Resveratrol ameliorates mitochondrial biogenesis and reproductive outcomes in women with polycystic ovary syndrome undergoing assisted reproduction: a randomized, triple-blind, placebo-controlled clinical trial.

BACKGROUND: This study was designed to examine the effect of resveratrol on mitochondrial biogenesis, oxidative stress (OS), and assisted reproductive technology (ART) outcomes in individuals with polycystic ovary syndrome (PCOS). METHODS: Fifty-six patients with PCOS were randomly assigned to receive 800 mg/day of resveratrol or placebo for 60 days. The primary outcome was OS in follicular fluid (FF). The secondary outcome involved assessing gene and protein expression related to mitochondrial biogenesis, mitochondrial DNA (mtDNA) copy number, and adenosine triphosphate (ATP) content in granulosa cells (GCs). ART outcomes were evaluated at the end of the trial. RESULTS: Resveratrol significantly reduced the total oxidant status (TOS) and oxidative stress index (OSI) in FF (P = 0.0142 and P = 0.0039, respectively) while increasing the total antioxidant capacity (TAC) (P < 0.0009). Resveratrol consumption also led to significant increases in the expression of critical genes involved in mitochondrial biogenesis, including peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and mitochondrial transcription factor A (TFAM) (P = 0.0032 and P = 0.0003, respectively). However, the effect on nuclear respiratory factor 1 (Nrf-1) expression was not statistically significant (P = 0.0611). Resveratrol significantly affected sirtuin1 (SIRT1) and PGC-1α protein levels (P < 0.0001 and P = 0.0036, respectively). Resveratrol treatment improved the mtDNA copy number (P < 0.0001) and ATP content in GCs (P = 0.0014). Clinically, the resveratrol group exhibited higher rates of oocyte maturity (P = 0.0012) and high-quality embryos (P = 0.0013) than did the placebo group. There were no significant differences between the groups in terms of chemical or clinical pregnancy rates (P > 0.05). CONCLUSIONS: These findings indicate that resveratrol may be a promising therapeutic agent for patients with PCOS undergoing assisted reproduction. TRIAL REGISTRATION NUMBER: http://www.irct.ir ; IRCT20221106056417N1; 2023 February 09.

The modulating effects of astaxanthin on apoptosis in women with polycystic ovarian syndrome: A randomized clinical trial.

Objective: Astaxanthin (ASX) is a lipid-soluble keto-carotenoid with several biological effects. These effects may benefit polycystic ovarian syndrome (PCOS) patients. Imbalanced apoptosis/anti-apoptosis signaling has been considered the major pathogenesis of PCOS. In a randomized clinical trial, we tested the impact of ASX on the apoptotic pathway in PCOS granulosa cells (GCs). The present study hypothesizes that ASX may improve apoptosis in PCOS patients. Materials and Methods: This trial recruited patients with confirmed PCOS. A total of 58 patients were randomly assigned to take ASX (12 mg) or placebo for 8 weeks. Aspirated follicular fluid (FF) and blood samples were taken from both groups to measure BAX and BCL2 protein expression. Following FF aspiration, GCs from both groups were obtained; Real-Time PCR and Western blotting were used to evaluate the apoptotic pathway's gene and protein expression levels in GCs.BAXBCL2. Results: In GCs analysis, ASX reduced DR5 gene and protein expression after 8 weeks compared to placebo(p<0.05). Also, Caspase8 (p>0.05) and BAX (p<0.05) gene expression declined, although the difference was not statistically significant for Caspase8. Besides,ASX treatment contributed to an elevated BCL2 gene expression in GCs(p<0.05). In FF and serum analysis, a statistically significant increase was found in BCL2 concentration in the ASX group (p<0.05). Moreover, a reduction in BAX level was confirmed in both FF and serum of the ASX group; however, this change was not significant in the serum (p>0.05). Conclusion: It seems that ASX consumption among women with PCOS improved serum and FF levels of apoptotic factors and modulated genes and protein expression of the apoptosis pathway in GCs. Nevertheless, further investigations are needed to reveal the potential role of this compound in PCOS treatment.

MTHFR as a Novel Candidate Marker for Litter Size in Rabbits.

