Primary Granulosa Cells Research Articles

The proteome and metabolome changes distinguish the effect of dietary energy levels on the development of ovary in chicken during sexual maturity

To deeply understanding the impact of peripheral energy level on the development of ovaries during the sexual maturation of chicken, in this study, the ovaries and serum of sexually mature and immature chickens at the same age from different energy level groups were collected, and the proteome and metabolome were detected. The results of ovarian and serum metabolomics revealed that dietary energy levels affected the energy metabolism and fatty acid oxidation of ovary in chicken, including the up-regulated expression of dihydroacetone phosphate and α-linolenic acid in high energy level groups. The results of proteomics showed that peripheral energy levels affected the catecholamine biosynthesis and metabolism in ovary before sexual maturation. The integrating analysis revealed that increased energy flux may influence ovarian development by regulating cholesterol reserves and steroid hormone synthesis in the ovaries. In vitro, the cultivation of chicken primary granulosa cells showed that sterol carrier protein 2 played a role in fatty acid synthesis and metabolism but did not significantly affect progesterone synthesis. Overall, dietary energy levels may be involved in the development of the ovaries during sexual maturation by influencing energy metabolism, biosynthesis of unsaturated fatty acids and steroid hormone within the ovaries.

Overuse of analgesics can affect the fertility biomarker Anti-Müllerian Hormone in females. A translational study.

Medication overuse headache is a prevalent secondary headache due to the overuse of analgesics, mainly over-the-counter analgesics. Over-the-counter analgesics have been associated with disrupted male endocrinology, while the effects on female endocrinology remain nearly unknown. The aim was to understand the effect of long-term analgesic exposure in females with medication overuse headache on Anti-Müllerian hormone, a surrogate measure of female fertility. Using a translational approach, an observational prospective clinical study was conducted to determine the effect of withdrawal therapy in females with medication overuse headache on Anti-Müllerian hormone levels, in combination with pre-clinical investigation of primary granulosa cells to understand the effects of analgesics on granulosa cell function. We included 21 females (mean-age 30.0 years; SD (7.3)) for Anti-Müllerian hormone -measurement. Anti-Müllerian Hormone increased by 21% from baseline (mean 20.1 pmol/L; SD (8.7)) after withdrawal of analgesics ((mean 24.3 pmol/L; SD (12.0)); p = 0.0023). Exposing primary granulosa cells to analgesics (acetaminophen (100 and 200 µM, n = 9-10) and ibuprofen (150 and 200 µM, n = 12-13)) did not reduce Anti-Müllerian hormone levels. In contrast, de novo DNA synthesis in GCs (n = 6) exposed to acetaminophen was reduced by 78% (p = 0.0036) compared to controls, suggesting that cellular proliferation was restricted. We found that frequent use of over-the-counter analgesics was associated with repressed Anti-Müllerian Hormone levels, likely through disruption of granulosa cell proliferation. Further research is crucial to investigate a potential effect of analgesics on adult female reproductive endocrinology.Trial registration: ClinicalTrials.gov Identifier NCT04090333.

Regulation of Bone Morphogenetic Protein Receptor Type II Expression by FMR1/Fragile X Mental Retardation Protein in Human Granulosa Cells in the Context of Poor Ovarian Response.

Fragile X mental retardation protein (FMRP) is a translational repressor encoded by FMR1. It targets bone morphogenetic protein receptor type II (BMPR2), which regulates granulosa cell (GC) function and follicle development. However, whether this interaction affects folliculogenesis remains unclear. Therefore, this study investigated the potential effect of FMRP-BMPR2 dysregulation in ovarian reserves and infertility. COV434 cells and patient-derived GCs were used to evaluate FMRP and BMPR2 expression. Similarly, FMR1, BMPR2, LIMK1, and SMAD expression were evaluated in GCs with normal (NOR) and poor (POR) ovarian responses. FMRP and BMPR2 were expressed in both cell types. They were co-localized to the nuclear membrane of COV434 cells and cytoplasm of primary GCs. FMR1 silencing increased the mRNA and protein levels of BMPR2. However, the mRNA levels of FMR1 and BMPR2 were significantly lower in the POR group. FMR1 and BMPR2 levels were strongly positively correlated in the NOR group but weakly correlated in the POR group. Additionally, SMAD9 expression was significantly reduced in the POR group. This study highlights the crucial role of FMR1/FMRP in the regulation of BMPR2 expression and its impact on ovarian function. These findings indicate that the disruption of FMRP-BMPR2 interactions may cause poor ovarian responses and infertility.

