Primordial Follicle Formation Research Articles

SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells.

The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1)in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.

H3K4me3 as a target of di(2-ethylhexyl) phthalate (DEHP) impairing primordial follicle assembly

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastics additive that growing evidence indicates as endocrine disruptor able to negatively affect various reproductive processes both in female and male animals, including humans. However, the precise molecular mechanism of such actions is not completely understood. In the present study, scRNA-seq was performed on the ovaries of offspring from mothers exposed to DEHP from 16.5 days post coitum to 3 days post-partum, when the primordial follicle (PF) stockpile is established. While the histological observations of the offspring ovaries from DEHP exposed mothers confirmed previous data about a distinct reduction of oocytes enclosed in PFs. Focusing on oocytes, scRNA-seq analyses showed that the genes that mostly changed by DEHP were enriched GO terms related to histone H3–K4 methylation. Moreover, we observed H3K4me3 level, an epigenetics modification of H3 that is crucial for chromatin transcription, decreased by 40.28% (P < 0.01) in DEHP-treated group compared with control. When the newborn ovaries were cultured in vitro, the DEHP effects were abolished by tamoxifen (an estrogen receptor antagonist) or overexpression of Smyd3 (one specific methyltransferase of H3K4me3), in particular, the percentage of oocyte enclosed in PF was increased by 15.39% in DEHP plus Smyd3 overexpression group than of DEHP group (P < 0.01), which was accompanied by the upregulation of H3K4me3. Collectively, the present results discover Smyd3-H3K4me3 as a novel target of the deleterious ER-mediated effect of DEHP on PF formation during early folliculogenesis in the mouse and highlight epigenetics changes as prominent targets of endocrine disruptors like DEHP.

Maternal ochratoxin A exposure impairs meiosis progression and primordial follicle formation of F1 offspring.

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium fungi, widely contaminates feed, food and their raw materials. OTA has been proved to have hepatotoxicity and nephrotoxicity. Its reproductive toxicity needs to be further explored. We found that OTA inhibited the progression of meiosis, keeping more germ cells at leptotene and zygotene. Furthermore, OTA impaired primordial follicle formation, keeping more germ cells in cysts. Increased γH2AX suggested that DNA damage occurred both at the stages of meiosis and primordial follicle formation. The expression of RAD51 increased with the concentration of OTA at the stage of meiosis, while decreased later, suggesting the activated DNA repair induced by DNA damage then inhibited by persistent and excessive stress of DNA damage, which further induced apoptosis. DEGs caused by OTA were also mainly enriched in DNA damage and repair through RNA-seq analysis. Higher level of reactive oxygen species (ROS) and increased degree of oxidative damage marker 8-OHdG were both found in the ovaries exposed to OTA. We concluded that maternal OTA exposure affected meiosis progression and primordial follicle formation via oxidative damage and DNA repair. Clarification of the mechanism of OTA will contribute to the development of more effective detoxification strategies.

Oocyte Casein kinase 1α deletion causes defects in primordial follicle formation and oocyte loss by impairing oocyte meiosis and enhancing autophagy in developing mouse ovary

Casein kinase 1α is a member of CK1 family, which is ubiquitously expressed and plays multiple functions, including its potential roles in regulating cell division. But the functions of CK1α in mammalian oogenesis and folliculogenesis remain elusive. In this study, we assayed the cell type of CK1α expression in the developing mouse ovary and confirmed that CK1α was highly expressed in ovaries after birth. The oocyte-specific CK1α knockout (cKO) mouse model was then established by crossing Ddx4-Cre mice with Csnk1a1-floxp mice, and the effects of CK1α deletion on oogenesis and folliculogenesis were identified. The results showed that oocyte CK1α deletion impaired the progression of oocyte meiosis and primordial follicle formation during meiotic prophase I, which subsequently caused oocyte loss and mouse infertility. Further, the in vivo CK1α deletion and in vitro inhibition of CK1 activity resulted in the defects of DNA double-strand break (DSB) repair, whereas apoptosis and autophagy were enhanced in the developing ovary. These may contribute to oocyte loss and infertility in cKO mice. It is thus concluded that CK1α is essential for mouse oogenesis and folliculogenesis by involving in regulating the processes of oocyte meiosis and DNA DSB repair during meiotic prophase I of mouse oocytes. However, the related signaling pathway and molecular mechanisms need to be elucidated further.

