Adult Mouse Ovaries Research Articles

Stem Cells in Adult Mice Ovaries Form Germ Cell Nests, Undergo Meiosis, Neo-oogenesis and Follicle Assembly on Regular Basis During Estrus Cycle.

Very small embryonic-like (VSELs) and ovarian (OSCs) stem cellsare located in adult mammalian ovary surface epithelium (OSE). OSCs can expand long-term and differentiate into oocyte-like structures in vitro and have resulted in birth of fertile pups. Lineage tracing studies have provided evidence to suggest OSCs differentiation into oocytes in vivo. But how these stem cells function under normal physiological conditions has not yet been well worked out. Besides studying STRA-8 and SCP-3 expression in enzymatically isolated OSE cells smears, mice were injected BrdU to track mitosis, meiosis and follicle assembly. H&E stained OSE cells during late diestrus and proestrus showed VSELs undergoing asymmetrical cell divisions to give rise to slightly bigger OSCs which in turn underwent symmetrical cell divisions followed by clonal expansion (rapid expansion with incomplete cytokinesis) during early estrus to form germ cell nests (GCN). OCT-4, SSEA-1, MVH and DAZL positive cells in GCN expressed Erα, Erβ and FSHR, were interconnected by ring canals (TEX-14), showed mitochondrial aggregation (Cytochrome C) and Balbiani Body (TRAL). Apoptosis in 'nurse' cells was marked by PARP and putative oocytes were clearly visualized. BrdU was detected in cells undergoing mitosis/meiosis and also in an oocyte of secondary follicle. FACS sorted, green fluorescent protein (GFP) positive VSELs upon transplantation resulted in GFP positive GCN suggesting crucial role for VSELs in adult ovaries. Results suggest that various events described during oogenesis and follicle assembly in fetal ovaries are recapitulated on regular basis in adult ovary and result in the formation of follicles.

O-157 Micro-Computed Tomography of the adult mouse ovary: an in-silico 3D reconstruction of folliculogenesis

Abstract Study question Which are the spatial dynamics of follicles recruitment and growth inside the ovary? Summary answer 3D micro-Computed Tomography (microCT) shows a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. What is known already In the mouse ovary, folliculogenesis progresses from the primordial type 1 (T1) to the fully-grown T8 follicle. Most of our knowledge of the folliculogenetic process has been obtained by disaggregating the ovary into its functional units (i.e., follicles and oocytes), thus losing the complexity of the whole histo-functional context. To date, few studies employed 3D imaging approaches to gain information on the inside 3D ovary organisation. MicroCT is the only technique that combines a high spatial resolution (down to ∼1 µm) with the production of a true 3D organ reconstruction, with cubic voxels and isotropic resolution. Study design, size, duration Three ovaries of three different adult mice were treated with the contrast agent and then imaged with microCT. A typical experiment required a total of 35 man/h from ovaries isolation to completion of X-ray scanning, and 24 man/h for follicles classification and mapping. Participants/materials, setting, methods Three ovaries of three different 8-week-old CD1 mice were fixed in 4% Paraformaldehyde and treated with Lugol’s solution for 3 hr at RT. Ovaries were scanned with Skyscan 1172 (Bruker) using a 1.5 µm/pixel resolution. MicroCT sections were processed with Fiji ImageJ (NIH), and 3D rendering of follicles and blood vessels were obtained with Avizo-9 (Thermo Fisher Scientific). ANOVA and Bonferroni post-­hocstatistical analyses were performed with RStudio, considering data significantly different when p < 0.05. Main results and the role of chance Using microCT we built the first in silico3D reconstruction of the tiny mouse ovary, identifying, mapping and counting follicles,from pre-antral secondary T4 (53.2 + 12.7 µm in diameter) to fully-grown antral T8 (321.0 + 21.3 µm), and the corpora lutea.MicroCTbrought up the main functional compartments of the growing follicle, i.e., granulosa and cumulus cells, the antrum, the zona pellucida, and the oocyte with its nucleus. Instead, primordial and primary follicles (T1–T3) could not be observed, perhaps due to the reduced size of their enclosed oocyte and to the absence of a well-formed zona pellucida around the germ cell. In addition, our analysis allowed the visualisation and 3D modelling of the main ovarian vasculature, from the largest vessel that enters the organ at the hilum site (∼150 µm size in diameter)to smaller branches present in the medulla region (∼35 µm). These results show that each of the eight ovarian sectors, virtually segmented along the dorsal-ventral axis,houses an equal number of each follicle type, suggesting a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. Limitations, reasons for caution To strengthen the results, the number of ovaries/individuals analysed should be increased. Wider implications of the findings This 3D mapping of follicles and vessels could contribute our understanding of folliculogenesis dynamics, not only under normal conditions, but also during ageing, after hormones or drugs administration, or in the presence of ovarian pathologies. Trial registration number not applicable

