Germ Cell Nests Research Articles

Dynamics of WNT signaling components in the human ovary from development to adulthood.

WNT signaling has been shown to play a pivotal role in mammalian gonad development and sex differentiation; however, its role in the developing human ovary has not been investigated. We analyzed a quantitative mass spectrometry dataset to determine the expression of WNT signaling components between 47 and 137 days of development and in adult ovarian cortex tissue. WNT signaling was identified within the top ten canonical pathways of proteins detected at every developmental stage examined. We further examined the specific localization of WNT signaling components glycogen synthase kinase 3 (GSK3B), frizzled 2 (FZD2), and β-catenin (CTNNB1) within ovarian tissue. GSK3B was nearly ubiquitously expressed during fetal development, while FZD2 was specific to germ cell nests during early development. β-catenin exhibited translocation from primarily membrane bound during early ovarian development to cytoplasmic and nuclear staining specifically in early primordial follicles in the fetal ovary. This cytoplasmic and nuclear β-catenin persisted in primordial follicles in adult ovarian tissue, but returned to membrane-bound localization in secondary follicles. We conclude that WNT signaling components are expressed in the human ovary from early to mid-gestation and remain in the adult ovary, and observed evidence for canonical WNT signaling only in the oocytes of primordial follicles. Together, these data are indicative of a role for canonical WNT signaling via β-catenin nuclear translocation during human follicle formation and follicle maintenance.

Further characterization of adult sheep ovarian stem cells and their involvement in neo-oogenesis and follicle assembly

BackgroundStem cells in the ovary comprise of two distinct populations including very small embryonic-like stem cells (VSELs) and slightly bigger progenitors termed ovarian stem cells (OSCs). They are lodged in ovary surface epithelium (OSE) and are expected to undergo neo-oogenesis and primordial follicle (PF) assembly in adult ovaries. The ovarian stem cells express follicle stimulating hormone (FSH) receptors and are directly activated by FSH resulting in formation of germ cell nests (GCN) in vitro. Present study was undertaken to further characterize adult sheep OSCs and to understand their role during neo-oogenesis and PF assembly.MethodsStem cells were collected by gently scraping the OSE cells and were characterized by H&E staining, immuno-localization, immuno-phenotyping and RT-PCR studies. Expression of FSH receptors and markers specific for stem cells (OCT-4, SSEA-4) and proliferation (PCNA) were studied on stem/progenitor cells in OSE culture and on adult sheep ovarian cortical tissue sections. Effect of FSH on stem cells was also studied in vitro. Asymmetric cell division (ACD) was monitored by studying expression of OCT-4 and NUMB.ResultsAdditional evidence was generated on the presence of two populations of stem cells in the OSE including VSELs and OSCs. FSHR expression was observed on both VSELs and OSCs by immuno-localization and immuno-phenotyping studies. FSH treatment in vitro stimulated VSELs that underwent ACD to self-renew and give rise to OSCs which divided rapidly by symmetric cell divisions (SCD) and clonal expansion with incomplete cytokinesis to form GCN. ACD was further confirmed by differential expression of OCT-4 in VSELs and NUMB in the OSCs. Immuno-histochemical expression of OCT-4, PCNA and FSHR was noted on stem cells located in the OSE in sheep ovarian sections. GCN and cohort of PF were observed in the ovarian cortex and provided evidence in support of neo-oogenesis from the stem cells.ConclusionResults of present study provide further evidence in support of two stem cells populations in adult sheep ovary. Both VSELs, OSCs and GCN express FSH receptors and FSH possibly regulates their function to undergo neo-oogenesis and primordial follicle assembly.

Non-suckling starvation of neonatal mice promotes primordial follicle formation with activation of ovarian autophagy.