The programmed death of fetal oocytes and the correlated surveillance mechanisms

Gamete production is essential for mammalian reproduction. In the ovaries, the primordial follicle, which is the basic reproductive unit, is formed either perinatally or during the second pregnancy stage in humans. However, some oocytes die before the establishment of the primordial follicle pool. Consequently, it is essential to uncover how the size of the primordial follicle pool is determined and how the programmed cell death of oocytes is performed under potential surveillance. According to recent studies, the fate of oocytes in the fetal ovary seems to be determined by different protective strategies through the timely control of apoptosis or autophagy. In this review, we discuss at least three oocyte-derived protective biomarkers, glycogen synthase kinase 3 beta, X-linked inhibitor of apoptosis, and Lysine-specific demethylase 1 (also known as KDM1A), responsible for surveilling the developmental quality of fetal oocytes to coordinate primordial follicle formation in the fetal ovary. This review contributes to a better understanding of the secrets of the female reproductive reserve under physiological conditions.

O-233 Cell-cell interactions within germ cell nests reveal the mechanism of primordial follicle formation in humans

Abstract Study question How interactions between human fetal female germ cells (hFGCs) and pre-granulosa cells (pre-GCs) affect the hFGCs transition from ovarian cord to primordial follicles (PFs). Summary answer Progressive engulfment of hFGCs by pre-GCs and establishment of cell-cell junctions and cell-cell adhesions between hFGCs and pre-GCs leads to PF formation in humans. What is known already In mice, development of FGCs is synchronized due to formation of cysts. Cysts comprise of sister FGCs that remain connected via intercellular bridges until germ cell nest breakdown is initiated and PFs are formed. In contrast, hFGCs differentiate asynchronously. In second trimester human ovaries, mitotic, RA-responsive, meiotic and oogonia hFGCs are present in designated parts of the ovary that corresponds to their differentiation stage. Meiotic hFGCs enter prophase I that is divided into 4 substages: leptotene, zygotene, pachytene, diplotene/dictyate. hFGCs in diplotene are ready to form a PF with pre-GCs and become dormant. Study design, size, duration We collected in total 17 second trimester human fetal ovaries between 14 and 22 weeks of gestation from elective abortions. We have analyzed cellular structures of hFGC and pre-GCs by transmission electron microscopy and investigated different cell adhesion and cell junction proteins by immunostaining. Participants/materials, setting, methods Materials: human fetal ovaries between 14 and 22 weeks of gestation. Methods: characterization of tissue morphology, immunofluorescence staining, whole mount staining, confocal microscopy, super-resolution microscopy, transmission electron microscopy (TEM), cell counting Main results and the role of chance Meiotic hFGCs formed germ cell cysts consisting of multiple sister hFGCs connected by intercellular bridges. Furthermore, we discovered that a subset of hFGCs formed syncytia containing several nuclei (2–4 nuclei). Syncytium formation was caused by cytokinesis failure when an in intercellular bridge failed to be formed. In addition, in some cases hFGC syncytia were connected to other syncytia or mono-nucleated hFGCs by an intercellular bridges. Those multinucleated syncytia did not display signs of apoptosis or morphological abnormalities. Investigation of physical interactions between hFGCs and pre-GCs revealed that as hFGCs enter prophase I, pre-GCs (FOXL2+, VIM+, CDH1+) form membrane protrusions (MPs) and progressively engulf them. First, CDH1+ and VIM+ domains within a pre-GC are mixed, but as engulfment progresses two distinct domains are formed. MP formation is possible due to mesenchymal-epithelial hybrid state and active Hippo pathway in pre-GCs. Inside the cords, hFGCs highly express CDH2, while CDH2 is lowly expressed in pre-GCs. At diplotene, hFGCs (TP63+) downregulate CDH2 and upregulate CDH1. Expression of CDH1 in both diplotene hFGC and pre-GCs causes increase of cell-cell adhesion and enables PF formation. In addition, progressive formation of tight junctions (TJP1+) and cellular interdigitations between pre-GCs and meiotic hFGCs reinforce PF structure. Limitations, reasons for caution Due to technical limitations and lack of in vitro culture protocols that support meiotic hFGCs development and survival as well as human PF formation in vitro, we cannot experimentally validate roles of cadherins and other cell junction and cell adhesion proteins in PF formation Wider implications of the findings In depth understanding of PF formation process in terms of physical interactions between hFGCs and pre-GCs will be crucial to design in vitro conditions that will support PF formation and our findings will be key to achieve assembling of PFs in artificial human oocytes (gametes) created from pluripotent stem cells. Trial registration number not applicable