Mechanisms and effects of AdipoRon, an adiponectin receptor agonist, on ovarian granulosa cells-a systematic review.

Granulosa cells, crucial components of ovarian follicles, play a fundamental role in follicle development, hormone production, and overall reproductive health. These cells are integral to steroidogenesis, including the synthesis and secretion of key hormones such as estrogen and progesterone. Dysregulation of granulosa cells can lead to reproductive disorders, including polycystic ovary syndrome and infertility. This systematic review provides a comprehensive evaluation of AdipoRon, a synthetic agonist of adiponectin receptors AdipoR1 and AdipoR2, and its effects on ovarian function, with a particular focus on granulosa cells. Due to the absence of clinical trials, the review centers on preclinical studies to explore AdipoRon's potential therapeutic benefits and to suggest future research directions. A detailed literature search across databases such as PubMed, Scopus, Web of Science, Embase, and Google Scholar was conducted using terms related to AdipoRon and ovarian function. The review encompasses four preclinical studies involving various models: primary granulosa cells from rats, laying hens' granulosa cells, human luteinized granulosa cells, and chicken ovary follicles. Findings indicate that AdipoRon enhances glucose absorption in rat granulosa cells by stimulating glucose transporter 1 expression, modulates steroid hormone secretion in laying hens' granulosa cells, and affects cell proliferation and steroidogenesis in human luteinized granulosa cells. Additionally, AdipoRon, in conjunction with recombinant chicken adiponectin, influences ovarian follicular cell proliferation and steroidogenesis in chicken ovary follicles. This review highlights the need for further investigation into AdipoRon's long-term effects and its potential applications in reproductive health and therapy.

CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1

Some patients with polycystic ovarian syndrome (PCOS) suffered from metabolic syndrome (MetS) including dyslipidemia, hyperinsulinism, but the underlying mechanism is unclear. Although C-terminal Binding Protein 1 (CTBP1) is a transcriptional co-repressor frequently involved in hormone secretion disorders and MetS-associated diseases, the role of CTBP1 in PCOS is rarely reported. In the present study, we found that CTBP1 expression was significantly elevated in primary granulosa cells (pGCs) derived from the PCOS with MetS patients and was positively associated with serum triglyceride, but negatively correlated with serum estradiol (E2) or high-density lipoprotein. Mechanistic study suggested that CTBP1 physically bound to the promoter II of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) to inhibit the aromatase gene transcription and expression, resulting in the reduced E2 synthesis. Moreover, CTBP1 interacted with the phosphorylated SREBP1a at S396 in nuclei, leading to the FBXW7-dependent protein degradation, resulting in the reduced lipid droplets formation in pGCs. Therefore, we conclude that CTBP1 in GCs dysregulates the synthesis of steroid hormones and lipids through suppression of aromatase expression and promotion of SREBP1a protein degradation in PCOS patients, which may offer some fresh insights into the potential pathological mechanism for this tough disease.

Effect of the LIF gene on the cell cycle and apoptosis of ovarian granulosa cells in white Muscovy ducks

Leukaemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) family, is a multifunctional cytokine. The maturation-to-ovulation process of poultry follicles is determined by granulosa cell proliferation and differentiation. Granulosa cell apoptosis and degeneration lead to follicular atresia, which reduces the number of normally developing follicles and leads to a decrease in the poultry egg production rate, thus affecting the large-scale development of poultry breeding. In this study, the LIF gene overexpression vector pCDH-CMV-LIF and a siRNA that inhibits LIF gene expression were transfected into primary granulosa cells from white Muscovy duck ovaries for functional study. Compared with that in the control group, LIF gene expression was confirmed to be significantly decreased or increased in the transfection groups (P < 0.01). After LIF overexpression, the expression of the cell cycle-related genes CCND1, CDK-1 and PCNA was decreased (P < 0.05); apoptosis was promoted; the proapoptotic genes Bax and caspase-3 were significantly upregulated (P < 0.01); and the antiapoptotic gene Bcl-2 was significantly downregulated (P < 0.01). After LIF interference, the expression of the cell cycle-related genes CCND1, CCNE1, CDK-1 and PCNA and the antiapoptotic gene Bcl-2 significantly increased (P < 0.01), whereas the expression of the proapoptotic genes Bax, caspase-3 and caspase-9 significantly decreased (P < 0.01). In summary, the LIF gene is involved in regulating the biological function of ovarian granulosa cells in white Muscovy ducks. LIF gene expression promotes granulosa cell apoptosis and inhibits cell cycle progression. These experimental results provide insights into the follicular development mechanism of white Muscovy ducks.

Quercetin ameliorates oxidative stress-induced apoptosis of granulosa cells in dairy cow follicular cysts by activating autophagy via the SIRT1/ROS/AMPK signaling pathway

BackgroundFollicular cysts contribute significantly to reproductive loss in high-yield dairy cows. This results from the death of follicular granulosa cells (GCs) caused by oxidative stress. Quercetin is known to have significant antioxidant and anti-apoptotic effects. However, the effect of quercetin on follicular cysts has yet been elucidated. Therefore, this study aimed to explore the anti-oxidant and anti-apoptosis effects and potential molecular mechanisms of quercetin in H2O2-induced primary cow GCs and 3-nitropropionic acid (3-NPA)-induced mouse model of oxidative stress and thus treat ovarian cysts in dairy cows.ResultsIn this study, compared with estrus cows, cows with follicular cysts showed heightened levels of oxidative stress and increased follicular cell apoptosis, while autophagy levels were reduced. A model of oxidative stress was induced in vitro by H2O2 and showed significant increases in apoptosis together with reduced autophagy. These effects were significantly ameliorated by quercetin. Effects similar to those of quercetin were observed after treatment of cells with the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC). Further investigations using chloroquine (autophagy inhibitor), rapamycin (autophagy activator), selisistat (SIRT1 inhibitor), and compound C (AMPK inhibitor) showed that chloroquine counteracted the effects of quercetin on oxidative stress-induced apoptosis, while rapamycin had the same effect as quercetin. In addition, the SIRT1/AMPK pathway inhibitors antagonized quercetin-mediated mitigation of the effects of oxidative stress on increased apoptosis and reduced autophagy. Consistent with the results in vitro, in mouse ovarian oxidative stress model induced by 3-NPA, quercetin activated autophagy through the SIRT1/AMPK signaling pathway, while alleviating oxidative stress damage and inhibiting apoptosis in mouse ovaries.ConclusionsThese findings indicate that quercetin can inhibit apoptosis in GCs and restore ovarian function by activating autophagy through the SIRT1/ROS/AMPK signaling pathway, suggesting a new direction for the treatment of ovarian follicular cysts in high-yield dairy cows.

Vitamin E alleviates glyphosate-based herbicide-induced progesterone secretion inhibition and oxidative stress increase in chicken primary granulosa cells

Glyphosate-based herbicides (GBH) are the most extensively used herbicides worldwide. Despite a presumed nondangerousness for animals, several studies reported negative effects after a GBH exposure in several animal models including birds, notably on reproductive functions. Several studies concerning the advantages of Vitamin E (VE) for antioxidant activity but also growth and reproduction have been reported in birds. However, it remains unclear whether VE could alleviate the negative effect of GBHs on chicken ovarian cells. Here we exposed chicken primary granulosa cells (GCs) from F1 and F3/4 follicles to growing doses of GBH (0.036, 0.36, 3.6, and 36 gly eq/L), with or without VE supplementation (1 mg/L) and investigated cell viability, proliferation, oxidative stress and steroidogenesis. GBH exposure did not affect F1 and F3 GCs viability but it increased cell proliferation only in F1 GCs and this effect was not altered by VE. In both F1 and F3/4 GCs, GBH exposure increased total oxidant status (TOS), reduced total antioxidant status (TAS) and consequently increased index of oxidative stress (OSI) in dose dependent manner. This latter effect for GBH 36 mg eq gly/L was totally abolished in response to VE. In both F1 and F3/4 GCs, GBH exposure reduced progesterone secretion in a dose dependent manner and this effect with GBH 0.36 and 1.8 mg eq glyphosate/L was alleviated by VE. However, we did not observe any effect of GBH and VE on the gene expression of several components of the steroidogenesis process. Taken together, these results show that GBH may have endocrine disruptor effects, and that these effects might be alleviated by antioxidant VE supplementation.

