Interlukin-22 improves ovarian function in polycystic ovary syndrome independent of metabolic regulation: a mouse-based experimental study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a reproductive endocrine disorder with multiple metabolic abnormalities. Most PCOS patients have concomitant metabolic syndromes such as insulin resistance and obesity, which often lead to the development of type II diabetes and cardiovascular disease with serious consequences. Current treatment of PCOS with symptomatic treatments such as hormone replacement, which has many side effects. Research on its origin and pathogenesis is urgently needed. Although improving the metabolic status of the body can alleviate reproductive function in some patients, there is still a subset of patients with metabolically normal PCOS that lacks therapeutic tools to address ovarian etiology. METHODS: The effect of IL-22 on PCOS ovarian function was verified in a non-metabolic PCOS mouse model induced by dehydroepiandrosterone (DHEA) and rosiglitazone, as well as granulosa cell -specific STAT3 knockout (Fshrcre+Stat3f/f) mice (10 groups totally and n = 5 per group). Mice were maintained under controlled temperature and lighting conditions with free access to food and water in a specific pathogen-free (SPF) facility. Secondary follicles separated from Fshrcre+Stat3f/f mice were cultured in vitro with DHEA to mimic the hyperandrogenic environment in PCOS ovaries (4 groups and n = 7 per group) and then were treated with IL-22 to investigate the specific role of IL-22 on ovarian function. RESULTS: We developed a non-metabolic mice model with rosiglitazone superimposed on DHEA. This model has normal metabolic function as evidenced by normal glucose tolerance without insulin resistance and PCOS-like ovarian function as evidenced by irregular estrous cycle, polycystic ovarian morphology (PCOM), abnormalities in sex hormone level. Supplementation with IL-22 improved these ovarian functions in non-metabolic PCOS mice. Application of DHEA in an in vitro follicular culture system to simulate PCOS follicular developmental block and ovulation impairment. Follicles from Fshrcre+Stat3f/f did not show improvement in POCS follicle development with the addition of IL-22. In DHEA-induced PCOS mice, selective ablation of STAT3 in granulosa cells significantly reversed the ameliorative effect of IL-22 on ovarian function. CONCLUSION: IL-22 can improve non-metabolic PCOS mice ovarian function. Granulosa cells deficient in STAT3 reverses the role of IL-22 in alleviating ovary dysfunction in non-metabolic PCOS mice.

Bisphenol A and its structural analogues exhibit differential potential to induce mitochondrial dysfunction and apoptosis in human granulosa cells.

Bisphenol A (BPA) is an endocrine disruptor strongly associated with ovarian dysfunction. BPA is being substituted by structurally similar chemicals, such as bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF). However, the toxicity of these analogues in female reproduction remains largely unknown. This study evaluated the effects of BPA and its analogues BPS, BPF, and BPAF on the mitochondrial mass and function, oxidative stress, and their potential to induce apoptosis of human granulosa cells (KGN cells). BPA and its analogues, especially BPA and BPAF, significantly decreased mitochondrial activity and cell viability. The potential of bisphenols to reduce mitochondrial mass and function differed in the following order: BPAF > BPA > BPF > BPS. Flow cytometry revealed that exposure to bisphenols significantly increased mitochondrial ROS levels and increased mitochondrial Ca2+ levels. Thus, bisphenols exposure causes mitochondrial stress in KGN cells. At the same time, bisphenols exposure significantly induced apoptosis. These results thus emphasize the toxicity of these bisphenols to cells. Our study suggests the action mechanism of BPA and its analogues in damage caused to ovarian granulosa cells. Additionally, these novel analogues may be regrettable substitutes, and the biological effects and potential risks of BPA alternatives must be evaluated.