Bone morphogenetic protein 2- and estradiol-17β-induced changes in ovarian transcriptome during primordial follicle formation†.

Estradiol-17β has been shown to promote primordial follicle formation and to involve bone morphogenetic protein 2 (BMP2) as a downstream effector to promote primordial follicle in hamsters. However, the molecular mechanism whereby these factors regulate ovarian somatic cells to pre-granulosa cells transition leading to primordial follicle formation remains unclear. The objective of this study was to determine whether BMP2 and/or estradiol-17β would regulate the expression of specific ovarian transcriptome during pre-granulosa cells transition and primordial follicle formation in the mouse ovary. BMP2 mRNA level increased during the period of primordial follicle formation with the concurrent presence of BMP2 protein in ovarian somatic cells. Estradiol-17β but not BMP2 exposure led to increased expression of ovarian BMP2 messenger RNA (mRNA), and the effect of estradiol-17β could not be suppressed by 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride (LDN) 193189. BMP2 or estradiol-17β stimulated primordial follicle formation without inducing apoptosis. Ribonucleic acid-sequence analysis (RNA-seq) of ovaries exposed to exogenous BMP2 or estradiol-17β revealed differential expression of several thousand genes. Most of the differentially expressed genes, which were common between BMP2 or estradiol-17β treatment demonstrated concordant changes, suggesting that estradiol-17β and BMP2 affected the same set of genes during primordial follicle formation. Further, we have identified that estradiol-17β, in cooperation with BMP2, could affect the expression of three major transcription factors, GATA binding protein 2, GATA binding protein 4 and Early growth response 2, and one serine protease, hepsin, in pre-granulosa cells during primordial follicle formation. Taken together, results of this study suggest that estradiol-17β and BMP2 may regulate ovarian gene expression that promote somatic cells to pre-granulosa cells transition and primordial follicle formation in the mouse ovary.

Impaired primordial follicle assembly in offspring ovaries from zearalenone-exposed mothers involves reduced mitochondrial activity and altered epigenetics in oocytes.

Previous works have shown that zearalenone (ZEA), as an estrogenic pollutant, has adverse effects on mammalian folliculogenesis. In the present study, we found that prolonged exposure of female mice to ZEA around the end of pregnancy caused severe impairment of primordial follicle formation in the ovaries of newborn mice and altered the expression of many genes in oocytes as revealed by single-cell RNA sequencing (scRNA-seq). These changes were associated with morphological and molecular alterations of mitochondria, increased autophagic markers in oocytes, and epigenetic changes in the ovaries of newborn mice from ZEA-exposed mothers. The latter increased expression of HDAC2 deacetylases was leading to decreased levels of H3K9ac and H4K12ac. Most of these modifications were relieved when the expression of Hdac2in newborn ovaries was reduced by RNA interference during in vitro culture in the presence of ZEA. Such changes were also alleviated in offspring ovaries from mothers treated with both ZEA and the coenzyme Q10 (CoQ10), which is known to be able to restore mitochondrial activities. We concluded that impaired mitochondrial activities in oocytes caused by ZEA are at the origin of metabolic alterations that modify the expression of genes controlling autophagy and primordial follicle assembly through changes in epigenetic histones.