Unique features of KGN granulosa-like tumour cells in the regulation of steroidogenic and antioxidant genes.

The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 μM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.

P-350 Recombinant human anti-Müllerian hormone reduces doxorubicin-induced reproductive aging by regulating Id3 expression and promoting DNA repair

Abstract Study question What is the mechanism of ovarian damage after doxorubicin (Dox) treatment and how recombinant human anti-Müllerian hormone (rhAMH) suppresses ovarian aging and prevents chemotherapy-induced damage? Summary answer Administration of rhAMH attenuates Dox-induced follicle depletion and protects the ovarian reserve in mice, primarily by reducing DNA damage via Id3 upregulation. What is known already Treatment with rhAMH results in reversible ovarian quiescence that can reduce primordial follicle depletion induced by Dox when it is used as an adjuvant. However, the exact mechanism of Dox-induced ovarian toxicity and the mechanisms by which rhAMH rescue this damage were unknown. Study design, size, duration For the in vivo experiments: 25-day-old mice were randomized into four treatment groups (Control, Dox, Dox+rhAMH, and rhAMH) for three different timepoints (n = 5 per group, total n = 60). Ovaries were removed for analysis at 4, 24 hours and and 1 week after treatment. For the in vitro studies: 21-day-old mice (n = 30) were treated with subcutaneous injection of diethylstilbestrol (DES) to enhance preantral follicle growth, and primary granulosa cells were collected by mechanical dissociation. Participants/materials, setting, methods Immunohistochemistry with DDX4 and counter staining with hematoxylin was used for differential follicle counts. DNA damage and apoptosis were measured by quantification of phosphorylated form of histone 2AX (γH2AX) and by TUNEL assay. MTS assay, clonogenic assay and siRNA Id3 RNA interference knockdown experiment were carried out to reveal the effects of doxorubicin and rhAMH on primary granulosa cell survival. Main results and the role of chance Treatment with Dox alone caused significant loss of PMF reserve (59% reduction, 5.25 versus 12.76 in control, P&amp;lt;0.0001) and this was significantly attenuated by coadministration with rhAMH (33% rescue, 9.47 P&amp;lt;0.001). The DNA damage marker γH2AX was elevated in primordial follicle (PMF) and primary follicle (PF) oocytes from Dox-treated ovaries 4 h after treatment, compared with controls (P&amp;lt;0.0001) and was significantly reduced (95% reduction) in rhAMH cotreated ovaries (P&amp;lt;0.001). Oocytes of secondary follicle (SF) and antral follicle (AF) were negative for γH2AX while granulosa cells (GCs) showed increased positivity for γH2AX after Dox treatment with significant reduction after rhAMH cotreatment. (24% reduction, P&amp;lt;0.001). Oocytes and somatic cells of PMF and PF rarely showed positive TUNEL staining at any of the timepoints evaluated.In vitro study showed reduced double stranded breaks (DSBs) in Dox-rhAMH cotreated GC compared to Dox alone indicated by number and signal intensity of γH2AX positive cells. Finally, while rhAMH treatment increased Id3 expression in early stage oocyte and granulosa cells, siRNA Id3 knock-down in granulosa cells significantly reduced the protective effect of rhAMH, as seen by the increased DSBs and pyknotic morphology of cells after Dox suggesting Id3 is downstream of AMH and enhancing Dox-induced DSB DNA repair. Limitations, reasons for caution This study failed to detect fate of PMF after DNA damage. PMF distinguished without apoptotic finding and this might be because experiment was conducted in limited time point or mechanism other than apoptotic cell death are responsible for PMF loss such as shedding, phagocytosis or necrosis. Wider implications of the findings Understanding of the mechanism behind rhAMH prevention of chemotherapy-induced reproductive aging is important for reproductive health and longevity. Possibility of rhAMH as non-invasive ferto-protective agent during chemotherapy may overcome limitation of current fertility preservation method which is limited by time requirement, cost and success rate. Trial registration number ’not applicable’