Around the time of oocyte meiotic arrest, germ cell nest breakdown occurs, and primordial follicle (PF) formation is initiated at the perinatal stage. Recently, autophagy was implicated in this process. Autophagy is induced by nutrient starvation. This study was conducted to understand how starvation affects PF formation and autophagy induction during neonatal life. Suckling of neonatal female mice was blocked immediately after birth for 12–36 h to induce starvation. The numbers of PFs at each stage were subsequently counted from serial sections of ovaries. The expression of autophagy-related proteins was also evaluated. The number of PFs peaked at 60 h after birth in the control group. The numbers for the starvation groups were significantly higher than those for the control groups at 12 and 36 h. LC3B was clearly present in the oocyte cytoplasm. At 36 h after birth, the starvation group showed a higher rate of LC3II/LC3-I expression as a marker for autophagy. Moreover, the expression of p62 as a selective substrate for autophagy decreased compared to the control group. The expression of caspase-9 as a marker for apoptosis tended to be lower at 36 h in the starvation groups. These results indicate that starvation promotes PF formation with a concomitant activation of autophagy in early neonatal ovaries, suggesting that autophagy induction during follicle assembly might increase the number of PFs.

Cell-based computational model of early ovarian development in mice.

Despite its importance to reproduction, certain mechanisms of early ovarian development remain a mystery. To improve our understanding, we constructed the first cell-based computational model of ovarian development in mice that is divided into two phases: Phase I spans embryonic day 5.5 (E5.5) to E12.5; and Phase II spans E12.5 to postnatal day 2. We used the model to investigate four mechanisms: in Phase I, (i) whether primordial germ cells (PGCs) undergo mitosis during migration; and (ii) if the mechanism for secretion of KIT ligand from the hindgut resembles inductive cell-cell signaling or is secreted in a static manner; and in Phase II, (iii) that changes in cellular adhesion produce germ cell nest breakdown; and (iv) whether localization of primordial follicles in the cortex of the ovary is due to proliferation of granulosa cells. We found that the combination of the first three hypotheses produced results that aligned with experimental images and PGC abundance data. Results from the fourth hypothesis did not match experimental images, which suggests that more detailed processes are involved in follicle localization. Phase I and Phase II of the model reproduce experimentally observed cell counts and morphology well. A sensitivity analysis identified contact energies, mitotic rates, KIT chemotaxis strength, and diffusion rate in Phase I and oocyte death rate in Phase II as parameters with the greatest impact on model predictions. The results demonstrate that the computational model can be used to understand unknown mechanisms, generate new hypotheses, and serve as an educational tool.

RNA-binding proteins in human oogenesis: Balancing differentiation and self-renewal in the female fetal germline

Primordial germ cells undergo three significant processes on their path to becoming primary oocytes: the initiation of meiosis, the formation and breakdown of germ cell nests, and the assembly of single oocytes into primordial follicles. However at the onset of meiosis, the germ cell becomes transcriptionally silenced. Consequently translational control of pre-stored mRNAs plays a central role in coordinating gene expression throughout the remainder of oogenesis; RNA binding proteins are key to this regulation. In this review we examine the role of exemplars of such proteins, namely LIN28, DAZL, BOLL and FMRP, and highlight how their roles during germ cell development are critical to oogenesis and the establishment of the primordial follicle pool.

Inhibition of mTOR Signaling Pathway Delays Follicle Formation in Mice

In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. Abnormalities in this process lead to a number of pathologies such as premature ovarian failure and infertility. As a conserved pathway regulating cell growth and metabolism in response to growth factors and nutrients, the roles of mTOR signaling in follicular development have been extensively studied in recent years. However, its functions during follicle formation remain unknown. In this study, the expression of p-rpS6 (phospho-ribosomal proteinS6), a downstream marker of mTORC1, showed dynamic changes in perinatal ovaries. When E18.5 ovaries, which mainly contained germ cell nests, were incubated with the mTOR inhibitors Rapamycin and Torin1 for 24 h, follicle assembly was delayed with differential somatic cell invasion into germ cell cyst among the groups. After transplanting treated or untreated ovaries into kidney capsules of recipient ovariectomized mice, follicular development was blocked in treated ovaries, as shown by fewer antral follicles and a higher proportion of primordial follicles. Further studies showed a significant decrease in somatic cell proliferation and the expression of marker genes related to follicular development (Kitl, Kit, Gdf9, Bmp15, Zp3, and Amhr2) in treated ovaries. Moreover, the addition of KITL, a growth factor that is mainly produced by pregranulosa cells during germ cell nest breakdown, rescued the extension of follicle formation induced by mTOR inhibitors. These results suggest that KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation. J. Cell. Physiol. 232: 585-595, 2017. © 2016 Wiley Periodicals, Inc.

