Oocyte Survival and Development During Follicle Formation and Folliculogenesis in Mice Lacking Aromatase

Abstract BackgroundAssembly of oocytes into primordial follicles is essential for establishing the ovarian reserve required for female fertility. In mice, this process begins during embryonic development. Primordial germ cells form cysts by incomplete mitosis until 13.5 days post coitum (dpc). These cysts break down just before birth. Some oocytes undergo apoptosis while surviving oocytes are enclosed by granulosa cells to form primordial follicles. Cyst breakdown and primordial follicle formation were previously shown to be inhibited by estradiol and estrogenic compounds in vitro, suggesting that estrogen is important for regulation of this process. MethodsTo determine the role of fetal estrogen in cyst breakdown and follicle formation these processes were quantified in aromatase deficient (ArKO) mice between 17.5 dpc and postnatal day (PND) 9. Ovaries of ArKO mice were also examined at 2-week intervals to determine if folliculogenesis is affected by lack of estrogen and the age at which the typical ArKO ovarian phenotype first appears. ResultsOocyte number, follicle assembly and follicle development in ArKO mice did not differ from controls between 17.5 dpc and PND9 except for a difference in the proportion of follicles at the primordial and primary stage at PND7. At 2 weeks, ArKO heterozygous and homozygous ovaries still had oocytes in cyst while all oocytes were enclosed in follicles in wildtype ovaries. From 2 to 8 weeks oocyte numbers were similar in all genotypes though there was a trend toward fewer total oocytes in ArKO homozygous females as compared to controls at 8 weeks and a significant reduction at 10 weeks. Abnormal structures such as hemorrhagic follicles and hemosiderin deposits were also observed starting at 6 weeks. ConclusionsThese results suggest that a lack of fetal estrogen does not affect the rate of cyst breakdown or primordial follicle formation perinatally, and maternal estrogen or other signals are the chief regulators. Furthermore, the typical ArKO ovarian phenotype occurs earlier than previously reported.

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Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide

Journal of Endocrinology ◽

10.1530/joe-18-0370 ◽

2019 ◽

Vol 240 (2) ◽

pp. 243-256 ◽

Cited By ~ 21

Author(s):

Yi Luan ◽

Maxwell E Edmonds ◽

Teresa K Woodruff ◽

So-Youn Kim

Keyword(s):

Germ Cells ◽

Ovarian Reserve ◽

Primordial Germ Cells ◽

Premenopausal Women ◽

Apoptotic Pathway ◽

Primordial Follicles ◽

Apoptosis Pathways ◽

Target Effects

Cancer therapy can cause off-target effects including ovarian damage, which may result in primary ovarian insufficiency in girls and premenopausal women. Loss of ovarian follicles within the ovarian reserve leads to ovarian endocrine dysfunction and impaired fertility. Cyclophosphamide (CPA), a commonly used chemotherapeutic and immunosuppressant agent, is a gonadotoxic agent that destroys ovarian cells by crosslinking DNA. To protect the ovary against CPA damage, we sought to precisely map the mechanism by which the ovarian reserve is depleted by CPA. We found that CPA specifically depletes primordial follicles without affecting primary and secondary follicles in three independent murine strains (CD-1, C57BL/6J and BALB/cJ) in vivo. We directly tested the effect of the active metabolite of CPA, 1 μM 4-hydroxyperoxycyclophophamide (4-HC), in vitro and confirmed the loss of primordial oocytes but no change in the number of primary and secondary follicles. We demonstrated that phospho-AKT (p-AKT) and cleaved PARP (cPARP) are present in primordial oocytes 3 days after CPA injection, consistent with the role of these markers as part of the apoptotic cascade. Interestingly, p-AKT positive primordial oocytes co-expressed cPARP. Treatment of animals with specific inhibitors of apoptotic pathway components, ETP46464 and CHK2, blocked 4-HC‒induced DNA damage in vitro. These data suggest that CPA targets primordial germ cells in the ovarian reserve by stimulating apoptosis pathways. Adjuvant therapies to protect primordial germ cells from the off-target effects of CPA may reduce the risk of POI.

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Single-Oocyte Gene Expression Suggests That Curcumin Can Protect the Ovarian Reserve by Regulating the PTEN-AKT-FOXO3a Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22126570 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6570

Author(s):

Yue Lv ◽

Rui-Can Cao ◽

Hong-Bin Liu ◽

Xian-Wei Su ◽

Gang Lu ◽

...

