p antiagomir had the opposite results. Using pmirGLO Dual-Luciferase miRNA Target Expression system, we confirmed both PDK1 and Caspase9 were targets of miR-378-3p, which suggested that miR-378-3p activated autophagy by targeting PDK1 and inhibited apoptosis by targeting Caspase9. MiR-378-3p could be used as a biomarker of diseases caused by abnormal size of primordial follicle pool for diagnosis, prevention, or therapy.

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Follistatin but not α or βA inhibin subunit mRNA is expressed in ovine fetal ovaries in late gestation

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0130001 ◽

1994 ◽

Vol 13 (1) ◽

pp. 1-9 ◽

Cited By ~ 12

Author(s):

R Braw-Tal ◽

D J Tisdall ◽

N L Hudson ◽

P Smith ◽

K P McNatty

Keyword(s):

Mrna Expression ◽

Granulosa Cells ◽

Cell Function ◽

Follicular Development ◽

Primordial Follicle ◽

Late Gestation ◽

Follicle Growth ◽

Primordial Follicles ◽

Fetal Ovary ◽

Ovine Fetal

ABSTRACT The aim of this study was to investigate the sites of follistatin and α and βA inhibin mRNA expression in the ovaries of female sheep fetuses at 90, 100, 120 and 135 days of gestation (term=day 147). At 90 and 100 days primordial follicles were formed, followed by the appearance of primary follicles at 100 days of gestation. At days 120 and 135, primordial, primary and preantral (i.e. secondary) follicles were present in the ovaries, but antral (i.e. tertiary) follicles were not observed at any of these gestational ages. Two Booroola genotypes were studied: homozygous carriers (BB) and non-carriers (++) of the fecundity gene (FecB). Irrespective of genotype no specific hybridization of the α and βA inhibin riboprobes was detected in any ovarian cells at days 90, 100, 120 or 135 of gestation. In control mature ovaries, on the other hand, strong hybridization in the granulosa cells of antral follicles was observed. In contrast to α and βA inhibin, follistatin antisense (but not sense) riboprobes hybridized specifically to the granulosa cells of preantral follicles with two or more layers of cells at days 120 and 135 of gestation. Moreover, hybridization was also evident in the cells of the ovarian rete at days 120 and 135, but not at 90 or 100 days. No follistatin mRNA expression was observed in the granulosa cells of primordial or primary follicles or in any other ovarian cell type at any of the gestational ages examined. No FecB-specific differences in follistatin expression were noted with respect to stage of preantral follicular development and there were no obvious differences in the intensity of expression. These results show that follistatin mRNA is expressed specifically in the granulosa cells and intraovarian rete. Expression of follistatin in rete cells was coincident with the increasing numbers of growing follicles within the fetal ovary, indicating that rete cell function may have a role in the ontogeny of early follicular growth. Our results suggest that follistatin and α and βA inhibin may not be important for the initiation of follicle growth in the sheep ovary, since these genes are not expressed during the transformation of a primordial follicle to a primary structure. However, the evidence for follistatin mRNA expression in the ovine fetal ovary implies that this hormone is likely to play a role during the early stages of follicle growth.

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Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

Biology of Reproduction ◽

10.1093/biolre/ioab193 ◽

2021 ◽

Author(s):

Emmalee A Ford ◽

Emily R Frost ◽

Emma L Beckett ◽

Shaun D Roman ◽

Eileen A McLaughlin ◽

...

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Expression Profiles ◽

Primordial Follicle ◽

Differentially Expressed ◽

Primary Follicle ◽

Primordial Follicles ◽

Time Points ◽

Primordial Follicle Activation ◽

Follicle Activation

Abstract 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 signalling 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 immunolocalisation 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 signalling pathway’s role in primordial follicle activation.

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Cytokine (IL16) and tyrphostin actions on ovarian primordial follicle development

Reproduction ◽

10.1530/rep-14-0246 ◽

2014 ◽

Vol 148 (3) ◽

pp. 321-331 ◽

Cited By ~ 3

Author(s):

Amanda Feeney ◽

Eric Nilsson ◽

Michael K Skinner

Keyword(s):

