Mouse Ovarian Granulosa Cells Research Articles

Corrigendum to “Differentiation of sow and mouse ovarian granulosa cells exposed to zearalenone in vitro using RNA-seq gene expression” [Toxicology and Applied Pharmacology, 350 (2018) 78–90]

Corrigendum to “Differentiation of sow and mouse ovarian granulosa cells exposed to zearalenone in vitro using RNA-seq gene expression” [Toxicology and Applied Pharmacology, 350 (2018) 78–90]

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles harboring IGF-1 improve ovarian function of mice with premature ovarian insufficiency through the Nrf2/HO-1 pathway

ObjectivePremature ovarian insufficiency (POI) is a disease with medical, psychological and reproductive implications, but its common therapies have limited efficacy and a likelihood of complications. This study delves into the therapeutic role of human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUC-MSCs-EVs) in POI mice through the insulin-like growth factor 1 (IGF-1)/nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/autophagy pathway.MethodshUC-MSCs were transfected with lentiviral short hairpin RNA of IGF-1 before EV extraction. Cyclophosphamide (CTX)-induced POI mouse models were administrated with hUC-MSCs-EVs. Mouse ovarian granulosa cells (GCs) were induced with CTX, then treated with hUC-MSCs-EVs and ML385. Ovarian histopathological changes were observed, changes in follicle number at all levels were counted and serum sex hormones were evaluated, as well as LC3II/I and Beclin-1 expression. GCs were subject to detection of proliferation, deaths, oxidative stress, and Nrf2 nuclear translocation.ResultsAfter CTX exposure, mice showed thinner GCs layer in the ovary, reduced number of GCs and follicles at all levels, disturbed serum sex hormones, enhanced oxidative stress and autophagy, and downregulated ovarian IGF-1; whereas, hUC-MSCs-EVs upregulated IGF-1 to improve the ovarian function. hUC-MSCs-EVs carrying IGF-1 activated Nrf2/HO-1 signaling to inhibit CTX-induced excessive autophagy of GCs, but this ameliorative effect was partially weakened by inhibiting Nrf2/HO-1 signaling. hUC-MSCs-EVs inhibited excessive autophagy of GCs and improved ovarian function of CTX-induced mice through IGF-1/Nrf2/HO-1 pathway.ConclusionhUC-MSCs-EVs activate the Nrf2/HO-1 signaling by carrying IGF-1, which in turn inhibits excessive autophagy and damage of GCs, thus improving ovarian function in POI mice.

Lactylation drives hCG-triggered luteinization in hypoxic granulosa cells

Hypoxia that occurs during the luteinization process of granulosa cells (GC) contributes to the formation of lactate in follicles. Lysine lactylation (Kla), a post-translational modification directly regulated by lactate levels, is a metabolic sensor that converts metabolic information into gene expression patterns. In this study, we employed human chorionic gonadotropin (hCG) to induce GCs luteinization and discovered that hypoxia enhances hCG-mediated GCs luteinization by stimulating lactate production/lactylation. The elevated levels of luteinization markers (including progesterone synthesis, expression of CYP11A1 and STAR) were accompanied by increased lactate production as well as enhanced lactylation in mouse ovarian GCs after the injection of hCG in vivo. By treating GCs with hypoxia in vitro, we found that hypoxia accelerated hCG-induced GCs luteinization, which was inhibited after blocking lactate production/lactylation. Further investigations revealed that H3K18la might contribute to hCG-induced luteinization in hypoxic GCs by upregulating CYP11A1 and STAR transcription. Additionally, we identified that CREB K136la is also required for hCG-induced GCs luteinization under hypoxia. Finally, the in vitro findings were verified in vivo, which showed impaired GCs luteinization and corpus luteum formation after blocking the lactate/lactylation by intraperitoneal injection of oxamate/C646 in mice. Taken together, this study uncovered a novel role of protein lactylation in the regulation of GCs luteinization.