From primordial germ cells to primordial follicles: a review and visual representation of early ovarian development in mice

BackgroundNormal development of reproductive organs is crucial for successful reproduction. In mice the early ovarian developmental process occurs during the embryonic and postnatal period and is regulated through a series of molecular signaling events. Early ovarian development in mice is a seventeen-day process that begins with the rise of six primordial germ cells on embryonic day five (E5) and ends with the formation of primordial follicles on postnatal day two (P2).ResultsWe reviewed the current literature and created a visual representation of early ovarian development that depicts the important molecular events and associated phenotypic outcomes based on primary data. The visual representation shows the timeline of key signaling interactions and regulation of protein expression in different cells involved in ovarian development. The major developmental events were divided into five phases: 1) origin of germ cells and maintenance of pluripotency; 2) primordial germ cell migration; 3) sex differentiation; 4) formation of germ cell nests; and 5) germ cell nest breakdown and primordial follicle formation.ConclusionsThis review and visual representation provide a summary of the current scientific understanding of the key regulation and signaling during ovarian development and highlights areas needing further study. The visual representation can be used as an educational resource to link molecular events with phenotypic outcomes; serves as a tool to generate new hypotheses and predictions of adverse reproductive outcomes due to perturbations at the molecular and cellular levels; and provides a comprehendible foundation for computational model development and hypothesis testing.

The effects of in utero bisphenol A exposure on the ovaries in multiple generations of mice.

Bisphenol A is used in polycarbonate plastics and epoxy resins. Previous studies show that in utero BPA exposure inhibits germ cell nest breakdown in the F1 generation of mice, but its effects on germ cell nest breakdown and on the ovary in the F2-F3 generations were unknown. Thus, we tested the hypothesis that BPA has transgenerational effects on the ovary. Mice were exposed to BPA in utero (BPA 0.5, 20, or 50μg/kg/day), and ovaries were collected at postnatal days (PND) 4 and 21 from the F1-F3 generations and subjected to histological evaluation and gene expression analyses. In utero BPA exposure did not have transgenerational effects on germ cell nest breakdown and gene expression on PND 4, but it caused transgenerational changes in expression in multiple genes on PND 21. Collectively, these data indicate that in utero BPA exposure has some transgenerational effects in mice.

Ovarian stem cells are always accompanied by very small embryonic-like stem cells in adult mammalian ovary.

BackgroundExisting dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies.ResultsFlow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02 ± 0.008, 0.03 ± 0.017 and 0.08 ± 0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary.ConclusionsVSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility.

Decontamination in pregnancy: The pregnant poisoned patient

Bisphenol A is a known endocrine disrupting chemical and reproductive toxicant. Previous studies indicate that in utero BPA exposure increases the percentage of germ cells in nests and decreases the percentage of primordial follicles. However, the mechanism by which BPA affects germ cell nest breakdown is unknown. Thus, we hypothesized that BPA inhibits germ cell nest breakdown by interfering with oxidative stress and apoptosis pathways. To test our hypothesis, ovaries from newborn mice were collected and cultured with vehicle (dimethyl sulfoxide, DMSO) or different doses of BPA (0.1, 1, 5, and 10 μg/mL). Ovaries then were subjected to histological evaluation of germ cell nests and primordial follicles or to measurements of factors that regulate oxidative stress and apoptosis. Our results indicate that in vitro BPA exposure significantly inhibits germ cell nest breakdown by altering the expression of key ovarian apoptotic genes, but not by interfering with the oxidative stress pathway.