High steroid content in conditioned medium of granulosa cells may disrupt primordial follicles formation in in vitro cultured one-day-old murine ovaries

This study was conducted to investigate the main and interactive effects of two methods of culture medium preparation [base medium vs granulosa cells conditioned medium (GCCM)] and two nutrient supplements [fetal bovine serum (FBS) vs knock-out serum replacement (KSR)] on formation and activation of primordial follicles and gene expression of corresponding factors during a seven-day culture period. One-day-old mouse ovaries were cultured with four different culture media including base medium containing FBS (BMF), base medium containing KSR (BMK), GCCM prepared with FBS (CMF) and GCCM prepared with KSR (CMK), and samples for histological and molecular assessments were collected on days 3 and 7 of culture. Further, steroid content of media was measured. Histological examination showed that KSR enhanced follicular formation and the number of follicular count was greater in BMK than CMF group (P < 0.05). Moreover, follicular activation was higher in CMK group than BMK and CMF groups (P < 0.05). Additionally, RT-PCR revealed that KSR upregulated Gdf9 gene expression (P < 0.05), while GCCM diminished expression of Gdf9, Bmp15, Notch2, Figla and Foxl2 (P < 0.05). GCCM decreased expression of Pten and increased expression of Pi3k (P < 0.05). Besides, hormonal assays indicated higher concentrations of estradiol and progesterone in GCCM compared with base media (P < 0.0001). In conclusion, the present study showed base medium containing KSR could serve as a proper medium for in vitro culture of neonatal mouse ovary since it could better support formation of primordial follicles. Yet BMK did not promote follicular activation as well as GCCM prepared with KSR did, and therefore, requires modifications.

Deletion of calponin 2 impairs primordial follicle formation and causes early exhausion of ovarian follicles in mice

Female reproductive aging includes progressive declining in ovarian follicle number. In human, premature ovarian failure (POF) is defined as menopause occurred before age 40 involves abnormal or failure of follicle formation. The mechanisms are largely unknown. Calponin is an actin filament associated regulatory protein. Calponin isoform 2 is known to regulate actin cytoskeleton related functions in various cell types. Our present study investigates the impact of calponin 2 deletion on ovarian development and aging.

A Single-Cell Omics Network Model of Cell Crosstalk during the Formation of Primordial Follicles

The fate of fetal germ cells (FGCs) in primordial follicles is largely determined by how they interact with the surrounding granulosa cells. However, the molecular mechanisms underlying this interactive process remain poorly understood. Here, we develop a computational model to characterize how individual genes program and rewire cellular crosstalk across FGCs and somas, how gene regulatory networks mediate signaling pathways that functionally link these two cell types, and how different FGCs diversify and evolve through cooperation and competition during embryo development. We analyze single-cell RNA-seq data of human female embryos using the new model, identifying previously uncharacterized mechanisms behind follicle development. The majority of genes (70%) promote FGC–soma synergism, only with a small portion (4%) that incur antagonism; hub genes function reciprocally between the FGC network and soma network; and germ cells tend to cooperate between different stages of development but compete in the same stage within a developmental embryo. Our network model could serve as a powerful tool to unravel the genomic signatures that mediate folliculogenesis from single-cell omics data.

The impact of oocyte death on mouse primordial follicle formation and ovarian reserve.