Casein kinase 1α mediates estradiol secretion via CYP19A1 expression in mouse ovarian granulosa cells

BackgroundCasein kinase 1α (CK1α), expressed in both ovarian germ and somatic cells, is involved in the initial meiosis and primordial follicle formation of mouse oocytes. Using in vitro and in vivo experiments in this study, we explored the function and mechanism of CK1α in estrogen synthesis in mice ovarian granulosa cells.MethodsA CK1α knockout (cKO) mouse model, targeted specifically to ovarian granulosa cells (GCs), was employed to establish the influence of CK1α on in vivo estrogen synthesis. The influence of CK1α deficiency on GCs was determined in vivo and in vitro by immunofluorescence analysis and Western blot assay. Transcriptome profiling, differentially expressed genes and gene functional enrichment analyses, and computation protein–protein docking, were further employed to assess the CK1α pathway. Furthermore, wild-type female mice were treated with the CK1α antagonist D4476 to elucidate the CK1α's role in estrogen regulation.ResultsOvarian GCs CK1α deficiency impaired fertility and superovulation of female mice; also, the average litter size and the estradiol (E2) level in the serum of cKO female mice were decreased by 57.3% and 87.4% vs. control mice, respectively.This deficiency disrupted the estrous cycle and enhanced the apoptosis in the GCs. We observed that CK1α mediated the secretion of estradiol in mouse ovarian GCs via the cytochrome P450 subfamily 19 member 1 (CYP19A1).ConclusionsThese findings improve the existing understanding of the regulation mechanism of female reproduction and estrogen synthesis.Trial registrationNot applicable.

The epithelial Na+ channel (ENaC) in ovarian granulosa cells modulates Ca2+ mobilization and gonadotrophin signaling for estrogen homeostasis and female fertility

Ovarian granulosa cells are essential to gonadotrophin-regulated estrogen production, female cycle maintenance and fertility. The epithelial Na+ channel (ENaC) is associated with female fertility; however, whether and how it plays a role in ovarian cell function(s) remained unexplored. Here, we report patch-clamp and Na+ imaging detection of ENaC expression and channel activity in both human and mouse ovarian granulosa cells, which are promoted by pituitary gonadotrophins, follicle stimulating hormone (FSH) or luteinizing hormone (LH). Cre-recombinase- and CRISPR-Cas9-based granulosa-specific knockout of ENaC α subunit (Scnn1a) in mice resulted in failed estrogen elevation at early estrus, reduced number of corpus luteum, abnormally extended estrus phase, reduced litter size and subfertility in adult female mice. Further analysis using technologies including RNA sequencing and Ca2+ imaging revealed that pharmacological inhibition, shRNA-based knockdown or the knockout of ENaC diminished spontaneous or stimulated Ca2+ oscillations, lowered the capacity of intracellular Ca2+ stores and impaired FSH/LH-stimulated transcriptome changes for estrogen production in mouse and/or human granulosa cells. Together, these results have revealed a previously undefined role of ENaC in modulating gonadotrophin signaling in granulosa cells for estrogen homeostasis and thus female fertility.

P-353 Perfluorooctanoic acid (PFOA) impacts murine ovarian follicle development through apoptotic signaling pathways and exhibits transgenerational effects