Three-Dimensional Micro-Computed Tomography of the Adult Mouse Ovary.

In the mouse ovary, folliculogenesis proceeds through eight main growth stages, from small primordial type 1 (T1) to fully grown antral T8 follicles. Most of our understanding of this process was obtained with approaches that disrupted the ovary three-dimensional (3D) integrity. Micro-Computed Tomography (microCT) allows the maintenance of the organ structure and a true in-silico 3D reconstruction, with cubic voxels and isotropic resolution, giving a precise spatial mapping of its functional units. Here, we developed a robust method that, by combining an optimized contrast procedure with microCT imaging of the tiny adult mouse ovary, allowed 3D mapping and counting of follicles, from pre-antral secondary T4 (53.2 ± 12.7 μm in diameter) to antral T8 (321.0 ± 21.3 μm) and corpora lutea, together with the major vasculature branches. Primordial and primary follicles (T1–T3) could not be observed. Our procedure highlighted, with unprecedent details, the main functional compartments of the growing follicle: granulosa, antrum, cumulus cells, zona pellucida, and oocyte with its nucleus. The results describe a homogeneous distribution of all follicle types between the ovary dorsal and ventral regions. Also, they show that each of the eight sectors, virtually segmented along the dorsal-ventral axis, houses an equal number of each follicle type. Altogether, these data suggest that follicle recruitment is homogeneously distributed all-over the ovarian surface. This topographic reconstruction builds sound bases for modeling follicles position and, prospectively, could contribute to our understanding of folliculogenesis dynamics, not only under normal conditions, but, importantly, during aging, in the presence of pathologies or after hormones or drugs administration.

Accurate Follicle Enumeration in Adult Mouse Ovaries

Accurate Follicle Enumeration in Adult Mouse Ovaries

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Epigallocatechin-3-gallate (EGCG) protects the oocytes from methyl parathion-induced cytoplasmic deformities by suppressing oxidative and endoplasmic reticulum stress

Methyl parathion (MP) is a commonly used organophosphorus insecticide in commercial farming. It is well known that MP exposure can affect the function of nervous, respiratory, cardiovascular and reproductive systems. In our previous report we have demonstrated that MP exposure results in poor oocyte maturation and defective embryo development which is mainly mediated through oxidative stress. The present investigation was designed to explore whether using a potent free radical scavenger like Epigallocatechin-3-gallate (EGCG) can help in reducing the detrimental effects of MP on the oocytes. For the study, germinal vesicle (GV) stage oocytes collected from the ovaries of adult Swiss albino mice were subjected to in vitro maturation (IVM) in the presence or absence of MP (100 μg/mL) and/or EGCG (0.25 μM). MP significantly reduced the nuclear maturation rate, and resulted in poor cytoplasmic organization which was evident from the altered distribution pattern of mitochondria, endoplasmic reticulum and abnormal spindle organization. These changes were associated with significant elevation in oxidative stress and expression of ER stress markers such as 78 kDa Glucose regulated protein (GRP78) as well as X-box binding protein-1 (XBP1) in the oocytes. Further, the oocytes exposed to MP had lower activation rate and developmental potential. Supplementation of EGCG during IVM not only improved the nuclear maturation rate but also reduced the cytoplasmic abnormalities. These beneficial effects appear to be due to mitigation of oxidative and ER stress in oocytes. In conclusion, results of our study indicate that EGCG can help in alleviating MP-induced oocyte abnormalities.