Ovaries, the source of oocytes, maintain the numbers of primordial follicles, develop oocytes for fertilization and embryonic development. Although it is well known that about two-thirds of oocytes are lost during the formation of primordial follicles through cyst fragmentation and the aggregation of oocytes within the cyst, the mechanism responsible for this remains unclear. We provide an overview of cell death that is associated with the oocyte cyst breakdown and primordial follicle assembly along with our recent findings for mice that had been treated with a TNFα ligand inhibitor. It is generally accepted that apoptosis is the major mechanism responsible for the depletion of germ cells. In fact, a gene deficiency or the overexpression of apoptosis regulators can have a great effect on follicle numbers and/or fertility. Apoptosis, however, may not be the only cause of the large-scale oocyte attrition during oocyte cyst breakdown, and other mechanisms, such as aggregation, may also be involved in this process. The continued study of oocyte death during primordial follicle formation could lead to the development of novel strategies for manipulating the primordial follicle pool, leading to improved fertility by enhancing the ovarian reserve.

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

ABSTRACT Background: Assembly 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 apart 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. Methods: To 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. Results: Oocyte number, follicle assembly, and follicle development in ArKO mice did not differ from controls between 17.5 dpc and PND 9. At 2 weeks, ArKO ovaries still had oocytes in cysts while all oocytes were enclosed in follicles in wild type ovaries. From 2 to 8 weeks oocyte numbers were similar in all genotypes with a significant reduction at 10 weeks in ovaries from homozygous mutants. Abnormal hemorrhagic follicles were observed starting at 6 weeks, earlier than previously reported and hemosiderin deposits were found starting at 8 weeks. Conclusions: These results suggest that a lack of fetal estrogen does not affect oocyte survival or the rate of primordial follicle formation perinatally, and maternal estrogen or other signals are the chief regulators. The appearance of abnormal hemorrhagic follicles observed as early as 6 weeks suggests that the lack of estrogen becomes problematic at this time.

Effects of Heat Stress on Follicular Physiology in Dairy Cows.

Simple SummaryEnvironmentally induced hyperthermia, also called heat stress (HS), compromises reproductive physiology in mammals. The number of oocytes is fixed after birth and they are stored in the ovary in a quiescent state (at the stage of the first meiotic prophase) in primordial follicles. There is evidence that HS alters the oocyte quality, the dynamics of follicular growth and ovulation. The dairy cow, submitted to the metabolic stress of high milk production, is a good model for studying the effects of HS on ovarian function. The aim of this review is to describe the influence of HS during the stages of follicular development in dairy cattle, from the activation of primordial follicles to ovulation. Some clinical aspects are also considered.Follicular organization starts during mid-to-late fetal life with the formation of primordial follicles. The bilateral interplay between the oocyte and adjoining somatic cells during follicular growth and ovulation may be sensitive to heat stress (HS). Mechanisms giving rise to pre-ovulatory temperature gradients across reproductive tissues are mostly regulated by the pre-ovulatory follicle, and because the cooling of the gonads and genital tract depends on a counter-current transfer system of heat, HS may be considered a major factor impairing ovulation, fertilization and early embryo development. There is evidence of a long-lasting influence of HS on oogenesis and final follicular maturation. Follicular stages that are susceptible to HS have not been precisely determined. Therefore, the aim of this review was to describe the influence of HS during the staged follicular development in dairy cattle, from the activation of primordial follicles to ovulation. Some clinical prospects are also considered.

Regulation of mouse primordial follicle formation by signaling through the PI3K pathway†.