Abstract Study question How does Perfluorooctanoic Acid (PFOA) exposure impact ovarian function and the physiological and reproductive health of subsequent offspring? Summary answer PFOA exposure leads to ovarian dysfunction, characterized by follicular depletion and hormonal imbalances, and adversely affects the reproductive health of offspring. What is known already Mass spectrometry reveals higher perfluorinated compound levels in premature ovarian failure patients compared to healthy individuals. PFOA, an environmental contaminant, is known for its endocrine-disrupting effects, but its specific impact on ovarian function and transgenerational health remains less explored. This study addresses the gap by examining PFOA’s direct ovarian effects and its consequential impacts on offspring, using both in vivo and in vitro models. Study design, size, duration This study employs mouse models and ovarian granulosa cells to assess PFOA’s impact. Mice were administered PFOA (50mg/Kg) and evaluated for ovarian function and offspring health. Ovarian tissue underwent histological analysis and hormone assays. RNA transcriptome sequencing identified activated apoptotic pathways post-PFOA exposure. Offspring physiological and reproductive parameters were also examined. Parallel in vitro experiments with ovarian granulosa cells at 50uM PFOA concentration provided insights into cellular apoptosis. Participants/materials, setting, methods Clinically, we collected serum samples from approximately 100 Premature Ovarian Insufficiency (POI) patients and 100 healthy individuals, analyzed using mass spectrometry. In murine experiments, we simulated human environmental exposure to PFOA (particularly in high-exposure professions) using a 50mg/Kg dose administered via gavage. Transcriptome sequencing analyzed potential signaling pathways in both experimental and control mice. Offspring underwent physiological metabolic assessments and fertility evaluations, including fertilization and blastocyst rates. Main results and the role of chance Mass spectrometry results from human samples revealed significantly higher levels of perfluorinated compounds in POI patients compared to healthy controls, suggesting a potential link between PFOA exposure and ovarian dysfunction. In murine models, PFOA administration at 50mg/Kg resulted in notable ovarian changes. Histological analysis showed a reduction in follicle counts and abnormalities in AMH and FSH levels, indicative of premature ovarian failure. Transcriptome sequencing of these mice highlighted the activation of apoptotic pathways, especially the PI3K/Akt/mTOR signaling, post-PFOA exposure. This suggests a mechanistic pathway through which PFOA may induce ovarian dysfunction. Furthermore, offspring of PFOA-exposed mice displayed significant reproductive health challenges. They exhibited irregularities in estrous cycles and a decrease in oocyte fertilization rates, pointing to potential transgenerational effects of PFOA exposure. These findings in murine models align with the elevated perfluorinated compound levels observed in human POI patients, reinforcing the hypothesis of PFOA’s detrimental impact on ovarian health and subsequent generational fertility. The consistency of results across human clinical data and animal models strengthens the argument for a causal relationship between PFOA exposure and reproductive health risks. Limitations, reasons for caution While these findings highlight PFOA’s detrimental effects on ovarian function and offspring health, translating results from animal models to humans necessitates caution. The specific mechanisms and potential variability in human response to environmental PFOA exposure require further investigation. Wider implications of the findings This research underscores the far-reaching impacts of environmental contaminants like PFOA on reproductive health. It emphasizes the need for stringent regulation of such pollutants and further studies to understand their long-term effects on human health, particularly regarding fertility and intergenerational wellbeing. Trial registration number not applicable

Decreased fertility in female mice lacking urokinase plasminogen activator

It is well established that mouse ovarian granulosa cells secrete urokinase plasminogen activator (uPA) under gonadotropin stimulation. The synthesis and secretion of the enzyme correlate well with the time of follicular rupture in vivo. Moreover, uPA is secreted by the trophoblast at the time of implantation. In the present study, we have analyzed whether the absence of uPA could influence follicular growth, ovulation, and embryo implantation. Our data show fewer preantral follicles in uPA-/- ovaries but no decrease in hormonally induced ovulation. However, we observed a significant decrease in the number of implanted embryos in uPA-/- animals and, therefore, a lower number of pups per family. Adding uPA to the epithelial and stromal uterine cell culture medium strongly upregulates the expression of prostaglandin-endoperoxide synthase 2 (Ptgs2), the enzyme required for prostaglandin production and embryo implantation. The uPA inhibitor amiloride abrogated this increase.

Rfamide-related peptide-3(RFRP-3) receptor gene is expressed in mouse ovarian granulosa cells: Potential role of RFRP-3 in steroidogenesis and apoptosis

RFRP-3 is a functional ortholog of avian GnIH and regulates reproductive activities in the gonads of animals. However, the role of RFRP-3 in the function of ovarian granulosa cells in mice remains unclear. First, we detected the expression of the RFRP-3 receptor (GPR147) in the ovarian granulosa cells of mice. Second, the effect of RFRP-3 treatment on estradiol and progesterone secretions from granulosa cells was tested by ELISA. Meanwhile, the expression of genes and proteins regulating steroid hormone synthesis was respectively examined by qPCR and western blot. Furthermore, the effect of RFRP-3 treatment on the apoptosis of granulosa cells was analyzed. The results revealed that the GPR147 protein (a RFRP-3 receptor) was expressed in the ovarian granulosa cells of mice. Low and medium doses RFRP-3 treatment significantly reduced progesterone secretion in the granulosa cells (P < 0.05), while RFRP-3 suppressed p450scc, 3β-HSD, StAR, and FSHR expression in a non-dose-dependent manner. Moreover, RFRP-3 treatment might induce the apoptosis of granulosa cells. Additionally, low doses RFRP-3 significantly reduced p-ERK1/2 protein expression (P < 0.05) in the ovarian granulosa cells. We here, for the first time, confirmed that GPR147 was expressed in the ovarian granulosa cells of mice. Our findings suggested that and RFRP-3 regulates the granulosa cell function through the ERK signaling pathway, which will lay the foundation for uncovering molecular mechanisms by which RFRP-3 regulates follicle development in future.