Hyperandrogenic environment regulates the function of ovarian granulosa cells by modulating macrophage polarization in PCOS.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder characterized by oligo-anovulation, hyperandrogenism, and polycystic ovaries, with hyperandrogenism being the most prominent feature of PCOS patients. However, whether excessive androgens also exist in the ovarian microenvironment of patients with PCOS, and their modulatory role on ovarian immune homeostasis and ovarian function, is not clear. METHODS: Follicular fluid samples from patients participating in their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment were collected. Androgen concentration of follicular fluid was assayed by chemiluminescence, and the macrophage M1:M2 ratio was detected by flow cytometry. In an in vitro model, we examined the regulatory effects of different concentrations of androgen on macrophage differentiation and glucose metabolism levels using qRT-PCR, Simple Western and multi-factor flow cytometry assay. In a co-culture model, we assessed the effect of a hyperandrogenic environment in the presence or absence of macrophages on the function of granulosa cells using qRT-PCR, Simple Western, EdU assay, cell cycle assay, and multi-factor flow cytometry assay. RESULTS: The results showed that a significantly higher androgen level and M1:M2 ratio in the follicular fluid of PCOS patients with hyperandrogenism. The hyperandrogenic environment promoted the expression of pro-inflammatory and glycolysis-related molecules and inhibited the expression of anti-inflammatory and oxidative phosphorylation-related molecules in macrophages. In the presence of macrophages, a hyperandrogenic environment significantly downregulated the function of granulosa cells. CONCLUSION: There is a hyperandrogenic microenvironment in the ovary of PCOS patients with hyperandrogenism. Hyperandrogenic microenvironment can promote the activation of ovarian macrophages to M1, which may be associated with the reprogramming of macrophage glucose metabolism. The increased secretion of pro-inflammatory cytokines by macrophages in the hyperandrogenic microenvironment would impair the normal function of granulosa cells and interfere with normal ovarian follicle growth and development.

Ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation mediates female reproductive dysfunction induced by cold exposure.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to ß3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

Ti3C2 nanosheet-induced autophagy derails ovarian functions.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have gained significant attention in various biomedical applications. Although previous studies have described the accumulation and associated damage of Ti3C2 nanosheets in the testes and placenta. However, it is currently unclear whether Ti3C2 nanosheets can be translocated to the ovaries and cause ovarian damage, thereby impairing ovarian functions. RESULTS: We established a mouse model with different doses (1.25, 2.5, and 5 mg/kg bw/d) of Ti3C2 nanosheets injected intravenously for three days. We demonstrated that Ti3C2 nanosheets can enter the ovaries and were internalized by granulosa cells, leading to a decrease in the number of primary, secondary and antral follicles. Furthermore, the decrease in follicles is closely associated with higher levels of FSH and LH, as well as increased level of E2 and P4, and decreased level of T in mouse ovary. In further studies, we found that exposure toTi3C2 nanosheets increased the levels of Beclin1, ATG5, and the ratio of LC3II/Ι, leading to autophagy activation. Additionally, the level of P62 increased, resulting in autophagic flux blockade. Ti3C2 nanosheets can activate autophagy through the PI3K/AKT/mTOR signaling pathway, with oxidative stress playing an important role in this process. Therefore, we chose the ovarian granulosa cell line (KGN cells) for in vitro validation of the impact of autophagy on the hormone secretion capability. The inhibition of autophagy initiation by 3-Methyladenine (3-MA) promoted smooth autophagic flow, thereby partially reduced the secretion of estradiol and progesterone by KGN cells; Whereas blocking autophagic flux by Rapamycin (RAPA) further exacerbated the secretion of estradiol and progesterone in cells. CONCLUSION: Ti3C2 nanosheet-induced increased secretion of hormones in the ovary is mediated through the activation of autophagy and impairment of autophagic flux, which disrupts normal follicular development. These results imply that autophagy dysfunction may be one of the underlying mechanisms of Ti3C2-induced damage to ovarian granulosa cells. Our findings further reveal the mechanism of female reproductive toxicity induced by Ti3C2 nanosheets.

P62 promotes FSH-induced antral follicle formation by directing degradation of ubiquitinated WT1.

In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitin‒proteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.

Cyclophosphamide induces ovarian granulosa cell ferroptosis via a mechanism associated with HO-1 and ROS-mediated mitochondrial dysfunction.