Cell signaling mediated by the KIT receptor is critical for many aspects of oogenesis including the proliferation and migration of primordial germ cells, as well as the survival, growth, and maturation of ovarian follicles. We previously showed that KIT regulates cyst breakdown and primordial follicle formation, and in this study, have investigated the mechanisms downstream of the receptor by modulating the activity of two downstream signaling cascades: the phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase pathways. E17.5 ovaries were cultured for 5days with a daily dose of media supplemented with either the PI3K inhibitor LY294002, the MEK inhibitor U0126, or a DMSO vehicle control. Our histological observations aligned with the established role of PI3K in oocyte growth and primordial follicle activation but also revealed that LY294002 treatment delayed the processes of cyst breakdown and primordial follicle formation. U0126 treatment also led to a reduction in oocyte growth and follicle development but did not appear to affect cyst breakdown. The delay in cyst breakdown was mitigated when ovaries were dually dosed with LY294002 and KITL, suggesting that while KIT may signal through PI3K to promote cyst breakdown, other signaling networks downstream of the receptor could compensate. These observations unearth a role for PI3K signaling in the establishment of the ovarian reserve and suggest that PI3K might be the primary mediator of KIT-induced cyst breakdown and primordial follicle formation in the mouse ovary.

Accelerated loss of oogonia and impaired folliculogenesis in females with Turner syndrome start during early fetal development.

How are germ cell numbers and initiation of folliculogenesis affected in fetal Turner syndrome (TS) ovaries? Germ cell development was severely affected already in early second trimester pregnancies, including accelerated oogonia loss and impaired initiation of primordial follicle formation in TS ovaries, while the phenotype in TS mosaic ovaries was less severe. Females with TS are characterized by premature ovarian insufficiency (POI). This phenotype is proposed to be a consequence of germ cell loss during development, but the timing and mechanisms behind this are not characterized in detail. Only few studies have evaluated germ cell development in fetal TS and TS mosaic ovaries, and with a sparse number of specimens included per study. This study included a total of 102 formalin-fixed and paraffin-embedded fetal ovarian tissue specimens. Specimens included were from fetuses with 45,X (N = 42 aged gestational week (GW) 12-20, except one GW 40 sample), 45,X/46,XX (N = 7, aged GW 12-20), and from controls (N = 53, aged GW 12-42) from a biobank (ethics approval # H-2-2014-103). The number of OCT4 positive germ cells/mm2, follicles (primordial and primary)/mm2 and cPARP positive cells/mm2 were quantified in fetal ovarian tissue from TS, TS mosaic and controls following morphological and immunohistochemical analysis. After adjusting for gestational age, the number of OCT4+ oogonia was significantly higher in control ovaries (N = 53) versus 45,X ovaries (N = 40, P < 0.001), as well as in control ovaries versus 45,X/46,XX mosaic ovaries (N = 7, P < 0.043). Accordingly, the numbers of follicles were significantly higher in control ovaries versus 45,X and 45,X/46,XX ovaries from GW 16-20 with a median range of 154 (N = 11) versus 0 (N = 24) versus 3 (N = 5) (P < 0.001 and P < 0.015, respectively). The number of follicles was also significantly higher in 45,X/46,XX mosaic ovaries from GW 16-20 compared with 45,X ovaries (P < 0.005). Additionally, the numbers of apoptotic cells determined as cPARP+ cells/mm2 were significantly higher in ovaries 45,X (n = 39) versus controls (n = 15, P = 0.001) from GW 12-20 after adjusting for GW. The analysis of OCT4+ cells/mm2, cPARP+ cells/mm2 and follicles (primordial and primary)/mm2 should be considered semi-quantitative as it was not possible to use quantification by stereology. The heterogeneous distribution of follicles in the ovarian cortex warrants a cautious interpretation of the exact quantitative numbers reported. Moreover, only one 45,X specimen and no 45,X/46,XX specimens aged above GW 20 were available for this study, which unfortunately made it impossible to assess whether the ovarian folliculogenesis was delayed or absent in the TS and TS mosaic specimens. This human study provides insights about the timing of accelerated fetal germ cell loss in TS. Knowledge about the biological mechanism of POI in girls with TS is clinically useful when counseling patients about expected ovarian function and fertility preservation strategies. This work was supported by the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC). N/A.

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans. Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Single-cell RNA-Seq reveals a highly coordinated transcriptional program in mouse germ cells during primordial follicle formation.