A fetal whole ovarian culture model for the evaluation of CrVI-induced developmental toxicity during germ cell nest breakdown

Prenatal exposure to endocrine disrupting chemicals (EDCs), including bisphenol A, dioxin, pesticides, and cigarette smoke, has been linked to several ovarian diseases such as premature ovarian failure (POF) and early menopause in women. Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries. As one of the world's leading producers of Cr compounds, the U.S. is facing growing challenges in protecting human health against adverse effects of CrVI. Our recent findings demonstrated that in vivo CrVI exposure during gestational period caused POF in F1 offspring. Our current research focus is three-fold: (i) to identify the effect of CrVI on critical windows of great vulnerability of fetal ovarian development; (ii) to understand the molecular mechanism of CrVI-induced POF; (iii) to identify potential intervention strategies to mitigate or inhibit CrVI effects. In order to accomplish these goals we used a fetal whole ovarian culture system. Fetuses were removed from the normal pregnant rats on gestational day 13.5. Fetal ovaries were cultured in vitro for 12days, and treated with or without 0.1ppm potassium dichromate (CrVI) from culture day 2–8, which recapitulated embryonic day 14.5–20.5, in vivo. Results showed that CrVI increased germ cell/oocyte apoptosis by increasing caspase 3, BAX, p53 and PUMA; decreasing BCL2, BMP15, GDF9 and cKIT; and altering cell cycle regulatory genes and proteins. This model system may serve as a potential tool for high throughput testing of various drugs and/or EDCs in particular to assess developmental toxicity of the ovary.

Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary.

In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β) family member activin (ACT) contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST), during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.

Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries.

Bisphenol A is a known endocrine disrupting chemical and reproductive toxicant. Previous studies indicate that in utero BPA exposure increases the percentage of germ cells in nests and decreases the percentage of primordial follicles. However, the mechanism by which BPA affects germ cell nest breakdown is unknown. Thus, we hypothesized that BPA inhibits germ cell nest breakdown by interfering with oxidative stress and apoptosis pathways. To test our hypothesis, ovaries from newborn mice were collected and cultured with vehicle (dimethyl sulfoxide, DMSO) or different doses of BPA (0.1, 1, 5, and 10μg/mL). Ovaries then were subjected to histological evaluation of germ cell nests and primordial follicles or to measurements of factors that regulate oxidative stress and apoptosis. Our results indicate that in vitro BPA exposure significantly inhibits germ cell nest breakdown by altering the expression of key ovarian apoptotic genes, but not by interfering with the oxidative stress pathway.

GDF9 is transiently expressed in oocytes before follicle formation in the human fetal ovary and is regulated by a novel NOBOX transcript.

During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Isolation and characterization of stem cells in the adult mammalian ovary.

Female mammals are born with a fixed pool of germ cells, which does not replenish during adult life. However, this has been recently challenged and adult ovaries produce oocytes throughout adult life just like sperm in the testes. Evidence is accumulating on the presence of ovarian stem cells, but the need for robust protocols to isolate, identify, further characterize, and subject them to various functionality tests is essential. Knowledge about the function and potential of ovarian stem cells is well demonstrated by various groups, but their true identity remains elusive because of the variability in the approaches used to identify them by different groups. In order to address this we have made attempts to compile our protocols to isolate, identify, characterize, and culture the stem cells using different animal models including human. Two distinct populations of stem cells exist in adult mammalian ovary, including very small embryonic-like stem cells (VSELs) and the progenitors termed ovarian germ stem cells (OGSCs). VSELs are relatively quiescent and undergo asymmetric cell division to give rise to OGSCs, which divide rapidly, occasionally form germ cell nests and undergo meiosis and differentiation into oocytes, which are surrounded by granulosa cells to assemble as primordial follicles.