Abnormal granulosa cell (GC) death contributes to cyclophosphamide (CTX) induced primary ovarian insufficiency (POI). To investigate the contribution of GCs to POI, gene profiles of GCs exposed to CTX were assessed using RNA-Seq and bioinformatics analysis. The results showed the differentially expressed genes (DEGs) were enriched in the ferroptosis-related pathway, which is correlated with upregulated heme oxygenase 1 (HO-1) and downregulated glutathione peroxidase-4 (GPX4). Using CTX-induced cell culture (COV434 and KGN cells), the levels of iron, reactive oxygen species (ROS), lipid peroxide, mitochondrial superoxide, mitochondrial morphology and mitochondrial membrane potential (MMP) were detected by DCFDA, MitoSOX, C11-BODIPY, MitoTracker, Nonylacridine Orange (NAO), JC-1 and transmission electron microscopy respectively. The results showed iron overload and disrupted ROS, including cytoROS, mtROS and lipROS homeostasis, were associated with upregulation of HO-1 and could induce ferroptosis via mitochondrial dysfunction in CTX-induced GCs. Moreover, HO-1 inhibition could suppress ferroptosis induced GPX4 depletion. This implies a role for ROS in CTX-induced ferroptosis and highlights the effect of HO-1 modulators in improving CTX-induced ovarian damage, which may provide a theoretical basis for preventing or restoring GC and ovarian function in patients with POI.

Cholecystokinin-mediated effect of insulin pathway on the steroidogenic activity of follicular granulosa cells in Camelus bactrianus: In vitro study.

The current study aimed to explore the molecular mechanism by which the cholecystokinin (CCK)-mediated CCKAR and CCKBR, as well as the molecular mechanisms of CCK-mediated insulin signalling pathway, regulate oestrogen in the granulosa cells. Also, the expression of CCK in ovaries, uterus, hypothalamus and pituitary gland was investigated in Camelus bactrianus. Ovaries, uterus, hypothalamus and pituitary gland were collected from six, three before ovulation (control) and three after ovulation, slaughtered Camelus bactrianus. Ovulation was induced by IM injection of seminal plasma before slaughtering in the ovulated group. The results showed that there were differences in the transcription and protein levels of CCK in various tissues before and after ovulation (p < .05, p < .01). After transfection with p-IRES2-EGFP-CCK, the mRNA and protein levels of CCK, CCKAR, CCKBR and ER in follicular granulosa cells were significantly upregulated (p < .05, p < .01), and the content of E2 was significantly upregulated (p < .01); On the contrary, after transfection with si-CCK, the mRNA and protein levels of CCK, CCKAR, CCKBR and ER in follicular granulosa cells were significantly downregulated (p < .05, p < .01), and the content of E2 was significantly downregulated (p < .01). Regulating CCK can affect the mRNA levels of INS, INSR, IGF and IGF-R. In summary, regulating the expression level of CCK can activate insulin-related signalling pathways by CCKR, thereby regulating the steroidogenic activity of granulosa cells.

Wine- and stir-frying processing of Cuscutae Semen enhance its ability to alleviate oxidative stress and apoptosis via the Keap 1-Nrf2/HO-1 and PI3K/AKT pathways in H2O2-challenged KGN human granulosa cell line.

BACKGROUND: Cuscutae Semen (CS) has been prescribed in traditional Chinese medicine (TCM) for millennia as an aging inhibitor, an anti-inflammatory agent, a pain reliever, and an aphrodisiac. Its three main forms include crude Cuscutae Semen (CCS), wine-processed CS (WCS), and stir-frying-processed CS (SFCS). Premature ovarian insufficiency (POI) is a globally occurring medical condition. The present work sought a highly efficacious multi-target therapeutic approach against POI with minimal side effects. Finally, it analyzed the relative differences among CCS, WCS and SFCS in terms of their therapeutic efficacy and modes of action against H2O2-challenged KGN human granulosa cell line. METHODS: In this study, ultrahigh-performance liquid chromatography (UPLC)-Q-ExactiveTM Orbitrap-mass spectrometry (MS), oxidative stress indices, reactive oxygen species (ROS), Mitochondrial membrane potential (MMP), real-time PCR, Western blotting, and molecular docking were used to investigate the protective effect of CCS, WCS and SFCS on KGN cells oxidative stress and apoptosis mechanisms. RESULTS: The results confirmed that pretreatment with CCS, WCS and SFCS reduced H2O2-induced oxidative damage, accompanied by declining ROS levels and malondialdehyde (MDA) accumulation in the KGN cells. CCS, WCS and SFCS upregulated the expression of antioxidative levels (GSH, GSH/GSSG ratio, SOD, T-AOC),mitochondrial membrane potential (MMP) and the relative mRNA(Nrf2, Keap1, NQO-1, HO-1, SOD-1, CAT). They inhibited apoptosis by upregulating Bcl-2, downregulating Bax, cleaved caspase-9, and cleaved caspase-3, and lowering the Bax/Bcl-2 ratio. They also exerted antioxidant efficacy by partially activating the PI3K/Akt and Keap1-Nrf2/HO-1 signaling pathways. CONCLUSIONS: The results of the present work demonstrated the inhibitory efficacy of CCS, WCS and SFCS against H2O2-induced oxidative stress and apoptosis in KGN cells and showed that the associated mechanisms included Keap1-Nrf2/HO-1 activation, P-PI3K upregulation, and P-Akt-mediated PI3K-Akt pathway induction.

Synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian granulosa cells.

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/ß-catenin signaling pathway, and ß-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1, levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1-silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

Leptin signalling regulates transcriptional differences in granulosa cells from genetically obese mice but not the activation of NLRP3 inflammasome.

Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16 weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity.

LncRNA SNHG12 promotes cell proliferation and inhibits apoptosis of granulosa cells in polycystic ovarian syndrome by sponging miR-129 and miR-125b.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is the most common endocrine disease in women of childbearing age which is often associated with abnormal proliferation or apoptosis of granulosa cells (GCs). Studies proved that long non-coding RNA SNHG12 (lncRNA SNHG12) is significantly increased in ovarian cancer and cervical cancer patients and cells. The inhibition of lncRNA SNHG12 restrains the proliferation, migration, and invasion in tumor cells. OBJECTIVE: This study explores the role of lncRNA SNHG12 in the apoptosis of GCs in PCOS and the underlying regulated mechanism. METHODS: In this study, the injection of dehydroepiandrosterone (DHEA) successfully induced the PCOS model in SD rats. The human granulosa-like tumor cell line KGN was incubated with insulin to assess the effects of lncRNA SNHG12 on GC proliferation and apoptosis. RESULTS: Overexpression of lncRNA SNHG12 influenced the body weight, ovary weight, gonadal hormone, and pathological changes, restrained the expressions of microRNA (miR)-129 and miR-125b, while downregulation of lncRNA SNHG12 exerted the opposite effects in PCOS rats. After silencing lncRNA SNHG12 in cells, the cell viability and proliferation were lessened whereas apoptosis of cells was increased. A loss-of-functions test was implemented by co-transfecting miR-129 and miR-125b inhibitors into lncRNA SNHG12-knocking down cells to analyze the effects on cell viability and apoptosis. Next, the existence of binding sites of SNHG12 and miR-129/miR-125b was proved based on the pull-down assay. CONCLUSION: lncRNA SNHG12 might be a potential regulatory factor for the development of PCOS by sponging miR-129 and miR-125b in GCs.

Genistein effect in cultured ovine ovarian granulosa cells.

Genistein, an isoflavone has the potential to mimic, augment, or dysregulate the steroid hormone production pathways. We hypothesized that genistein affects the granulosa cell (GCs) functions through a series of biochemical, molecular, and genomic cascades. The present study was conducted to evaluate the impact of genistein exposure on GCs viability, apoptosis, and steroidogenesis. The present study involved 3/5 days of exposure to genistein on GCs collected from abattoir-derived ovine ovaries at doses of 0, 1, 10, 25, 50, and 100 µM. The harvested GCs were used for growth, cytotoxicity, and gene expression studies related to apoptosis, growth, and steroidogenesis. We observed that genistein had both stimulatory at 10 and 25 µM levels as well as inhibitory effects at 50 and 100 µM levels on the growth and proliferation of GCs. Genistein significantly decreased the levels of 17ß-estradiol at higher exposure (50 and 100 µM), whereas the progesterone level increased significantly as the genistein exposure increased. Additionally, genistein could also alter the mRNA expression of the steroidogenic receptor, enzymes, proteins, and growth-related genes suggesting that genistein could potentially alter the steroidogenic pathways. We conclude that genistein can interfere with cell survival and steroidogenesis by exhibiting a dose-dependent biphasic response on the viability, growth-related parameters, and the synthesis of 17ß-estradiol in the cultured GCs.

Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

EPAS1 expression contributes to maintenance of the primordial follicle pool in the mouse ovary.