FSH preserves the viability of hypoxic granulosa cells viaactivating the HIF-1α-GAS6-Axl-Akt pathway.

The developmental fate of ovarian follicles is primarily determined by the survival status (proliferation or apoptosis) of granulosa cells (GCs). Owing to the avascular environment within follicles, GCs are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH) has been reported to improve GCs survival by governing hypoxia-inducible factor-1α (HIF-1α)-dependent hypoxia response, but the underlying mechanisms remain poorly understood. Growth arrest-specific gene 6 (GAS6) is a secreted ligand of tyrosine kinase receptors, and has been documented to facilitate tumor growth. Here, we showed that the level of GAS6 was markedly increased in mouse ovarian GCs after the injection of FSH. Specifically, FSH-induced GAS6 expression was accompanied by HIF-1α accumulation under conditions of hypoxia both in vivo and in vitro, whereas inhibition of HIF-1αwith smallinterfering RNAs/antagonist repressed both expression and secretion of GAS6. As such, Luciferase reporter assay and chromatin immunoprecipitation assay showed that HIF-1α directly bound to a hypoxia response element site within the Gas6 promoter and contributed to the regulation of GAS6 expression in response to FSH. Notably, blockage of GAS6 and/or its receptor Axl abrogated the pro-survival effects of FSH under hypoxia. Moreover, phosphorylation of Axl by GAS6 is required for FSH-mediated Akt activation and the resultant pro-survival phenotypes. Finally, the in vitro findings were verified in vivo, which showed that FSH-induced proliferative and antiapoptotic effects in ovarian GCs were diminished after blocking GAS6/Axl using HIF-1α antagonist. These findings highlight a novel function of FSH in preserving GCs viability against hypoxic stress by activating the HIF-1a-GAS6-Axl-Akt pathway.

Scutellarin targets Wnt5a against zearalenone-induced apoptosis in mouse granulosa cells in vitro and in vivo

Zearalenone (ZEA) poses severe reproductive toxicity to both humans and animals. Scutellarin has been demonstrated to rescue ZEA-induced apoptosis in mouse ovarian granulosa cells (GCs), but its specific targets remain unclear. In the present study, the potential targets of scutellarin were determined to clarify the mechanisms of scutellarin against ZEA-induced ovarian damage. 287 targets of scutellarin in mouse ovarian GCs were obtained by magnetic nano-probe-based fishing assay and liquid chromatography-tandem mass spectrometry. Wnt5a had the lowest binding free energy with scutellarin at − 8.3 kcal/mol. QRT-PCR and western blot showed that scutellarin significantly increased the Wnt5a and β-catenin expression compared with the ZEA-treated group, and cleaved-caspase-3 expression was significantly increased in the scutellarin-treated group after interfering with the expression of Wnt5a. The affinity constant (KD) of Wnt5a and scutellarin was 1.7 × 10−5 M. The pull-down assay also demonstrated that scutellarin could specifically bind to Wnt5a protein. Molecular docking results showed that scutellarin could form hydrogen bonds with TRY52, GLN56, and SER90 on Wnt5a protein, and western blot assay confirmed SER90 was an important site for the binding. Scutellarin significantly increased Wnt5a and β-catenin expression and decreased cleaved-caspase-3 expression in ovarian tissues of mice. In conclusion, scutellarin exerted anti-apoptotic effects on ZEA-induced mouse ovarian GCs by targeting Wnt5a.

Fertility restoration in mice with chemotherapy induced ovarian failure using differentiated iPSCs.

