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

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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Hypoxia induces the dormant state in oocytes through expression of Foxo3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817223116 ◽

2019 ◽

Vol 116 (25) ◽

pp. 12321-12326 ◽

Cited By ~ 8

Author(s):

So Shimamoto ◽

Yohei Nishimura ◽

Go Nagamatsu ◽

Norio Hamada ◽

Haruka Kita ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Analysis ◽

Hypoxic Condition ◽

Primordial Follicle ◽

Primordial Follicles ◽

Dormant State ◽

Precise Understanding ◽

Reproductive Life

In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further gene-expression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state.

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Implications of environmental toxicants on ovarian follicles: how it can adversely affect the female fertility?

Environmental Science and Pollution Research ◽

10.1007/s11356-021-16489-4 ◽

2021 ◽

Author(s):

Keerthi Priya ◽

Manjunath Setty ◽

Uddagiri Venkanna Babu ◽

Karkala Sreedhara Ranganath Pai

Keyword(s):

Polycystic Ovary ◽

Endocrine Disrupting Chemicals ◽

Epidemiological Studies ◽

Female Fertility ◽

Environmental Toxicants ◽

Primordial Follicles ◽

Reproductive Life ◽

Sanitary Product

AbstractThe pool of primordial follicles formed in the ovaries during early development determines the span and quality of fertility in the reproductive life of a woman. As exposure to occupational and environmental toxicants (ETs) has become inevitable, consequences on female fertility need to be established. This review focuses on the ETs, especially well-studied prototypes of the classes endocrine disrupting chemicals (EDCs), heavy metals, agrochemicals, cigarette smoke, certain chemicals used in plastic, cosmetic and sanitary product industries etc that adversely affect the female fertility. Many in vitro, in vivo and epidemiological studies have indicated that these ETs have the potential to affect folliculogenesis and cause reduced fertility in women. Here, we emphasize on four main conditions: polycystic ovary syndrome, primary ovarian insufficiency, multioocytic follicles and meiotic defects including aneuploidies which can be precipitated by ETs. These are considered main causes for reduced female fertility by directly altering the follicular recruitment, development and oocytic meiosis. Although substantial experimental evidence is drawn with respect to the detrimental effects, it is clear that establishing the role of one ET as a risk factor in a single condition is difficult as multiple conditions have common risk factors. Therefore, it is important to consider this as a matter of public and wildlife health.

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

Reproduction ◽

10.1530/rep-15-0282 ◽

2016 ◽

Vol 151 (2) ◽

pp. 105-115 ◽

Cited By ~ 10

Author(s):

Zhen Teng ◽

Chao Wang ◽

Yijing Wang ◽

Kun Huang ◽

Xi Xiang ◽

...

Keyword(s):

Gap Junction ◽

Granulosa Cells ◽

Primordial Follicle ◽

Underlying Mechanism ◽

Temporal Window ◽

Primordial Follicles ◽

Reproductive Ability ◽

Ovarian Cells ◽

Gap Junction Communication ◽

Reproductive Life

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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Estradiol, Progesterone, and Genistein Inhibit Oocyte Nest Breakdown and Primordial Follicle Assembly in the Neonatal Mouse Ovary in Vitro and in Vivo

Endocrinology ◽

10.1210/en.2007-0088 ◽

2007 ◽

Vol 148 (8) ◽

pp. 3580-3590 ◽

Cited By ~ 173

Author(s):

Ying Chen ◽

Wendy N. Jefferson ◽

Retha R. Newbold ◽

Elizabeth Padilla-Banks ◽

Melissa E. Pepling

Keyword(s):