Oxygen availability can have profound effects on cell fate decisions and survival, in part by regulating expression of hypoxia-inducible factors (HIFs). In the ovary, HIF expression has been characterised in granulosa cells, however, any requirement in oocytes remains relatively undefined. Here we developed a Hif2a/Epas1 germline-specific knockout mouse line in which females were fertile, however produced 40% fewer pups than controls. No defects in follicle development were detected, and quality of MII oocytes was normal, as per assessments of viability, intracellular reactive oxygen species, and spindle parameters. However, a significant diminishment of the primordial follicle pool was evident in cKO females that was attributed to accelerated follicle loss from postnatal day 6 onwards, potentially via disruption of the autophagy pathway. These data demonstrate the importance of HIF signalling in oocytes, particularly at the primordial follicle stage, and lend to the importance of controlling oxygen tension in the development of in vitro growth and maturation approaches for assisted reproduction.

GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Methyl 3,4-dihydroxybenzoate alleviates oxidative damage in granulosa cells by activating Nrf2 antioxidant pathway.

Oxidative damage induced granulosa cells (GCs) apoptosis was considered as a significant cause of compromised follicle quality, antioxidants therapy has emerged as a potential method for improving endometriosis pregnancy outcomes. Here, we found that GCs from endometriosis patients show increased oxidative stress level. Methyl 3,4-dihydroxybenzoate (MDHB), a small molecule compound that is extracted from natural plants, reversed tert-butyl hydroperoxide (TBHP) induced GCs oxidative damage. Therefore, the aim of this study was to assess the protective effect of MDHB for GCs and its potential mechanisms. TUNEL staining and immunoblotting of cleaved caspase-3/7/9 showed MDHB attenuated TBHP induced GCs apoptosis. Mechanistically, MDHB treatment decreased cellular and mitochondria ROS production, improved the mitochondrial function by rescuing the mitochondrial membrane potential (MMP) and ATP production. Meanwhile, MDHB protein upregulated the expression of vital antioxidant transcriptional factor Nrf2 and antioxidant enzymes SOD1, NQO1 and GCLC to inhibited oxidative stress state, further beneficial to oocytes and embryos quality. Therefore, MDHB may represent a potential drug candidate in protecting granulosa cells in endometriosis, which can improve pregnancy outcomes for endometriosis-associated infertility.

Saturated fatty acids inhibit unsaturated fatty acid induced glucose uptake involving GLUT10 and aerobic glycolysis in bovine granulosa cells.

Fatty acids have been shown to modulate glucose metabolism in vitro and in vivo. However, there is still a need for substantial evidence and mechanistic understanding in many cell types whether both saturated and unsaturated fatty acids (SFAs and UFAs) pose a similar effect and, if not, what determines the net effect of fatty acid mixes on glucose metabolism. In the present study, we asked these questions by treating granulosa cells (GCs) with the most abundant non-esterified fatty acid species in bovine follicular fluid. Results revealed that oleic and alpha-linolenic acids (UFAs) significantly increased glucose consumption compared to palmitic and stearic acids (SFAs). A significant increase in lactate production, extracellular acidification rate, and decreased mitochondrial activity indicate glucose channeling through aerobic glycolysis in UFA treated GCs. We show that insulin independent glucose transporter GLUT10 is essential for UFA driven glucose consumption, and the induction of AKT and ERK signaling pathways necessary for GLUT10 expression. To mimic the physiological conditions, we co-treated GCs with mixes of SFAs and UFAs. Interestingly, co-treatments abolished the UFA induced glucose uptake and metabolism by inhibiting AKT and ERK phosphorylation and GLUT10 expression. These data suggest that the net effect of fatty acid induced glucose uptake in GCs is determined by SFAs under physiological conditions.

Spatiotemporal transcriptomic changes of human ovarian aging and the regulatory role of FOXP1.

Limited understanding exists regarding how aging impacts the cellular and molecular aspects of the human ovary. This study combines single-cell RNA sequencing and spatial transcriptomics to systematically characterize human ovarian aging. Spatiotemporal molecular signatures of the eight types of ovarian cells during aging are observed. An analysis of age-associated changes in gene expression reveals that DNA damage response may be a key biological pathway in oocyte aging. Three granulosa cells subtypes and five theca and stromal cells subtypes, as well as their spatiotemporal transcriptomics changes during aging, are identified. FOXP1 emerges as a regulator of ovarian aging, declining with age and inhibiting CDKN1A transcription. Silencing FOXP1 results in premature ovarian insufficiency in mice. These findings offer a comprehensive understanding of spatiotemporal variability in human ovarian aging, aiding the prioritization of potential diagnostic biomarkers and therapeutic strategies.