Treatment options for premature ovarian insufficiency (POI) are limited to hormone replacement and donor oocytes. A novel induced pluripotent stem cell (iPSC) transplant paradigm in a mouse model has potential translational applications for management of POI. Mouse ovarian granulosa cell derived-iPSCS were labelled with green fluorescent protein (GFP) reporter and differentiated invitro into oocytes. Differentiated cells were assayed for estradiol and progesterone secretion by enzyme-linked immunosorbent assays. After Fluorescence-Activated Cell Sorting (FACS) for the cell surface marker anti-Mullerian hormone receptor (AMHR2), enriched populations of differentiated cells were surgically transplanted into ovaries of mice that had POI secondary to gonadotoxic pre-treatment with alkylating agents. A total of 100 mice were used in these studies in five separate experiments with 56 animals receiving orthotopic ovarian injections of either FACS sorted or unsorted differentiated iPSCSs and the remaining animals receiving sham injections of PBS diluent. Following transplantation surgery, mice were stimulated with gonadotropins inducing oocyte development and underwent oocyte retrieval. Nine transplanted mice were cross bred with wild-type mice to assess fertility. Lineage tracing of resultant oocytes, F1 (30 pups), and F2 (42 pups) litters was interrogated by GFP expression and validation by short tandem repeat (STR) lineage tracing. [1] iPSCs differentiate into functional oocytes and steroidogenic ovarian cells which [2] express an ovarian (GJA1) and germ cell (ZP1) markers. [3] Endocrine function and fertility were restored in mice pretreated with gonadotoxic alkylating agents via orthotopic transplantation of differentiated iPSCS, thus generating viable, fertile mouse pups. iPSC-derived ovarian tissue can reverse endocrine and reproductive sequelae of POI. Center for Infertility and Reproductive Surgery Research Award, Siezen Foundation award (RMA). Reproductive Scientist Development Program, Marriott Foundation, Saltonstall Foundation, Brigham Ovarian Cancer Research Fund (K.E).

Bisphenol A interferes with lncRNA Fhadlos2 and RUNX3 association in adolescent mouse ovary

Bisphenol A (BPA) has a number of adverse effects on the reproductive development of females. In particular, the mechanism of disruption of ovarian development in adolescent mice is still unclear. Based on transcriptome sequencing results, a differentially expressed lncRNA, Fhad1os2, was detected in the ovaries of BPA-exposed pubertal mice. In our study, the lncRNA Fhad1os2, localized in the ovarian granulosa cell cytoplasm, could regulate the proliferation of mouse ovarian granulosa cells. Mechanistically, the results of RNA pull-down experiments as well as mass spectrometry analysis showed that ERα, an interfering signaling molecule of BPA, could directly bind lncRNA Fhad1os2 and decrease the transcription of lncRNA Fhad1os2 in response to the estrogen-like effect of BPA. BPA exposure also caused abnormal lncRNA Fhad1os2 pulldown protein-related signaling pathways in the ovaries of adolescent mice. Furthermore, lncRNA Fhad1os2 interacted with RUNX3, a transcription factor related to follicle development and hormone synthesis. As a negative regulator, lncRNA Fhad1os2 transactivated the expression of Runx3, which in turn induced RUNX3 to positively regulate aromatase (Cyp19a1) expression in mouse ovarian granulosa cells and promote estrogen synthesis. In conclusion, our study indicates that BPA exposure interferes with ERα-regulated lncRNA Fhad1os2 interactions with RUNX3 in pubertal mice, affecting estrogen synthesis in mouse granulosa cells and contributing to premature ovarian maturation in pubertal mice.

Iron deposition in ovarian endometriosis evaluated by magnetic resonance imaging R2* correlates with ovarian function