Organ Culture ◽

Primordial Follicle ◽

Estrogen Signaling ◽

Primordial Follicles ◽

Oocyte Number ◽

Genistein Treatment ◽

Normal Environment ◽

Synthetic Estrogens

In developing mouse ovaries, oocytes develop as clusters of cells called nests or germ cell cysts. Shortly after birth, oocyte nests dissociate and granulosa cells surround individual oocytes forming primordial follicles. At the same time, two thirds of the oocytes die by apoptosis, but the link between oocyte nest breakdown and oocyte death is unclear. Although mechanisms controlling breakdown of nests into individual oocytes and selection of oocytes for survival are currently unknown, steroid hormones may play a role. Treatment of neonatal mice with natural or synthetic estrogens results in abnormal multiple oocyte follicles in adult ovaries. Neonatal genistein treatment inhibits nest breakdown suggesting multiple oocyte follicles are nests that did not break down. Here we investigated the role of estrogen signaling in nest breakdown and oocyte survival. We characterized an ovary organ culture system that recapitulates nest breakdown, reduction in oocyte number, primordial follicle assembly, and follicle growth in vitro. We found that estradiol, progesterone, and genistein inhibit nest breakdown and primordial follicle assembly but have no effect on oocyte number both in organ culture and in vivo. Fetal ovaries, removed from their normal environment of high levels of pregnancy hormones, underwent premature nest breakdown and oocyte loss that was rescued by addition of estradiol or progesterone. Our results implicate hormone signaling in ovarian differentiation with decreased estrogen and progesterone at birth as the primary signal to initiate oocyte nest breakdown and follicle assembly. These findings also provide insight into the mechanism of multiple oocyte follicle formation.

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Development of Primordial Follicles in the Hamster: Role of Estradiol-17β

Endocrinology ◽

10.1210/en.2006-1193 ◽

2007 ◽

Vol 148 (4) ◽

pp. 1707-1716 ◽

Cited By ~ 29

Author(s):

Cheng Wang ◽

Shyamal K. Roy

Keyword(s):

Primordial Follicle ◽

Serum Levels ◽

Primordial Follicles ◽

Ici 182,780 ◽

Higher Doses ◽

Estradiol 17Β ◽

Modest Reduction

The role of E2 on primordial follicle formation was examined by treating neonatal hamsters with 1 or 2 μg estradiol cypionate (ECP) at age postnatal d 1 (P1) and P4 or by in vitro culture of embryonic d 15 (E15) ovaries with 1, 5, or 10 ng/ml estradiol-17β (E2). The specificity of E2 action was examined by ICI 182,780. One microgram of ECP maintained serum levels of E2 within the physiological range, significantly reduced apoptosis, and stimulated the formation and development of primordial follicles. In contrast, 2 μg ECP increased serum E2 levels to 400 pg/ml and had significantly less influence on primordial follicle formation. In vivo, ICI 182,780 significantly increased apoptosis and caused a modest reduction in primordial follicle formation. The formation and development of primordial follicles in vitro increased markedly with 1 ng/ml E2, and the effect was blocked by ICI 182,780. Higher doses of E2 had no effect on primordial follicle formation but significantly up-regulated apoptosis, which was blocked by ICI 182,780. CYP19A1 mRNA expression occurred by E13 and increased with the formation of primordial follicles. P4 ovaries synthesized E2 from testosterone, which increased further by FSH. Both testosterone and FSH maintained ovarian CYP19A1 mRNA, but FSH up-regulated the expression. These results suggest that neonatal hamster ovaries produce E2 under FSH control and that E2 action is essential for the survival and differentiation of somatic cells and the oocytes leading to the formation and development of primordial follicles. This supportive action of E2 is lost when hormone levels increase above a threshold.

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The in vitro growth and maturation of follicles

Reproduction ◽

10.1530/rep-08-0290 ◽

2008 ◽

Vol 136 (6) ◽

pp. 703-715 ◽

Cited By ~ 150

Author(s):

H M Picton ◽

S E Harris ◽

W Muruvi ◽

E L Chambers

Keyword(s):