Signaling Pathways ◽

Signaling Pathway ◽

Granulosa Cells ◽

Ovarian Follicle ◽

Primordial Follicle ◽

Cytokine Signaling ◽

Follicle Development ◽

Primary Follicle ◽

Primordial Follicles ◽

Network Analyses

An ovarian follicle is composed of an oocyte and surrounding theca and granulosa cells. Oocytes are stored in an arrested state within primordial follicles until they are signaled to re-initiate development by undergoing primordial-to-primary follicle transition. Previous gene bionetwork analyses of primordial follicle development identified a number of critical cytokine signaling pathways and genes potentially involved in the process. In the current study, candidate regulatory genes and pathways from the gene network analyses were tested for their effects on the formation of primordial follicles (follicle assembly) and on primordial follicle transition using whole ovary organ culture experiments. Observations indicate that the tyrphostin inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased follicle assembly significantly, supporting a role for the MAPK signaling pathway in follicle assembly. The cytokine interleukin 16 (IL16) promotes primordial-to-primary follicle transition as compared with the controls, where as Delta-like ligand 4 (DLL4) and WNT-3A treatments have no effect. Immunohistochemical experiments demonstrated the localization of both the cytokine IL16 and its receptor CD4 in the granulosa cells surrounding each oocyte within the ovarian follicle. The tyrphostin LDN193189 (LDN) is an inhibitor of the bone morphogenic protein receptor 1 within the TGFB signaling pathway and was found to promote the primordial-to-primary follicle transition. Observations support the importance of cytokines (i.e., IL16) and cytokine signaling pathways in the regulation of early follicle development. Insights into regulatory factors affecting early primordial follicle development are provided that may associate with ovarian disease and translate to improved therapy in the future.

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microRNA 376a regulates follicle assembly by targeting Pcna in fetal and neonatal mouse ovaries

Reproduction ◽

10.1530/rep-13-0508 ◽

2014 ◽

Vol 148 (1) ◽

pp. 43-54 ◽

Cited By ~ 32

Author(s):

Huan Zhang ◽

Xiaohua Jiang ◽

Yuanwei Zhang ◽

Bo Xu ◽

Juan Hua ◽

...

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Primordial Follicle ◽

Neonatal Mouse ◽

Nuclear Antigen ◽

Mrna Levels ◽

Microrna Target ◽

Ovarian Insufficiency ◽

Primordial Follicles ◽

Reproductive Life ◽

Proliferating Cell

In mammals, the primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. Dysregulation of primordial follicle assembly results in female reproductive diseases, such as premature ovarian insufficiency and infertility. Female mice lackingDicer1(Dicer), a gene required for biogenesis of microRNAs, show abnormal morphology of follicles and infertility. However, the contribution of individual microRNAs to primordial follicle assembly remains largely unknown. Here, we report that microRNA 376a (miR-376a) regulates primordial follicle assembly by modulating the expression of proliferating cell nuclear antigen (Pcna), a gene we previously reported to regulate primordial follicle assembly by regulating oocyte apoptosis in mouse ovaries. miR-376a was shown to be negatively correlated withPcnamRNA expression in fetal and neonatal mouse ovaries and to directly bind toPcnamRNA 3′ untranslated region. Cultured 18.5 days postcoitum mouse ovaries transfected with miR-376a exhibited decreasedPcnaexpression both in protein and mRNA levels. Moreover, miR-376a overexpression significantly increased primordial follicles and reduced apoptosis of oocytes, which was very similar to those in ovaries co-transfected with miR-376a and siRNAs targetingPcna. Taken together, our results demonstrate that miR-376a regulates primordial follicle assembly by modulating the expression ofPcna. To our knowledge, this is the first microRNA–target mRNA pair that has been reported to regulate mammalian primordial follicle assembly and further our understanding of the regulation of primordial follicle assembly.

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A role for orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in primordial follicle activation

Scientific Reports ◽

10.1038/s41598-020-80178-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marie-Charlotte Meinsohn ◽

Camilla H. K. Hughes ◽

Anthony Estienne ◽

Hatice D. Saatcioglu ◽

David Pépin ◽

...

Keyword(s):

Granulosa Cells ◽

Primordial Follicle ◽

Conditional Knockout ◽

Cellular Migration ◽

Orphan Nuclear Receptor ◽

Ki 67 ◽

Mesenchymal Transition ◽

Primordial Follicles ◽

Follicle Activation

AbstractLiver receptor homolog-1 (NR5A2) is expressed specifically in granulosa cells of developing ovarian follicles where it regulates the late stages of follicle development and ovulation. To establish its effects earlier in the trajectory of follicular development, NR5A2 was depleted from granulosa cells of murine primordial and primary follicles. Follicle populations were enumerated in neonates at postnatal day 4 (PND4) coinciding with the end of the formation of the primordial follicle pool. The frequency of primordial follicles in PND4 conditional knockout (cKO) ovaries was greater and primary follicles were substantially fewer relative to control (CON) counterparts. Ten-day in vitro culture of PND4 ovaries recapitulated in vivo findings and indicated that CON mice developed primary follicles in the ovarian medulla to a greater extent than did cKO animals. Two subsets of primordial follicles were observed in wildtype ovaries: one that expressed NR5A2 and the second in which the transcript was absent. Neither expressed the mitotic marker. KI-67, indicating their developmental quiescence. RNA sequencing on PND4 demonstrated that loss of NR5A2 induced changes in 432 transcripts, including quiescence markers, inhibitors of follicle activation, and regulators of cellular migration and epithelial-to-mesenchymal transition. These experiments suggest that NR5A2 expression poises primordial follicles for entry into the developing pool.