Simultaneous Gene Deletion of Gata4 and Gata6 Leads to Early Disruption of Follicular Development and Germ Cell Loss in the Murine Ovary1

Granulosa cell formation and subsequent follicular assembly are important for ovarian development and function. Two members of the GATA family of transcription factors, GATA4 and GATA6, are expressed in ovarian somatic cells early in development, and their importance in adult ovarian function has been recently highlighted. In this study, we demonstrated that the embryonic loss of Gata4 and Gata6 expression within the ovary results in a strong down-regulation of genes involved in the ovarian developmental pathway (Fst and Irx3) as well as diminished expression of the pregranulosa and granulosa cell markers SPRR2 and FOXL2, respectively. Postnatal ovaries deficient in both Gata genes show impaired somatic cell proliferation and arrested follicular development at the primordial stage, where oocytes are either enclosed by one layer of squamous granulosa cells or remain in germ cell nests/clusters. Furthermore, germ cell nests and primordial follicles are predominantly localized to the central region of the Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) ovaries, where the boundary between the medulla and cortex is almost nonexistent. Lastly, most of the oocytes are lost early in development in conditional double mutant ovaries, which confirms the importance of normally differentiated granulosa cells as supporting cells for oocyte survival. Thus, both GATA4 and GATA6 proteins are fundamental regulators of granulosa cell differentiation and proliferation, and consequently of proper follicular assembly during normal ovarian development and function.

PUMA regulates germ cell loss and primordial follicle endowment in mice

The number of primordial follicles initially established within the ovary is influenced by the extent of germ cell death during foetal ovarian development, but the mechanisms that mediate this death have not been fully uncovered. In this study, we identified BBC3 (PUMA) (p53 upregulated modulator of apoptosis, also known as BCL2-binding component 3), a pro-apoptotic BH3-only protein belonging to the BCL2 family, as a critical determinant of the number of germ cells during ovarian development. Targeted disruption of the Bbc3 gene revealed a significant increase in the number of germ cells as early as embryonic day 13.5. The number of germ cells remained elevated in Bbc3(-/-) female mice compared with WT female mice throughout the remainder of embryonic and early postnatal life, resulting in a 1.9-fold increase in the number of primordial follicles in the ovary on postnatal day 10. The increase in the number of germ cells observed in the ovaries of Bbc3(-/-) mice could not be attributed to the altered proliferative activity of germ cells within the ovaries. Furthermore, BBC3 was found to be not required for the massive germ cell loss that occurs during germ cell nest breakdown. Our data indicate that BBC3 is a critical regulator of germ cell death that acts during the migratory phase of oogenesis or very soon after the arrival of germ cells in the gonad and that BBC3-mediated cell death limits the number of primordial follicles established in the initial ovarian reserve.

Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro

BackgroundRecent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro.MethodsOvarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging.ResultsDifferential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming.ConclusionsPresence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine.

In utero bisphenol A exposure disrupts germ cell nest breakdown and reduces fertility with age in the mouse

Bisphenol A (BPA) is a known reproductive toxicant in rodents. However, the effects of in utero BPA exposure on early ovarian development and the consequences of such exposure on female reproduction in later reproductive life are unclear. Thus, we determined the effects of in utero BPA exposure during a critical developmental window on germ cell nest breakdown, a process required for establishment of the finite primordial follicle pool, and on female reproduction. Pregnant FVB mice (F0) were orally dosed daily with tocopherol-striped corn oil (vehicle), diethylstilbestrol (DES; 0.05μg/kg, positive control), or BPA (0.5, 20, and 50μg/kg) from gestational day 11 until birth. Ovarian morphology and gene expression profiles then were examined in F1 female offspring on postnatal day (PND) 4 and estrous cyclicity was examined daily after weaning for 30days. F1 females were also subjected to breeding studies with untreated males at three to nine months. The results indicate that BPA inhibits germ cell nest breakdown via altering expression of selected apoptotic factors. BPA also significantly advances the age of first estrus, shortens the time that the females remain in estrus, and increases the time that the females remain in metestrus and diestrus compared to controls. Further, F1 females exposed to low doses of BPA exhibit various fertility problems and have a significantly higher percentage of dead pups compared to controls. These results indicate that in utero exposure to low doses of BPA during a critical ovarian developmental window interferes with early ovarian development and reduces fertility with age.