Culture Media ◽

Ovarian Tissue ◽

Cell Communication ◽

Primordial Follicle ◽

Primordial Follicles ◽

Follicle Culture ◽

Preantral Follicles ◽

Large Animals

The development of technologies to grow oocytes from the most abundant primordial follicles to maturity in vitro holds many attractions for clinical practice, animal production technology and research. The production of fertile oocytes and live offspring has been achieved in mice following the long-term culture of oocytes in primordial follicles from both fresh and cryopreserved ovarian tissue. In contrast, in non-rodent species advances in follicle culture are centred on the growth of isolated preantral follicles. As a functional unit, mammalian preantral follicles are well-suited to culture but primordial and primary follicles do not grow well after isolation from the ovarian stroma. The current challenges for follicle culture are numerous and include: optimisation of culture media and the tailoring of culture environments to match the physiological needs of the cell in vivo; the maintenance of cell–cell communication and signalling during culture; and the evaluation of the epigenetic status, genetic health and fertility of in vitro derived mature oocytes. In large animals and humans, the complete in vitro growth and maturation of oocytes is only likely to be achieved following the development of a multistage strategy that closely mimics the ovary in vivo. In this approach, primordial follicle growth will be initiated in situ by the culture of ovarian cortex. Isolated preantral follicles will then be grown to antral stages before steroidogenic function is induced in the somatic cells. Finally, cytoplasmic and nuclear maturation will be induced in the in vitro derived oocytes with the production of fertile metaphase II gametes.

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Altered germline cyst and oocyte differentiation in Tex14 mutant mice reveal a new mechanism underlying female reproductive life-span

10.1101/814384 ◽

2019 ◽

Author(s):

Nafisa Nuzhat ◽

Kanako Ikami ◽

Haley Abbott ◽

Heather Tanner ◽

Allan C. Spradling ◽

...

Keyword(s):

Germ Cells ◽

Ovarian Function ◽

Primordial Follicle ◽

Wild Type ◽

Developmental Potential ◽

Primordial Follicles ◽

Intercellular Bridges ◽

Reproductive Life ◽

Reproductive Life Span ◽

Oocyte Differentiation

AbstractIn adult mammalian females, primordial follicles that form in the fetal/neonatal ovary are the only source to sustain adult ovarian function. Our previous studies revealed that during oocyte differentiation and primordial follicle formation in mouse fetal ovaries, primary oocytes form via gaining cytoplasm and organelles from sister germ cells that are connected to them by intercellular bridges within germline cysts. To better understand the role of intercellular bridges in oocyte differentiation, we analyzed mutant females lacking testis-expressed 14 (Tex14), a gene involved in cytokinesis and bridge formation. In Tex14-/- fetal ovaries, germ cells divide to form a reduced number of cysts in which sister germ cells are still connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wildtype cysts, Tex14-/- cysts fragment at a higher frequency and produce a greatly reduced number of primary oocytes with highly precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14+/- germline cysts are less fragmented and generate primary oocytes that are smaller than wild type. Interestingly, enlarged Tex14-/- primary oocytes are much more stable than wild type oocytes and more efficiently sustain folliculogenesis, whereas undersized Tex14+/- primary oocytes turn over at an accelerated rate. Our observations directly link the nature of fetal germ cell connectivity to cytoplasmic enrichment during oocyte differentiation and to oocyte developmental potential in the adult ovary. Our results imply that the duration of adult ovarian function is strongly influenced by the number of primary oocytes acquiring highly enriched cytoplasm during oocyte differentiation in fetal ovaries, rather than just by the size of the primordial follicle pool.

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The Follistatin-288 Isoform Alone Is Sufficient for Survival But Not for Normal Fertility in Mice

Endocrinology ◽

10.1210/en.2009-1176 ◽

2009 ◽

Vol 151 (3) ◽

pp. 1310-1319 ◽

Cited By ~ 32

Author(s):

Fuminori Kimura ◽

Yisrael Sidis ◽

Lara Bonomi ◽

Yin Xia ◽

Alan Schneyer

Keyword(s):