The direct effect of SARS-CoV-2 virus vaccination on human ovarian granulosa cells explains menstrual irregularities

Following administration of the SARS-CoV-2 vaccine, many women worldwide reported short-term menstrual irregularities. Although menstrual bleeding, “the fifth vital sign”, is experienced by more than 300 million people on any given day worldwide, these changes were only partially studied. Irregular periods are important well beyond fertility and the discomfort they impose; they are associated with the risk of cardiovascular morbidity, chronic diseases, and premature mortality. Pre-clinical examination of the vaccine polymeric envelope indicates its accumulation in the ovaries. The somatic endocrine cells of the ovarian follicle - the granulosa cells (GCs)—participate in the strict hypothalamic-pituitary-ovarian (HPO) feedback loop that governs the menstrual cycle via endocrine and paracrine regulators, as AMH and Inhibins. We aimed to unravel the direct effect of the COVID-19 vaccine on GCs and link their post-vaccine activity to changes in menstrual patterns. Human primary GCs exposed in-vitro to the Pfizer COVID-19 vaccine BNT162b2, demonstrated no change in their viability but altered mRNA transcripts, specifically of the regulatory key factors: InhibinB was upregulated, whereas AMH was downregulated. We further examined pre- and post-vaccination blood samples from individual women and found a 2–3 folds change in the post-vaccination FSH/InhibinB protein level ratio, compared to their pre-vaccination values. This altered expression of InhibinB could significantly impact the HPO axis in vaccinated women and may ultimately influence the endometrium cyclicity, manifested clinically by the commonly reported changes in menstrual bleeding patterns.

Klotho accelerates the progression of polycystic ovary syndrome through promoting granulosa cell apoptosis and inflammation.

The morbidity of polycystic ovary syndrome (PCOS) is in highly increasing rate nowadays. PCOS not only affects the fertility in women, but also threatens the health of whole life. Hence, to find the prognostic risk factors is of great value. However, the effective predictors in clinical practice of PCOS are still in blackness. In this study, we found Klotho was increased in FF (Follicular Fluid) and primary luteinized granulosa cells (GCs) from PCOS patients with hyperandrogenism. Furthermore, we found follicular Klotho was negatively correlated with numbers of mature oocytes, and positively correlated with serum testosterone, LH, and LH/FSH levels menstrual cycle and number of total antral follicles in PCOS patients. In primary luteinized GCs, the increased Klotho was accompanied with upregulation of cell apoptosis and inflammation-related genes. In ovaries of PCOS mice and cultured human KGN cell line, Klotho was up-regulated and accompanied by apoptosis, inflammation and mitochondrial dysfunction. Therefore, our findings suggest new mechanisms for granulosa cell injury and revealed to target inhibit Klotho maybe a new therapeutic strategy for treatment of PCOS.

Regulation and Role of Adiponectin Secretion in Rat Ovarian Granulosa Cells.