Research questionDoes iron overload in patients with endometriosis affect ovarian function? Can a method be developed to visually reflect this? DesignMagnetic resonance imaging (MRI) R2* was used to evaluate the correlation between iron deposition of ovarian and anti-Müllerian hormone (AMH) in patients with endometriosis. All patients underwent T2* MRI scanning. Serum AMH levels were measured preoperatively. The area of focal iron deposition, iron content of the cystic fluid and AMH levels between the endometriosis and control groups were compared using non-parametric tests. The effects of iron overload on AMH secretion in mouse ovarian granulosa cells were investigated by adding different concentrations of ferric citrate to the medium. ResultsA significant difference was found between endometriosis and control groups in area of iron deposition (P < 0.0001), cystic fluid iron content (P < 0.0001), R2* of lesions (P < 0.0001) and R2* of the cystic fluid (P < 0.0001). Negative correlations were found between serum AMH levels and R2* of cystic lesions in patients with endometriosis aged 18–35 years (rs = –0.6484, P < 0.0001), and between serum AMH levels and R2* of cystic fluid (rs = –0.5074, P = 0.0050). Transcription level (P < 0.0005) and secretion level (P < 0.005) of AMH significantly decreased with the increase in iron exposure. ConclusionIron deposits can impair ovarian function, which is reflected in MRI R2*. Serum AMH levels and R2* of cystic lesions or fluid in patients aged 18–35 years had a negative correlation with endometriosis. R2* can be used to reflect the changes of ovarian function caused by iron deposition.

CLPP inhibition triggers apoptosis in human ovarian granulosa cells via COX5A abnormality-Mediated mitochondrial dysfunction.

Premature ovarian insufficiency (POI) is characterized by early loss of ovarian function before the age of 40years. It is confirmed to have a strong and indispensable genetic component. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a key inducer of mitochondrial protein quality control for the clearance of misfolded or damaged proteins, which is necessary to maintain mitochondrial function. Previous findings have shown that the variation in CLPP is closely related to the occurrence of POI, which is consistent with our findings. This study identified a novel CLPP missense variant (c.628G > A) in a woman with POI who presented with secondary amenorrhea, ovarian dysfunction, and primary infertility. The variant was located in exon 5 and resulted in a change from alanine to threonine (p.Ala210Thr). Importantly, Clpp was mainly localized in the cytoplasm of mouse ovarian granulosa cells and oocytes, and was relatively highly expressed in granulosa cells. Moreover, the overexpression of c.628G > A variant in human ovarian granulosa cells decreased the proliferative capacity. Functional experiments revealed that the inhibition of CLPP decreased the content and activity of oxidative respiratory chain complex IV by affecting the degradation of aggregated or misfolded COX5A, leading to the accumulation of reactive oxygen species and reduction of mitochondrial membrane potential, ultimately activating the intrinsic apoptotic pathways. The present study demonstrated that CLPP affected the apoptosis of granulosa cells, which might be one of the mechanisms by which CLPP aberrations led to the development of POI.

Human Pluripotent Stem Cell-Mesenchymal Stem Cell-Derived Exosomes Promote Ovarian Granulosa Cell Proliferation and Attenuate Cell Apoptosis Induced by Cyclophosphamide in a POI-like Mouse Model.

Premature ovarian insufficiency (POI) is a complex disease which causes amenorrhea, hypergonadotropism and infertility in patients no more than 40 years old. Recently, several studies have reported that exosomes have the potential to protect ovarian function using a POI-like mouse model induced by chemotherapy drugs. In this study, the therapeutic potential of exosomes derived from human pluripotent stem cell-mesenchymal stem cells (hiMSC exosomes) was evaluated through a cyclophosphamide (CTX)-induced POI-like mouse model. POI-like pathological changes in mice were determined by serum sex-hormones levels and the available number of ovarian follicles. The expression levels of cellular proliferation proteins and apoptosis-related proteins in mouse ovarian granulosa cells were measured using immunofluorescence, immunohistochemistry and Western blotting. Notably, a positive effect on the preservation of ovarian function was evidenced, since the loss of follicles in the POI-like mouse ovaries was slowed. Additionally, hiMSC exosomes not only restored the levels of serum sex hormones, but also significantly promoted the proliferation of granulosa cells and inhibited cell apoptosis. The current study suggests that the administration of hiMSC exosomes in the ovaries can preserve female-mouse fertility.