Protective effects of Avena sativa against oxidative stress-induced kidney damage resulting from an estrogen deficiency in ovariectomized Swiss mice model.

The aim of this study was to evaluate the antioxidant capacity of Avena sativa and to confirm its protective effects on estrogen deficiency in ovariectomized mice (OVX) model. Animals were treated during 21 and 60days as follows: negative control, positive control treated with oat, ovariectomized, and ovariectomized receiving hormonal or oat treatments, respectively. 21days post-surgery, our results showed that ovariectomy increases weight gain and urea level in plasma. After 60days of treatment, OVX showed a gain in weight, high increases in relative kidney weight, plasmatic creatinine, glutathione peroxidase (GPX) activity, and protein oxidation as shown by elevated protein carbonyl (PCO) and advanced oxidation protein products (AOPP) levels. On the other side, a decrease was detected in catalase activity. Meanwhile, no significant changes in urea level, lipid peroxidation, SOD and GSH activity were detected. Conversely, supplementation with oat reduced weight gain, as well as oxidative stress and oxidation protein in the kidney of OVX mice. PRACTICAL APPLICATIONS: In our study, we removed the two ovaries of adult female mice in order to eliminate the essential source of steroid hormones. Deficiency in these sexual hormones, especially estrogen, leads to several physiological and morphological disturbances that are found in post-menopausal women. The gain in weight and oxidative kidney damages were the main health problems detected in ovariectomized animals. This work explored for the first time the use of oat grains as a safe natural remedy to treat all previous health problems and to avoid carcinogenic effects of the menopausal hormone therapy. Owing to its health benefits, this cereal species has recently gained attention as an important source of antioxidants and fibers. Thanks to these two components, it can be able to restore all indicated disturbances. Therefore, this cereal can be very useful in the prevention of obesity and kidney oxidative damage occurring during menopause.

Quantification of viable granulosa cells in murine ovarian follicles

ABSTRACT Ovarian follicle growth and oocyte maturation depend on the viability of granulosa cells (GC). We quantified GC in whole mouse follicles. Single follicles were isolated from adult mouse ovaries and stained with DAPI or Live-Dead stain before fixation. An objective image analysis protocol for counting fluorescent labeled GC was developed that used Image J software to measure GC cytoplasmic and nuclear areas. These data were compared to the number of GC obtained by disaggregating 96 follicles with enzymes to produce a suspension of GC, which then was stained with trypan blue and assessed using a hemocytometer. We found a linear relation between GC/follicle and follicle diameter. Viability of GC/follicle ranged from 40 ± 11 to 72 ± 7%. The coefficient of variation for image analysis of DAPI stained GC by different assessors was 4%, but the number of GC obtained from image analysis was approximately 50% less than from disaggregated follicles. The number of GC in intact mouse follicles was greater than the number reported earlier for fixed ovarian sections. We found that the number of GC was less in fluorescence labeled follicles; it is possible that the three-dimensional structure of the intact follicles obscured the fluorescent signal. Direct quantification of viable GC isolated from follicles appears to be the most accurate method.

Combined iDISCO and CUBIC tissue clearing and lightsheet microscopy for in toto analysis of the adult mouse ovary†.

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and performed qualitative assessments of follicles at all stages of development. This study is an important step toward developing quantitative computational models that allow rapid and accurate assessments of growing and quiescent primordial follicles, and to investigate the integrity of extrinsic ovarian components including vascular and neuronal networks.