Adiponectin is an important adipokine involved in glucose and lipid metabolism, but its secretion and potential role in regulating glucose utilization during ovarian development remains unclear. This study aims to investigate the mechanism and effects of follicle-stimulating hormones (FSHs) on adiponectin secretion and its following impact on glucose transport in the granulosa cells of rat ovaries. A range of experimental techniques were utilized to test our research, including immunoblotting, immunohistochemistry, immunofluorescence, ELISA, histological staining, real-time quantitative PCR, and transcriptome analysis. The immunohistochemistry results indicated that adiponectin was primarily located in the granulosa cells of rat ovaries. In primary granulosa cells cultured in vitro, both Western blot and immunofluorescence assays demonstrated that FSH significantly induced adiponectin secretion within 2 h of incubation, primarily via the PKA signaling pathway rather than the PI3K/AKT pathway. Concurrently, the addition of the AdipoR1/AdipoR2 dual agonist AdipoRon to the culture medium significantly stimulated the protein expression of GLUT1 in rat granulosa cells, resulting in enhanced glucose absorption. Consistent with these in vitro findings, rats injected with eCG (which shares structural and functional similarities with FSH) exhibited significantly increased adiponectin levels in both the ovaries and blood. Moreover, there was a notable elevation in mRNA and protein levels of AdipoRs and GLUTs following eCG administration. Transcriptomic analysis further revealed a positive correlation between the expression of the intraovarian adiponectin system and glucose transporter. The present study represents a novel investigation, demonstrating that FSH stimulates adiponectin secretion in ovarian granulosa cells through the PKA signaling pathway. This mechanism potentially influences glucose transport (GLUT1) and utilization within the ovaries.

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 β3-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.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here, we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, results in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of polycystic ovary syndrome (PCOS) induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

PPAR-α inhibits DHEA-induced ferroptosis in granulosa cells through upregulation of FADS2

BackgroundPolycystic ovary syndrome (PCOS), a prevalent endocrine disorder among women of reproductive age, is characterized by disturbances in hormone levels and ovarian dysfunction. Ferroptosis, a unique form of regulated cell death characterized by iron-dependent lipid peroxidation. Emerging evidence indicates that ferroptosis may have a significant role in the pathogenesis of PCOS, highlighting the importance of studying this mechanism to better understand the disorder and potentially develop novel therapeutic interventions. MethodsTo create an in vivo PCOS model, mice were injected with dehydroepiandrosterone (DHEA) and the success of the model was confirmed through further assessments. Ferroptosis levels were evaluated through detecting ferroptosis-related indicators. Ferroptosis-related genes were found through bioinformatic analysis and identified by experiments. An in vitro PCOS model was also established using DHEA treated KGN cells. The molecular binding relationship was confirmed using a chromatin immunoprecipitation (ChIP) assay. ResultsIn PCOS model, various ferroptosis-related indicators such as MDA, Fe2+, and lipid ROS showed an increase, while GSH, GPX4, and TFR1 exhibited a decrease. These findings indicate an elevated level of ferroptosis in the PCOS model. The ferroptosis-related gene FADS2 was identified and validated. FADS2 and PPAR-α were shown to be highly expressed in ovarian tissue and primary granulosa cells (GCs) of PCOS mice. Furthermore, the overexpression of both FADS2 and PPAR-α in KGN cells effectively suppressed the DHEA-induced increase in ferroptosis-related indicators (MDA, Fe2+, and lipid ROS) and the decrease in GSH, GPX4, and TFR1 levels. The ferroptosis agonist erastin reversed the suppressive effect, suggesting the involvement of ferroptosis in this process. Additionally, the FADS2 inhibitor SC26196 was found to inhibit the effect of PPAR-α on ferroptosis. Moreover, the binding of PPAR-α to the FADS2 promoter region was predicted and confirmed. This indicates the regulatory relationship between PPAR-α and FADS2 in the context of ferroptosis. ConclusionsOur study indicates that PPAR-α may have an inhibitory effect on DHEA-induced ferroptosis in GCs by enhancing the expression of FADS2. This discovery provides valuable insights into the pathophysiology and potential therapeutic targets for PCOS.

Analysis of Toxic Effects of Fluoride on Ovine Follicular Granulosa Cells Using RNA-Seq.