Polystyrene-microplastics and DEHP co-exposure induced DNA damage, cell cycle arrest and necroptosis of ovarian granulosa cells in mice by promoting ROS production

The joint pollution of microplastics (MPs) and di-(2-ethylhexyl) phthalic acid (DEHP) often occurs, and consequently poses a serious threat to human and animal health, which has attracted widespread attention. However, the damage to the female mammalian ovary caused by the single exposure and co-exposure of MPs and DEHP and its specific mechanisms are not clear. Here, we established mouse models of single and co-exposures to polystyrene-microplastics (PS-MPs) and DEHP. The results showed that exposed to 100 mg/L PS-MPs and 200 mg/kg DEHP for 35 days destroyed the ovarian granulosa cell layer of mice, leading to follicular fragmentation and atresia. We cultured ovary granulosa cells in vitro to perform further mechanism studies and found that PS-MPs and DEHP had synergistic effects. Both of them promoted the excessive production of ROS and induced oxidative stress by triggering the CNR1/CRBN/YY1/CYP2E1 signaling axis, which in turn caused DNA oxidative damage. Additionally, we provided compelling evidence that oxidative stress mediated-hippo signaling pathway played a critical role in PS-MPs and DEHP caused ovary damage, resulting in ovarian granulosa cell cycle arrest and necroptosis. Using oxidative stress inhibitor AM251 or DAS could reverse these changes markedly and alleviate the reproductive toxicity caused by PS-MPs and DEHP, effectively. Overall, these results demonstrated that co-exposure of PS-MPs and DEHP adversely affected the integrity of ovary granulosa cell layer, resulting in DNA oxidative damage, cell cycle arrest and increased necroptosis of mouse ovarian granulosa cells by inducing oxidative stress. Our study shed new light on the co-exposure toxicity of PS-MPs and DEHP, provided novel insights for the reproductive toxicity of PS-MPs combined exposure with DEHP in female animals from a new free radical generation pathway perspective.

Study on the mechanism of scutellarin's protective effect against ZEA-induced mouse ovarian granulosa cells injury.

Zearalenone (ZEA), a mycotoxin produced by Fusarium, can cause reproductive disorders by targeting ovarian granulosa cells (GCs). We previous showed that scutellarin (Scu) rescues ZEA-induced GCs damage in mice. In this study, we employed iTRAQ-based proteomics to investigate the mechanism underlying the restorative effects of Scu in this model. Compared to the model group, we identified 415 differentially expressed proteins (DEPs) in both the control and Scu-treated groups, and found that these were enriched mainly in the biosynthesis and metabolism, drug metabolism, and pentose phosphate pathway. Moreover, the MAPK and heat shock protein-necroptosis pathway were implicated in regulating ZEA toxicity and the protective effect of Scu. Receptor-interacting serine threonine-protein kinase 1 (RIPK1) showed the highest fold-change in expression in the Scu-treated group. Small-interfering RNA-mediated RIPK1 knockdown further promoted the increase in cleaved-caspase-3 expression induced by ZEA, but not in the cells treated with Scu. These data indicated the involvement of multiple targets and pathways in the protective effect of Scu against ZEA-induced damage. Our findings also indicated that RIPK1 may be involved in the inhibition of GCs apoptosis induced by ZEA.

The Corticosterone-Glucocorticoid Receptor-AP1/CREB Axis Inhibits the Luteinizing Hormone Receptor Expression in Mouse Granulosa Cells.

Under stress conditions, luteinizing hormone (LH)-mediated ovulation is inhibited, resulting in insufficient oocyte production and excretion during follicular development. When the body is stressed, a large amount of corticosterone (CORT) is generated, which will lead to a disorder of the body’s endocrine system and damage to the body. Our previous work showed that CORT can block follicular development in mice. Since LH acts through binding with the luteinizing hormone receptor (Lhcgr), the present study aimed to investigate whether and how corticosterone (CORT) influences Lhcgr expression in mouse ovarian granulosa cells (GCs). For this purpose, three-week-old ICR female mice were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG). In addition, the treatment group was injected with CORT (1 mg/mouse) at intervals of 8 h and the control group was injected with the same volume of methyl sulfoxide (DMSO). GCs were collected at 24 h, 48 h, and 55 h after PMSG injection. For in vitro experiments, the mouse GCs obtained from healthy follicles were treated with CORT alone, or together with inhibitors against the glucocorticoid receptor (Nr3c1). The results showed that the CORT caused a downregulation of Lhcgr expression in GCs, which was accompanied by impaired cell viability. Moreover, the effect of the CORT was mediated by binding to its receptor (Nr3c1) in GCs. Further investigation revealed that Nr3c1 might regulate the transcription of Lhcgr through inhibiting the expression of Lhcgr transcription factors, including AP1 and Creb. Taken together, our findings suggested a possible mechanism of CORT-induced anovulation involving the inhibition of Lhcgr expression in GCs by the CORT–Nr3c1–AP1/Creb axis.