Keyword(s):

Gene Expression ◽

Ovarian Reserve ◽

Primordial Follicle ◽

Term Treatment ◽

New Drugs ◽

Gene Profiling ◽

Primordial Follicles ◽

Long Term Treatment ◽

Follicle Activation

A better understanding of the mechanism of primordial follicle activation will help us better understand the causes of premature ovarian insufficiency (POI), and will help us identify new drugs that can be applied to the clinical treatment of infertility. In this study, single oocytes were isolated from primordial and primary follicles, and were used for gene profiling with TaqMan array cards. Bioinformatics analysis was performed on the gene expression data, and Ingenuity Pathway Analysis was used to analyze and predict drugs that affect follicle activation. An ovarian in vitro culture system was used to verify the function of the drug candidates, and we found that curcumin maintains the ovarian reserve. Long-term treatment with 100 mg/kg curcumin improved the ovarian reserve indicators of AMH, FSH, and estradiol in aging mice. Mechanistic studies show that curcumin can affect the translocation of FOXO3, thereby inhibiting the PTEN-AKT-FOXO3a pathway and protecting primordial follicles from overactivation. These results suggest that curcumin is a potential drug for the treatment of POI patients and for fertility preservation.

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Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells

Development ◽

10.1242/dev.122.4.1235 ◽

1996 ◽

Vol 122 (4) ◽

pp. 1235-1242 ◽

Cited By ~ 3

Author(s):

U. Koshimizu ◽

T. Taga ◽

M. Watanabe ◽

M. Saito ◽

Y. Shirayoshi ◽

...

Keyword(s):

Germ Cells ◽

Intracellular Signaling ◽

Primordial Germ Cells ◽

Cell Formation ◽

Embryonic Stem ◽

Oncostatin M ◽

Receptor Complexes ◽

Binding Subunit

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

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The activin-follistatin system and in vitro early follicle development in goats

Journal of Endocrinology ◽

10.1677/joe.1.06487 ◽

2006 ◽

Vol 189 (1) ◽

pp. 113-125 ◽

Cited By ~ 35

Author(s):

J R V Silva ◽

T Tharasanit ◽

M A M Taverne ◽

G C van der Weijden ◽

R R Santos ◽

...

Keyword(s):

Ovarian Tissue ◽

Primordial Follicle ◽

Activin A ◽

Terminal Deoxynucleotidyl Transferase ◽

Follicle Development ◽

Cortical Tissue ◽

Primary Follicle ◽

Growth And Survival ◽

Primordial Follicles

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

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Hyperactivation of dormant primordial follicles in ovarian endometrioma patients

Reproduction ◽

10.1530/rep-20-0265 ◽

2020 ◽

Vol 160 (6) ◽

pp. R145-R153

Author(s):

Sachiko Matsuzaki ◽

Michael W Pankhurst

Keyword(s):

Surgical Treatment ◽

Ovarian Reserve ◽

Severe Pain ◽

Primordial Follicle ◽

Primordial Follicles ◽

Relative Contribution ◽

Ovarian Endometrioma ◽

Main Factor ◽

Surgical Injury

Serum anti-Müllerian hormone (AMH) levels decrease after surgical treatment of ovarian endometrioma. This is the main reason that surgery for ovarian endometrioma endometriosis is not recommended before in vitro fertilization, unless the patient has severe pain or suspected malignant cysts. Furthermore, it has been suggested that ovarian endometrioma itself damages ovarian reserve. This raises two important challenges: (1) determining how to prevent surgical damage to the ovarian reserve in women with ovarian endometrioma and severe pain requiring surgical treatment and (2) deciding the best treatment for women with ovarian endometrioma without pain, who do not wish to conceive immediately. The mechanisms underlying the decline in ovarian reserve are potentially induced by both ovarian endometrioma and surgical injury but the relative contribution of each process has not been determined. Data obtained from various animal models and human studies suggest that hyperactivation of dormant primordial follicles caused by the local microenvironment of ovarian endometrioma (mechanical and/or chemical cues) is the main factor responsible for the decreased primordial follicle numbers in women with ovarian endometrioma. However, surgical injury also induces hyperactivation of dormant primordial follicles, which may further reduce ovarian reserve after removal of the endometriosis. Although further studies are required to elucidate the mechanisms underlying diminished ovarian reserve in women with ovarian endometrioma, the available data strongly suggests the need to prevent/minimize hyperactivation of dormant primordial follicles, regardless of whether surgery is performed, for better clinical management of ovarian endometrioma.