Basal characterization and in vitro differentiation of putative stem cells derived from the adult mouse ovary

Lately, stem cell approaches have provided new information on reproductive organ function and additionally recommended novel treatment possibilities. The type(s) and differentiation potential of stem cells present in the mammalian ovary are largely unknown; while oogonial stem cells have been reported, we explored the possibility that multipotent stem cells may reside in the ovary and have wide differentiation potential. In this experimental study, homogenates of whole mouse ovaries were sorted using the stem cell surface markers stem cell antigen-1 and stage specific embryonic antigen-1/CD15. Viable double-positive cells 3-10μm in diameter were evaluated immediately after sorting and after culture using differentiation conditions. Ovarian-derived stem cells were differentiated into the three main cell types: adipocytes, chondrocytes, or osteocytes. The subsequent culture was performed in media containing bone morphogenetic protein 4 (BMP-4) and/or retinoic acid (RA). RA, BMP-4 or the two agents in combination, consistently stimulated germ cell gene expression. RA treatment strongly stimulated germline gene expression and also the development of cells that were morphologically reminiscent of oocytes. The germ cell genes Dazl, Ddx4, Figla, Gdf-9, Nobox, Prdm9, and Sycp-1 were all detected at low levels. Remarkably, treatment with BMP-4 alone significantly increased protein expression of the granulosa cell product anti-Müllerian hormone (AMH). We have shown that an inclusive isolation protocol results in the consistent derivation of multipotent stem cells from the adult ovary; these cells can be differentiated towards the germ cell fate (RA alone), somatic ovarian cell fate as indicated by AMH production (BMP-4 alone), or classical mesenchymal cell types. Taken together, these data suggest the presence of multipotent mesenchymal stem cells in the murine ovary.

Functional Oocytes Derived from Granulosa Cells

The generation of genomically stable and functional oocytes has great potential for preserving fertility and restoring ovarian function. It remains elusive whether functional oocytes can be generated from adult female somatic cells through reprogramming to germline-competent pluripotent stem cells (gPSCs) by chemical treatment alone. Here, we show that somatic granulosa cells isolated from adult mouse ovaries can be robustly induced to generate gPSCs by a purely chemical approach, with additional Rock inhibition and critical reprogramming facilitated by crotonic sodium or acid. These gPSCs acquired high germline competency and could consistently be directed to differentiate into primordial-germ-cell-like cells and form functional oocytes that produce fertile mice. Moreover, gPSCs promoted by crotonylation and the derived germ cells exhibited longer telomeres and high genomic stability like PGCs invivo,providing additional evidence supporting the safety and effectiveness of chemical induction, which is particularly important for germ cells in genetic inheritance.

The effect of GnRH antagonist cetrorelix on Wnt signaling members in pubertal and adult mouse ovaries.

Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C-8C) and sham groups (5S-8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Polycystic ovary syndrome (PCOS) is a hypothalamic-pituitary-gonadal (HPG) axis disorder. PCOS symptoms most likely result from a disturbance in the complex feedback regulation system of the HPG axis, which involves gonadotrophic hormones and ovarian steroid hormones. However, the nature of this complex and interconnecting feedback regulation makes it difficult to dissect the molecular mechanisms responsible for PCOS phenotypes. Global estrogen receptor α (ERα) knockout (KO) mice exhibit a disruption of the HPG axis, resulting in hormonal dysregulation in which female ERα KO mice have elevated levels of serum estradiol (E2), testosterone, and LH. The ERα KO females are anovulatory and develop cystic hemorrhagic ovaries that are thought to be due to persistently high circulating levels of LH from the pituitary. However, the role of ERα in the pituitary is still controversial because of the varied phenotypes reported in pituitary-specific ERα KO mouse models. Therefore, we developed a mouse model where ERα is reintroduced to be exclusively expressed in the pituitary on the background of a global ERα-null (PitERtgKO) mouse. Serum E2 and LH levels were normalized in PitERtgKO females and were comparable to wild-type serum levels. However, the ovaries of PitERtgKO adult mice displayed a more overt cystic and hemorrhagic phenotype when compared with ERα KO littermates. We determined that anomalous sporadic LH secretion caused the severe ovarian phenotype of PitERtgKO females. Our observations suggest that pituitary ERα is involved in the estrogen negative feedback regulation, whereas hypothalamic ERα is necessary for the precise control of LH secretion. Uncontrolled, irregular LH secretion may be the root cause of the cystic ovarian phenotype with similarities to PCOS.-Arao, Y., Hamilton, K. J., Wu, S.-P., Tsai, M.-J., DeMayo, F. J., Korach, K. S. Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Extracellular matrix signaling activates differentiation of adult ovary-derived oogonial stem cells in a species-specific manner