Primordial Follicle ◽

Protein Isoforms ◽

Differential Distribution ◽

Primordial Follicles ◽

C Terminus ◽

Old Females ◽

Tgfβ Superfamily

Follistatin (FST) is a natural antagonist of activin and related TGFβ superfamily ligands that exists as three protein isoforms differing in length at the C terminus. The longest FST315 isoform is found in the circulation, whereas the shortest FST288 isoform is typically found in or on cells and tissues, and the intermediate FST303 isoform is found in gonads. We recently demonstrated that the FST isoforms have distinct biological actions in vitro that, taken together with the differential distribution, suggests they may also have different roles in vivo. To explore the specific role of individual FST isoforms, we created a single-isoform FST288-only mouse. In contrast to the neonatal death of FST global knockout mice, FST288-only mice survive to adulthood. Although they appear normal, FST288-only mice have fertility defects including reduced litter size and frequency. Follicles were counted in ovaries from 8.5- to 400-d-old females. Significantly fewer morphologically healthy antral follicles were found in 100- to 250-d FST288-only ovaries, but there were significantly more secondary, primary, and primordial follicles detected at d 8.5 in FST288-only ovaries. However, depletion of this primordial follicle pool is more rapid in FST288-only females resulting in a deficit by 250 d of age and early cessation of reproduction. Superovulated FST288-only females have fewer ovulated eggs and embryos. These results indicate that the FST isoforms have different activities in vivo, that the FST288-only isoform is sufficient for development, and that loss of FST303 and FST315 isoforms results in fertility defects that resemble activin hyperactivity and premature ovarian failure.

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Reproductive Longevity and Aging: Geroscience Approaches to Maintain Long-Term Ovarian Fitness

The Journals of Gerontology Series A ◽

10.1093/gerona/glaa204 ◽

2020 ◽

Author(s):

Natalia Llarena ◽

Christopher Hine

Keyword(s):

Ovarian Reserve ◽

Ovarian Function ◽

Reproductive Function ◽

Primordial Follicle ◽

Oocyte Quality ◽

Nutrient Sensing ◽

Primordial Follicles ◽

Age Related ◽

Reproductive Life ◽

Reproductive Life Span

Abstract Increases in delayed childbearing worldwide have elicited the need for a better understanding of the biological underpinnings and implications of age-related infertility. In women 35 years and older the incidences of infertility, aneuploidy, and birth defects dramatically increase. These outcomes are a result of age-related declines in both ovarian reserve and oocyte quality. In addition to waning reproductive function, the decline in estrogen secretion at menopause contributes to multisystem aging and the initiation of frailty. Both reproductive and hormonal ovarian function are limited by the primordial follicle pool, which is established in utero and declines irreversibly until menopause. Because ovarian function is dependent on the primordial follicle pool, an understanding of the mechanisms that regulate follicular growth and maintenance of the primordial follicle pool is critical for the development of interventions to prolong the reproductive life span. Multiple pathways related to aging and nutrient-sensing converge in the mammalian ovary to regulate quiescence or activation of primordial follicles. The PI3K/PTEN/AKT/FOXO3 and associated TSC/mTOR pathways are central to the regulation of the primordial follicle pool; however, aging-associated systems such as the insulin-like growth factor-1/growth hormone pathway, and transsulfuration/hydrogen sulfide pathways may also play a role. Additionally, sirtuins aid in maintaining developmental metabolic competence and chromosomal integrity of the oocyte. Here we review the pathways that regulate ovarian reserve and oocyte quality, and discuss geroscience interventions that leverage our understanding of these pathways to promote reproductive longevity.

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Regulation of Ovarian Primordial Follicle Assembly and Development by Estrogen and Progesterone: Endocrine Model of Follicle Assembly

Endocrinology ◽

10.1210/en.2002-0131 ◽

2003 ◽

Vol 144 (8) ◽

pp. 3329-3337 ◽

Cited By ~ 182

Author(s):

Phillip Kezele ◽

Michael K. Skinner

Keyword(s):

Granulosa Cells ◽

Calf Serum ◽

Single Layer ◽

Primordial Follicle ◽

Follicle Development ◽

Newborn Rat ◽

Primary Follicle ◽

Primordial Follicles

Abstract The assembly of the developmentally arrested primordial follicle and the subsequent transition of the primordial follicle to the primary follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian follicles do not proliferate and the primordial follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated follicle development. Abnormalities in primordial follicle assembly, arrest, and development (i.e. primordial to primary follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial follicle transition to the primary follicle was found to be 3 times greater in culture. This abnormal rate of primary follicle development in culture suggests the primordial follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary follicle transition. Approximately 60% of follicles make the primordial to primary follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial follicle assembly. All viable oocytes assembled into primordial follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial follicle assembly in vivo with 10% of the total follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial follicle assembly and primordial to primary follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial follicle assembly and early follicular development.

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