The assembly of primordial follicles in mammals represents one of the most critical processes in ovarian biology. It directly affects the number of oocytes available to a female throughout her reproductive life. Premature depletion of primordial follicles contributes to the ovarian pathology primary ovarian insufficiency (POI). To delineate the developmental trajectory and regulatory mechanisms of oocytes during the process, we performed RNA‐seq on single germ cells from newborn (P0.5) ovaries. Three cell clusters were classified which corresponded to three cell states (germ cell cyst, cyst breakdown, and follicle) in the newborn ovary. By Monocle analysis, a uniform trajectory of oocyte development was built with a series of genes showed dynamic changes along the pseudo‐timeline. Gene Ontology term enrichment revealed a significant decrease in meiosis‐related genes and a dramatic increase in oocyte‐specific genes which marked the transition from a germ cell to a functional oocyte. We then established a network of regulons by using single‐cell regulatory network inference and clustering (SCENIC) algorithm and identified possible candidate transcription factors that may maintain transcription programs during follicle formation. Following functional studies further revealed the differential regulation of the identified regulon Id2 and its family member Id1, on the establishment of primordial follicle pool by using siRNA knockdown and genetic modified mouse models. In summary, our study systematically reconstructed molecular cascades in oocytes and identified a series of genes and molecular pathways in follicle formation and development.

Roles of the Notch Signaling Pathway in Ovarian Functioning.

The Notch signaling pathway regulates cell invasion, adhesion, proliferation, apoptosis, and differentiation via cell-to-cell interactions and plays important physiological roles in the ovary. This review summarizes current knowledge about the Notch signaling pathway in relation to ovarian functions and reveals the potential underlying mechanisms. We conducted an in-depth review of relevant literature to determine the current status of research into the Notch signaling pathway in relation to ovarian functioning and reveal potential underlying mechanisms. The activation of different Notch receptors promotes the formation of primordial follicles and proliferation of granulosa cells and inhibits steroid secretion. Abnormal regulation of the Notch signaling pathway or direct mutations might lead to over-activation or under-activation of the receptors, resulting in Notch upregulation or downregulation. It can also disrupt the normal physiological functions of the ovary. The lncRNA HOTAIR and growth hormones improved premature ovarian failure (POF) and promoted follicle maturation in a mouse model of POF by upregulating Notch1 expression. They also stimulated the Notch1 signaling pathway, increased the level of plasma estradiol, and decreased the level of plasma follicle-stimulating hormone. Thus, Notch1 could serve as a novel therapeutic target for POF. Several studies have reported multiple roles of Notch in regulating female primordial follicle formation and follicle maturation. Direct mutations in Notch-related molecules or abnormal gene regulation in the signaling pathway can lead to ovarian dysfunction. However, the underlying mechanisms are not fully understood.

Decoding the transcriptome of pre-granulosa cells during the formation of primordial follicles in the mouse

Primordial follicles, a finite reservoir of eggs in mammalian ovaries, are composed of a single oocyte and its supporting somatic cells, termed granulosa cells. Although their formation may require reciprocal interplay between oocytes and pre-granulosa cells, precursors of granulosa cells, little is known about the underlying mechanisms. We addressed this issue by decoding the transcriptome of pre-granulosa cells during the formation of primordial follicles. We found that marked gene expression changes, including extracellular matrix, cell adhesion, and several signaling pathways, occur along with primordial follicle formation. Importantly, differentiation of Lgr5-EGFP-positive pre-granulosa cells to FOXL2-positive granulosa cells was delayed in mutant ovaries of the germ cell-specific genes Nanos3 and Figla, accompanied by perturbed gene expression in mutant pre-granulosa cells. These results suggest that proper development of oocytes is required for the differentiation of pre-granulosa cells. Our data provide a valuable resource for understanding the gene regulatory networks involved in the formation of primordial follicles.