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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 Role of Anti-Müllerian Hormone (AMH) During Follicle Development in a Monovulatory Species (Sheep)

Endocrinology ◽

10.1210/en.2012-1158 ◽

2012 ◽

Vol 153 (9) ◽

pp. 4533-4543 ◽

Cited By ~ 72

Author(s):

Bruce K. Campbell ◽

M. Clinton ◽

R. Webb

Keyword(s):

Functional Relationship ◽

Primordial Follicle ◽

Active Immunization ◽

Follicle Development ◽

Antral Follicles ◽

Therapeutic Value ◽

Clinical Interest

Knockout studies in mice have suggested that anti-Müllerian hormone (AMH) modulates primordial follicle recruitment and the response of growing follicles to FSH. Little is known of the physiology of AMH in monovular species, despite intense clinical interest in this factor. Using sheep as a model, we sought to investigate the functional role of AMH in modulating follicle development in monovular species. In contrast to the rodent, the results indicate that AMH does not affect the rate of primordial follicle recruitment but appears to regulate the rate at which follicles progress through the gonadotropin-responsive phase, during which it is maximally expressed. Thus, knockdown of AMH bioactivity by active immunization lead to a decline in the population of gonadotropin-responsive preantral and small antral follicles (P < 0.01) and increases in both the number of gonadotropin-dependent antral follicles (P < 0.01) and ovulation rate (P < 0.05). These in vivo findings were consistent with the results of other studies examining the pattern of expression of AMH, which was negatively correlated with aromatase (P < 0.001), and in vitro supplementation experiments, which supported an inhibitory role for AMH in modulating the response of both theca and granulosa cells to LH and FSH, respectively. The elucidation of a functional relationship between AMH and LH-stimulated thecal androgen production may be significant in terms of the etiology of common forms of anovulatory infertility in women. Furthermore, the observed increase in both the number of recruitable antral follicles and ovulatory quota in response to AMH knockdown may have therapeutic value in women who respond poorly to ovarian stimulation.

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Fetal estrogens are not involved in sex determination but critical for early ovarian differentiation in rabbits

Endocrinology ◽

10.1210/endocr/bqab210 ◽

2021 ◽

Author(s):

Geneviève Jolivet ◽

Nathalie Daniel-Carlier ◽

Erwana Harscoët ◽

Eloïse Airaud ◽

Aurélie Dewaele ◽

...

Keyword(s):

Gene Expression ◽

Sex Determination ◽

Germ Cells ◽

Ovarian Reserve ◽

Sexual Differentiation ◽

Primordial Follicles ◽

Ovarian Differentiation ◽

Estrogen Synthesis ◽

Proliferation And Differentiation

Abstract AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced three lines of TALEN genetically edited CYP19A1 KO rabbits, that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small-sized ovaries in adulthood, an almost empty reserve of primordial follicles and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.

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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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Disorders of follicle development and steroidogenesis in ovaries of androgenised foetal sheep

Journal of Endocrinology ◽

10.1530/joe-14-0150 ◽

2015 ◽

Vol 225 (1) ◽

pp. 39-46 ◽

Cited By ~ 13

Author(s):

Fabio V Comim ◽

Kate Hardy ◽

Jane Robinson ◽

Stephen Franks

Keyword(s):

Germ Cells ◽

Polycystic Ovary ◽

Primordial Follicle ◽

In Utero ◽

Metabolic Effects ◽

Specific Marker ◽

Primordial Follicles ◽

Significant Difference ◽

And Control

The prenatally androgenised (PA) sheep is a well-recognised model for the study of developmental programming of adult polycystic ovary syndrome (PCOS). Most of the studies to date have involved examination of the reproductive and metabolic effects in the offspring after puberty, but more recently, it has been reported that there is disruption of follicle formation and steroid gene expression in ovaries of foetal sheep after exposure of the mother to excess androgen. Our study examines evidence for precocious primordial follicle formation at day 90 of gestation in ovaries of foetal Poll Dorset sheep. Using a specific marker of germ cells (VASA homologue protein) in ovarian sections, we found that androgenised sheep had nearly double the proportion of germ cells enclosed in follicles compared with control animals. When analysed by follicle stage, there was no significant difference between groups in the proportion of primordial follicles and growing (transitional and primary) follicles. Differences between PA and control foetal sheep were found in both mRNA and in protein expression of steroidogenic enzymes and androgen receptor. Our results in Dorset ewes are complementary to previous reports, but suggest that the timing of follicle formation and steroidogenic activity may vary between different breeds as well as in response to androgen. These data show that granulosa cells constitute a specific target for programming by androgenin uteroand raise key questions about the role of exposure to androgenin uteroin developmental origins of PCOS.

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