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

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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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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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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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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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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132. ACTIVIN A HAS A STIMULATORY EFFECT IN VITRO ON EARLY FOLLICLE DEVELOPMENT IN RAT OVARIES

Reproduction Fertility and Development ◽

10.1071/srb10abs132 ◽

2010 ◽

Vol 22 (9) ◽

pp. 50

Author(s):

D. A. Cossigny ◽

J. K. Findlay ◽

A. E. Drummond

Keyword(s):

Treatment Group ◽

Real Time ◽

Granulosa Cells ◽

Combined Treatment ◽

Activin A ◽

Tunel Staining ◽

Follicle Development ◽

Primordial Follicles ◽

Preantral Follicles

Activins are dimers of inhibin β subunits and are growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily (1). Both βA and βB subunits are highly expressed in rat granulosa cells, while theca cells express little or no β subunit mRNAs (2). Oocytes lack expression of either subunit (3, 4). Activin is suggested to facilitate the responsiveness of granulosa cells to FSH (5). We hypothesized that activin, with or without FSH, could enhance the transition from the primordial to later preantral stages of follicle development. In two independent experiments, day 4 rat ovaries (n = 3 from different rats per treatment) were randomly assigned and cultured (6, 7) for 10 days in DMEM/Hams F-12 media with either no additives, FSH (100 ng/mL), activin A (50 ng/mL), or both. Day 4 fresh ovaries were also used as controls. Media and treatments were refreshed every alternate day. Ovaries were fixed andsectioned, or placed into Ultraspec for RNA extraction and real-time PCR analysis. Follicle numbers were counted as described previously (7). The proportion of atretic follicles (TUNEL staining) was determined in 3 randomly selected sections per ovary. Primordial follicles in all treatment groups were approximately 20% of those in Day 4 fresh ovaries. Primary follicles increased significantly (P < 0.05) only in the combined treatment group, where preantral follicles increased significantly (P < 0.0001) only when treated with activin A alone. Activin A alone decreased the proportion of atretic follicles in the primary and preantral classes, where the combined treatment increased the proportion of atretic preantral follicles. Real-time analysis revealed that expression levels of follistatin, FSH receptor and activin βA and βB subunits were all expressed at significantly higher levels in the Activin A-only treated group (P < 0.05). In summary, there was no effect on primordial follicle activation by any treatment. Activin alone had a stimulatory effect in vitro on subsequent folliculogenesis, but in the presence of FSH its effect was counteracted shown by an increase in atresia. Reasons for an increase in atretic preantral follicles in the combined treatment group are unclear. These studies support a stimulatory role for activin A in early follicle development and confirm the in vivo effects of activin on folliculogenesis (4). NHMRC program grant # 494802 and Fellowship (# 441101) provided financial support. (1) Vale W et al. 1986. Nature 321: 776–779.(2) Meunier H et al. 1988. Proc Natl Acad Sci USA 85: 547–251.(3) Roberts V et al. 1993. Journal of Clinical Endocrinology & Metabolism 7: 1402–1410.(4) Sidis Y et al. 1998. Biology of Reproduction 59(4): 807–812.(5) Drummond A et al. 2002. Endocrinology 143 (4): 1423–1433.(6) Nilsson E et al. 2001. Molecular and Cellular Endocrinology 182 (2): 145–155.(7) Rosairo D et al. 2008. Reproduction 136: 799–809.

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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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Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro

Zygote ◽

10.1017/s0967199411000074 ◽

2011 ◽

Vol 20 (1) ◽

pp. 73-78 ◽

Cited By ~ 9

Author(s):

Janduí Escarião da Nóbrega ◽

Paulo Bayard Dias Gonçalves ◽

Roberta Nogueira Chaves ◽

Deborah de Melo Magalhães ◽

Rafael Rossetto ◽

...

Keyword(s):

Leukemia Inhibitory Factor ◽

Fluorescence Analysis ◽

Primordial Follicle ◽

Inhibitory Factor ◽

Primary Follicle ◽

Primordial Follicles ◽

Preantral Follicles ◽

Primordial Follicle Activation

SummaryThe aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control – FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control – CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.

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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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In vitro and in vivo mouse follicle development in ovaries and reaggregated ovaries

Reproduction ◽

10.1530/rep-18-0115 ◽

2019 ◽

pp. 135-148 ◽

Cited By ~ 2

Author(s):

Belinda K M Lo ◽

Sairah Sheikh ◽

Suzannah A Williams

Keyword(s):

Cell Function ◽

Somatic Cells ◽

Primordial Follicle ◽

Culture Technique ◽

Fertility Treatment ◽

Follicle Development ◽

Ovarian Insufficiency ◽

Host Mouse

Follicle development requires complex and coordinated interactions between both the oocyte and its associated somatic cells. In ovarian dysfunction, follicle development may be abnormal due to defective somatic cell function; for example, premature ovarian insufficiency or malignancies. Replacing defective somatic cells, using the reaggregated ovary (RO) technique, may ‘rescue’ follicle development. ROs containing mature follicles have been generated when transplanted to a host mouse to develop. We have developed a RO culture technique and the aims were to determine how follicle development differed between transplanted and cultured ROs, and the influence of ovarian age (P2 vs P6). Mouse ROs were cultured for 14 days; P2 and P6 ovaries cultured as Controls. Follicle development was compared to ROs transplanted for 14 days and ovaries from P16 and P20 mice. ROs generated from either P2 or P6 exhibited similar follicle development in culture whereas in vivo follicle development was more advanced in P6 ROs. Follicles were more developed in cultured ROs than transplanted ROs. However, follicles in cultured ROs and ovaries had smaller oocytes with fewer theca and granulosa cells than in vivo counterparts. Our results demonstrate the fluidity of follicle development despite ovary dissociation and that environment is more important to basal lamina formation and theca cell development. Furthermore, follicle development within cultured ROs appears to be independent of oocyte nest breakdown and primordial follicle formation in source ovaries. Our results highlight the need for understanding follicle development in vitro, particularly in the development of the RO technique as a potential fertility treatment.

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