Di-isononyl phthalate induces apoptosis and autophagy of mouse ovarian granulosa cells via oxidative stress

Di-isononyl phthalate (DINP) has been widely utilized in industrial, commercial and medical applications for the past few years. Therefore, more attention should be paid to the toxicity of DINP. DINP can cause damage to female reproductive system; however, the potential mechanism remains to be further investigated. In this study, female mice were orally administered with 0, 2, 20 and 200 mg DINP/kg/day for 14 days. We found that DINP significantly affected the arrangement of granulosa cells in ovarian follicles. In addition, DINP could induce apoptosis, autophagy and oxidative stress of the ovary tissue. Meanwhile, the serum estradiol concentration distinctly decreased in the 20 and 200 mg/kg DINP-treated groups, suggesting that DINP might affect the function of ovarian granulosa cells. Primary mouse ovarian granulosa cells were utilized for further investigation after the cells were treated with 0, 100, 200, 400 μM DINP for 24 h. Similar to the in vivo experiment, DINP could also induce apoptosis and autophagy of ovarian granulosa cells, as well as oxidative stress; while inhibition of oxidative stress by NAC could alleviate DINP-induced apoptosis and autophagy. Furthermore, inhibition of autophagy by 3-MA could also rescue the induction of apoptosis by DINP. Taken together, these results indicated that DINP induced apoptosis and autophagy of mouse ovarian granulosa cells via oxidative stress, and autophagy played a cytotoxic role in DINP-induced apoptosis.

Cell-free fat extract improves ovarian function and fertility in mice with premature ovarian insufficiency

BackgroundPremature ovarian insufficiency (POI) is a refractory disease that seriously affects the reproductive health of women and is increasing in incidence and prevalence globally. There is enormous demand to improve fertility in women with POI, while there is still lack of effective therapeutic methods in clinic. Cell-free fat extract (CEFFE) has been reported to contain thousands of active proteins which possess the ability to promote tissue repair in other diseases. In our study, we aimed to observe the efficacy and biosecurity of CEFFE on the repair of ovarian function and fertility of mice with POI and further explore the underlying mechanism.MethodsIn vivo, POI mice model, established by cyclophosphamide (CTX, 120 mg/kg) and busulfan (BUS, 12 mg/kg), was treated with CEFFE via the tail vein every two days for 2 weeks. Then, the weight of ovaries, estrous cycle and follicle count by H&E staining were measured. The content of AMH, E2 and FSH in serum was measured by Enzyme-linked immunosorbent assay. Fertility was evaluated by the number of oocytes retrieved, the development of embryos in vitro and the litter size. Biosecurity of parent mice and their pups were examined by body mass and visceral index. The proliferation and apoptosis of cells in ovaries were examined by immunohistochemistry and transmission electron microscopy. Furthermore, the mRNA-Seq of mouse ovarian granulosa cells was performed to explore underlying mechanism of CEFFE. In vitro, KGN cell line and human primary ovarian granulosa cells (hGCs) were treated with 250 μM CTX for 48 h with/without CEFFE. The proliferative ability of cells was detected by cell counting kit-8 assay (CCK-8) and EDU test; the apoptosis of cells was detected by TUNEL and flow cytometry.ResultsCEFFE recovered the content of AMH, E2 and FSH in serum, increased the number of follicles and the retrieved oocytes of POI mice (P < 0.05). CEFFE contributed to the development of embryos and improved the litter size of POI mice (P < 0.05). There was no side effect of CEFFE on parent mice and their pups. CEFFE contributed to the proliferation and inhibited the apoptosis of mouse granulosa cells in ovary, as well as in human ovarian granulosa cells (including KGN cell line and hGCs) (P < 0.05).ConclusionsThe treatment of CEFFE inhibited the apoptosis of granulosa cells and contributed to the recovery of ovarian function, as well as the fertility of mice with POI.