To test if ovarian microenvironmental cues affect oogonial stem cell (OSC) function in a species-specific manner. Animal and human study. Research laboratory. Human ovarian cells obtained from cryopreserved ovarian cortical tissue of reproductive-age women, and ovarian cells and tissues from female C57BL/6 mice. Mouse ovarian tissue, mouse OSCs (mOSCs) and human OSCs (hOSCs) were analyzed for extracellular matrix (ECM) protein expression, and OSCs isolated from adult mouse and human ovaries were cultured in the absence or presence of ECM proteins without or with an integrin signaling inhibitor. Gene expression and invitro derived (IVD) oocyte formation. Culture of mOSCs on a collagen-based ECM significantly elevated the rate of differentiation of the cells into IVD oocytes. Mouse OSCs expressed many integrins, including Arg-Gly-Asp (RGD)-binding subunits, and ECM-mediated increases in mOSC differentiation were blocked by addition of integrin-antagonizing RGD peptides. In comparison, hOSCs expressed a different pattern of integrin subunits compared with mOSCs, and hOSCs were unresponsive to a collagen-based ECM; however, hOSCs exhibited increased differentiation into IVD oocytes when cultured on laminin. These data, along with in silico analysis of ECM protein profiles in human ovaries, indicate that ovarian ECM-based niche components function in a species-specific manner to control OSC differentiation.

Stem Cells in theMammalian Gonads.

Besides spermatogonial stem cells (SSCs) and ovarian stem cells (OSCs), a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) has been reported in both adult mouse and human testes and ovaries. VSELs and SSCs/OSCs are developmentally linked to each other. VSELs are relatively quiescent, small-sized stem cells that undergo asymmetrical cell divisions (ACDs) whereby they self-renew and give rise to the slightly bigger SSCs/OSCs which in turn undergo symmetrical cell divisions (SCDs) and clonal expansion to form germ cell chains/nests before further differentiation into gametes. Comparison of VSELs and SSCs/OSCs for their potential to differentiate into sperm/oocytes is irrelevant since VSELs only undergo ACD to give rise to SSCs/OSCs that further differentiate into gametes. Being relatively quiescent, VSELs survive oncotherapy and can be manipulated to regenerate nonfunctional gonads of cancer survivors, and thus there is possibly no need to bank testicular/ovarian tissue prior to oncotherapy. Being developmentally linked to the primordial germ cells (PGCs) which are the natural precursors to the gametes, VSELs differentiate into haploid sperm/oocyte-like structures in vitro when cultured on appropriate feeder support, in the absence of a cocktail of growth factors. VSELs express receptors for pituitary and sex hormones (FSHR, ER) and thus get directly stimulated/affected by their circulating levels. Excessive self-renewal of VSELs in the gonads may initiate testicular and ovarian cancers. To conclude, VSELs can be targeted to regenerate the gonads of patients with gonadal insufficiency including cancer survivors and are excellent candidates to differentiate into gametes in vitro.

Intrafollicular fibroblast growth factor 13 in polycystic ovary syndrome: relationship with androgen levels and oocyte developmental competence