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HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Cell Death and Disease ◽

10.1038/s41419-021-03842-1 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Tuo Zhang ◽

Meina He ◽

Lihua Zhao ◽

Shaogang Qin ◽

Zijian Zhu ◽

...

Keyword(s):

Granulosa Cells ◽

Primordial Follicle ◽

Mtor Signaling ◽

Negative Regulator ◽

Expression Level ◽

Female Reproduction ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

Follicle Activation

AbstractPrimordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.

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Expression of E-Cadherin and N-Cadherin in Perinatal Hamster Ovary: Possible Involvement in Primordial Follicle Formation and Regulation by Follicle-Stimulating Hormone

Endocrinology ◽

10.1210/en.2009-1489 ◽

2010 ◽

Vol 151 (5) ◽

pp. 2319-2330 ◽

Cited By ~ 28

Author(s):

Cheng Wang ◽

Shyamal K. Roy

Keyword(s):

Granulosa Cells ◽

Hormonal Regulation ◽

Primordial Follicle ◽

Amino Acid Sequences ◽

Transient Increase ◽

Ovary Development ◽

Mrna Levels ◽

Altered Expression ◽

Primordial Follicles ◽

E Cadherin

We examined the expression and hormonal regulation of E-cadherin (CDH1) and N-cadherin (CDH2) with respect to primordial follicle formation. Hamster Cdh1 and Cdh2 cDNA and amino acid sequences were more than 90% similar to those of the mouse, rat, and human. Although CDH1 expression remained exclusively in the oocytes during neonatal ovary development, CDH2 expression shifted from the oocytes to granulosa cells of primordial follicles on postnatal day (P)8. Subsequently, strong CDH2 expression was restricted to granulosa cells of growing follicles. Cdh2 mRNA levels in the ovary decreased from embryonic d 13 through P10 with a transient increase on P7, which was the day before the appearance of primordial follicles. Cdh1 mRNA levels decreased from embryonic d 13 through P3 and then showed a transient increase on P8, coinciding with the formation of primordial follicles. CDH1 and CDH2 expression were consistent with that of mRNA. Neutralization of FSH in utero impaired primordial follicle formation with an associated decrease in Cdh2 mRNA and CDH2, but an increase in Cdh1 mRNA and CDH1 expression. The altered expression was reversed by equine chorionic gonadotropin treatment on P1. Whereas a CDH2 antibody significantly reduced the formation of primordial and primary follicles in vitro, a CDH1 antibody had the opposite effect. This is the first evidence to suggest that primordial follicle formation requires a differential spatiotemporal expression and action of CDH1 and CDH2. Further, FSH regulation of primordial follicle formation may involve the action of CDH1 and CDH2.

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Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes

Science Advances ◽

10.1126/sciadv.aav9960 ◽

2019 ◽

Vol 5 (6) ◽

pp. eaav9960 ◽

Cited By ~ 14

Author(s):

Go Nagamatsu ◽

So Shimamoto ◽

Nobuhiko Hamazaki ◽

Yohei Nishimura ◽

Katsuhiko Hayashi

Keyword(s):

Extracellular Matrix ◽

Mechanical Stress ◽

Granulosa Cells ◽

Nuclear Export ◽

Clinical Importance ◽

Primordial Follicles ◽

Dormant State ◽

Forkhead Box ◽

Reproductive Life ◽

Nuclear Rotation

The most immature oocytes remain dormant in primordial follicles in the ovary, ensuring the longevity of female reproductive life. Despite its biological and clinical importance, knowledge of mechanisms regulating the dormant state remains limited. Here, we show that mechanical stress plays a key role in maintaining the dormant state of the oocytes in primordial follicles in mice. Transcriptional and histological analyses revealed that oocytes were compressed by surrounding granulosa cells with extracellular matrix. This environmental state is functionally crucial, as oocytes became activated upon loosening the structure and the dormancy was restored by additional compression with exogenous pressure. The nuclei of oocytes in primordial follicles rotated in response to the mechanical stress. Pausing the rotation triggered activation of oocytes through nuclear export of forkhead box O3 (FOXO3). These results provide insights into the mechanisms by which oocytes are kept dormant to sustain female reproductive life.

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