Fluoride is abundant in the environment and is an essential trace element in living organisms. However, prolonged excessive fluoride intake can lead to fluorosis, which poses a threat to the reproductive health of animals and humans. Although previous research has mainly focused on animal models, the impact of fluoride on ovine follicular granulosa cells (GCs) has not been comprehensively elucidated. This study employed RNA-Seq technology to elucidate the toxic effects of fluoride on ovine follicular GCs and its mechanism of action. Culturing primary ovine follicular GCs in vitro and subjecting them to fluoride treatment revealed 3218 differentially expressed genes (DEGs), with 2278 upregulated and 940 downregulated. Significantly, this study unveiled fluoride's induction of endoplasmic reticulum (ER) stress in cells, triggering a cascade involving the PERK pathway factor ATF4, leading to cell death via DDIT3/CHOP activation and the subsequent upregulation of CHAC1, ATF3, ERO1α, and TRIB3. These findings provide crucial insights into the toxicity of fluoride in ovine, offering a foundation for mitigating fluoride-related losses in the farming industry.

Indole-3-carbinol ameliorates ovarian damage in female old mice through Nrf2/HO-1 pathway activation

Ovarian aging leads to infertility and birth defects. We aimed to clarify the role of Indole-3-carbinol (I3C) in resistance to oxidative stress, apoptosis, and fibrosis in ovarian aging. I3C was administered via intraperitoneal injection for 3 weeks in young or old mice. Immunohistochemistry; Masson, Sirius red, and TUNEL staining; follicle counting; estrous cycle analysis; and Western blotting were used for validating the protective effect of I3C against ovarian senescence. Human granulosa-like tumor cell line and primary granulosa cells were used for in vitro assay. The results indicated that I3C inhibited ovarian fibrosis and apoptosis while increasing the number of primordial follicles. Mechanistic studies have shown that I3C promoted the nuclear translocation of nuclear factor-erythroid 2-related factor (Nrf2) and upregulated the expression of heme oxygenase 1 (HO-1). Additionally, I3C increased cell viability and decreased lactate dehydrogenase, malondialdehyde, reactive oxygen species and JC-1 levels. Furthermore, the antioxidant effect of I3C was found to be dependent on the activation of Nrf2 and HO-1, as demonstrated by the disappearance of the effect upon inhibition of Nrf2 expression. In conclusion, I3C can alleviate the ovarian damage caused by aging and may be a protective agent to delay ovarian aging.

Pigment epithelium-derived factor expression and role in follicular development

Research QuestionFolliculogenesis is the process of follicles assembly and growth. The process requires a tight dialogue between the oocyte and its supporting granulosa cells (GCs) and is mediated by endocrine and paracrine factors. Pigment epithelium-derived factor (PEDF), an important player in the female reproductive tract, is highly expressed in the GCs and in various ovarian components. Our aim was to characterize the involvement of PEDF in CGs during folliculogenesis. DesignWe examined mRNA expression of PEDF and other key factors, (Cyp19, anti-Müllerian hormone receptor (AMHR), and VEGF), in mice follicles to typify the expression of PEDF in growing follicles and in human primary GCs (hpGCs); and to follow the interplay between PEDF and other folliculogenesis leading players, follicle-stimulating hormone (FSH) and AMH. ResultsPEDF expression increases through folliculogenesis (≥9 follicles/group), though, different than other examined genes, affecting the follicular angiogenic balance. In hpGCs, FSH stimulates the upregulation of PEDF mRNA. Furthermore, a negative correlation between AMH and PEDF was observed, as AMH stimulation reduced the level of PEDF mRNA and PEDF stimulation reduced AMHR level. ConclusionsOur findings suggest that PEDF participates in folliculogenesis and places it as a pro-folliculogenesis player that interacts with the main participants in the process of follicle growth; however, the mechanism of this process is yet to be determined.

Kisspeptin stimulates oestradiol biosynthesis by upregulating steroidogenic transcripts and proliferation markers in the bubaline granulosa cells in vitro

AbstractKisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E2) and progesterone (P4) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6–10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E2 and P4 was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3β‐HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal‐regulated kinase 1/2, ERK1/2 and p‐ERK1/2) was studied in the GCs by qPCR. Significant E2 production was found in the Kp 50 and 100 nM groups (p &lt; .05), whereas P4 production was reduced in Kp 100 nM group (p &lt; .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p‐ERK1/2 was observed in the Kp‐treated GCs. It was concluded that Kp at 100 nM concentration stimulated E2 production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp‐mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.