BackgroundFibroblast growth factor 13 (FGF13) is one of the most highly expressed FGF family members in adult mouse ovary. However, its precise roles in ovarian function remain largely unknown. We sought to evaluate the associations between FGF13 in follicular fluid and oocyte developmental competence in patients with polycystic ovary syndrome (PCOS).MethodsA cross-sectional study was conducted on 43 patients with PCOS and 32 non-PCOS patients who underwent in vitro fertilization/intracytoplasmic sperm injection treatments. The highest quartiles of follicular fluid (FF)-FGF13 (≥117.51 pg/mL) and FF-total testosterone (FF-TT) (≥51.90 nmol/L) were defined as “elevated” FF-FGF13 levels and “elevated” FF-TT levels, respectively.ResultsThe levels of FF-FGF13 were skewed, with a median of 82.97 pg/mL (59.79–117.51 pg/mL) in 75 patients. The prevalence of elevated FF-TT levels was significantly higher in the PCOS patients with elevated FF-FGF13 levels than in those without (64.3% vs. 35.7%, adjusted P = 0.0096). FF-TT and increased ovarian volume (> 10 mL for one or both ovaries) were positively correlated with FF-FGF13 in PCOS patients (r = 0.37, P = 0.013; r = 0.33, P = 0.032). A negative association was evident between FF-FGF13 and the MII oocyte rate in the multiple linear regression analysis (β = − 0.10, SE = 0.045, adjusted P = 0.027). However, the associations were not evident in the non-PCOS patients.ConclusionsOur study suggests the presence of intrafollicular FGF13 in PCOS patients and implies that FGF13 might be involved in the pathophysiological process of PCOS.

The ovarian estrogen synthesis function was impaired in Y123F mouse and partly restored by exogenous FSH supplement

BackgroundLepR tyrosine site mutation mice (Y123F) exhibit decreased serum E2 levels, immature reproductive organs, infertility as well as metabolic abnormalities. Although the actions of leptin and lepR in the control of reproductive function are thought to be exerted mainly via the hypothalamic-pituitary-gonadal axis, relatively less is known regarding their local effects on the peripheral ovary, especially on steroid hormone synthesis. Meanwhile, whether the decreased fertility of Y123F mouse could be restored by gonadotropin has not been clear yet.MethodsThe serum levels of E2, P4, FSH, LH, T and leptin of Y123F and WT mice at the age of 12 weeks were measured by enzyme-linked immunosorbent assay. Immunohistochemistry was used to compare the distribution of hormone synthases (STAR, CYP11A1, CYP19A1, HSD17B7) and FSHR in adult mouse ovaries of two genotypes. Western blot and real-time PCR were used to detect the expression levels of four ovarian hormone synthases and JAK2-STAT3 / STAT5 signaling pathway in 4 and 12 weeks old mice, as well as the effects of exogenous hFSH stimulation on hormone synthases and FSHR.ResultsCompared with WT mice, the serum levels of FSH, LH and E2 in 12-week-old Y123F mice were significantly decreased; T and leptin levels were significantly increased; but there was no significant difference of serum P4 levels. STAR, CYP11A1, HSD17B7 expression levels and the phosphorylation levels of JAK2 and STAT3 were significantly decreased in adult Y123F mice, while the expression of CYP19A1 and phospho-STAT5 were significantly increased. No significant differences were found between 4-week-old Y123F and WT mice. After exogenous hFSH stimulation, E2 levels and expression of CYP19A1 and HSD17B7 were significantly higher than that in the non-stimulated state, but significant differences still existed between Y123F and WT genotype mice under the same condition.ConclusionsAbnormal sex hormone levels of Y123F mice were due to not only decreased gonadotropin levels in the central nervous system, but also ovarian hormone synthase abnormalities in the peripheral gonads. Both FSH signaling pathway and JAK2-STAT3/STAT5 signaling pathway were involved in regulation of ovarian hormone synthases expression. Exogenous FSH just partly improved the blood E2 levels and ovarian hormone synthase expression.

Circular RNA expression profiles of mouse ovaries during postnatal development and the function of circular RNA epidermal growth factor receptor in granulosa cells

PurposeCircular RNAs (circRNAs) are a class of noncoding RNAs that can regulate gene expression at the post-transcriptional level. The contribution of circRNAs in the regulation of granulosa cells (GCs) functions is not yet clear. The aim of this study was to analyze circRNA expression in adult and neonate ovaries, uncover the biological roles of circ_0002861 (circEGFR) and identify the mechanism by which it modulates follicular development. Basic ProceduresThe circRNA expression profiles of adult and neonatal mouse ovaries were explored by high-throughput sequencing. The function of circEGFR was measured by RNA fluorescence in situ hybridization, overexpression, knockdown, RNA immunoprecipitation and luciferase reporter assays in GCs. Main FindingsNumerous differentially expressed circRNAs were identified in adult and neonatal ovaries. Through circRNAs expression patterns and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, estrogen signaling was found to be upregulated in adult ovaries compared with neonate ovaries. Further analysis revealed that the expression of circEGFR (circ_0002861, ID: mmu_circ_0002861 in circBase) was increased in adult ovaries compared with neonate ovaries. circEGFR is formed by splicing from exons 14 and 15 of the epidermal growth factor receptor (EGFR) gene to produce a covalently linked 249-nucleotide circRNA. Overexpression of circEGFR increased estradiol (E2) production and GCs growth, whereas circEGFR knockdown enhanced progesterone production and inhibited (E2) secretion by GCs. Bioinformatic screening identified several binding sites for miR-125a-3p in the circEGFR sequence. RNA immunoprecipitation and luciferase reporter assays demonstrated that circEGFR may act as a sponge for miR-125a-3p, thus modulating Fyn expression. Principal ConclusionsThese findings illustrate that circEGFR may play a vital role in ovarian GCs by modulating Fyn via competitive binding with miR-125a-3p. Our results suggest potential applications of circEGFR in reproductive and steroid-related disorder therapy.

Characterization of female germline stem cells from adult mouse ovaries and the role of rapamycin on them.

Germline stem cells (GSCs) play an indispensable role in establishing the fertility of an organism. The isolation and culture of adult female GSCs (FGSCs) have provided a robust foundation to study the development of female germ cells in rodents. However, many problems still need to be identified, such as the origin and location of FGSCs and the specific markers for screening. In this study, we acquired FGSCs that stably expressed Oct4 from Oct4 promoter-GFP transgenic mouse ovarian surface epithelium and cortical layer, and identified the cells possessing the representative features including the expression of GSCs marker genes and the potentiality of differentiation into all three germ layers in vitro. Moreover, rapamycin was confirmed to promote proliferation of mouse FGSCs and inhibit the differentiation capability in vivo. In addition to the reported disinfection function, rapamycin inhibited the activation of primordial follicles, as the inhibitor of mechanistic target of rapamycin pathway. These results will contribute to the study on folliculogenesis or oogenesis mechanism and have important implications on developing new technology and therapeutic approach in medicine for premature ovarian failure, infertility and even ovary remodelling in future.

Putative germline and pluripotent stem cells in adult mouse ovary and their in vitro differentiation potential into oocyte-like and somatic cells.

According to classical knowledge of reproductive biology, in the ovary of female mammals there is a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in recent years, the results of some studies on renewal and formation of oocytes and follicles in the adult mammalian ovary have led to the questioning of this opinion. The aim of our study is to demonstrate the presence of putative germline and pluripotent stem cells in the adult mouse ovary and their differentiation potential into germ and somatic cells. In ovary tissues and cells harvested from pre-differentiation step, the expression of pluripotent and germline stem cell markers was analysed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and western blotting. Embryoid bodies that formed in this step were analysed using immunofluorescence staining and transmission electron microscopy. Ovarian stem cells were induced to differentiate into oocyte, osteoblast, chondrocyte and neural cells. Besides morphological observation, differentiated cells were analysed by RT-PCR, histochemical and immunofluorescence staining. Expression of germline and pluripotent stem cell markers both in mRNA and at the protein level were detected in the pre-differentiated cells and ovary tissues. As a result of the differentiation process, the formation of oocyte-like cells, osteoblasts, chondrocytes and neural cells was observed and characteristics of differentiated cells were confirmed using the methods mentioned above. Our study results revealed that the adult mouse ovary contains germline and pluripotent stem cells with the capacity to differentiate into oocyte-like cells, osteoblasts, chondrocytes and neural cells.