Litter size is a significant economic trait during animal reproduction. This current study attempted to decipher whether MTHFR promotes the apoptosis of granulosa cells (GCs) and inhibits their proliferation by investigating the effects of the MTHFR gene using flow cytometry and a Cell Counting Kit-8 (CCK-8) assay. MTHFR is linked with ovarian follicle development in the reproductive performance of 104 female New Zealand rabbits. We observed that MTHFR could regulate the mRNA of follicular development-related genes (TIMP1, CITED1, FSHR, GHR, HSD17B1, and STAR) with a qRT-PCR, and we observed the protein expression of CITED1 and GHR using a western blot (WB) analysis. The dual luciferase activity assays helped identify the core promoter region of the MTHFR gene, and the polymorphism of the MTHFR promoter region was studied using Sanger sequencing. The results indicated four single nucleotide polymorphisms (SNPs) within the core promoter region, among which the g.-680C>A locus was significantly associated with both the total and alive litter sizes. Additionally, the CC genotype was associated with the largest total and alive litter sizes, compared to the CA and AA genotypes (p < 0.05). In conclusion, this study investigated the effects of MTHFR on ovarian granulosa cells and its association with selected reproductive parameters in rabbits. The results provide a theoretical foundation for the use of MTHFR as a molecular marker in rabbits.

Fat Mass and Obesity-Associated Protein Regulates Granulosa Cell Aging by Targeting Matrix Metalloproteinase-2 Gene Via an N6-Methyladenosine-YT521-B Homology Domain Family Member 2-Dependent Pathway in Aged Mice.

In this study, we aimed to investigate the molecular mechanisms of RNA N6-methyladenosine (m6A) modification and how its associated proteins affect granulosa cell aging. A granulosa cell senescence model was constructed to detect the differences in total RNA m6A modification levels and the expression of related enzymes. Changes in downstream molecular expression and the effects on the cellular senescence phenotype were explored by repeatedly knocking down and overexpressing the key genes fat mass and obesity-associated protein (FTO), YT521-B homology domain family member 2 (YTHDF2), and matrix metalloproteinase-2 (MMP2). There was an increased total RNA m6A modification and decreased expression of the demethylase FTO and target gene MMP2 in senescent granulosa cells. FTO and MMP2 knockdown promoted granulosa cell senescence, whereas FTO and MMP2 overexpression retarded it. YTHDF2 and FTO can bind to the messenger RNA of MMP2. The extracellular signal-regulated kinase (ERK) pathway, which is downstream of MMP2, retarded the process of granulosa cell senescence through ERK activators. In granulosa cells, FTO can regulate the expression of MMP2 in an m6A-YTHDF2-dependent manner, influencing the activation status of the ERK pathway and contributing to the aging process of granulosa cells.

Mitochondrial Quality Control in Ovarian Function: From Mechanisms to Therapeutic Strategies.

Mitochondrial quality control plays a critical role in cytogenetic development by regulating various cell-death pathways and modulating the release of reactive oxygen species (ROS). Dysregulated mitochondrial quality control can lead to a broad spectrum of diseases, including reproductive disorders, particularly female infertility. Ovarian insufficiency is a significant contributor to female infertility, given its high prevalence, complex pathogenesis, and profound impact on women's health. Understanding the pathogenesis of ovarian insufficiency and devising treatment strategies based on this understanding are crucial. Oocytes and granulosa cells (GCs) are the primary ovarian cell types, with GCs regulated by oocytes, fulfilling their specific energy requirements prior to ovulation. Dysregulation of mitochondrial quality control through gene knockout or external stimuli can precipitate apoptosis, inflammatory responses, or ferroptosis in both oocytes and GCs, exacerbating ovarian insufficiency. This review aimed to delineate the regulatory mechanisms of mitochondrial quality control in GCs and oocytes during ovarian development. This study highlights the adverse consequences of dysregulated mitochondrial quality control on GCs and oocyte development and proposes therapeutic interventions for ovarian insufficiency based on mitochondrial quality control. These insights provide a foundation for future clinical approaches for treating ovarian insufficiency.

Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells.

Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4-/- mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4-/- mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis.

Per2 mediated steroid hormone synthesis by regulating transcription of StAR in porcine granulosa cells.

Steroidogenesis is associated with circadian clock genes. However, the regulation of steroid hormone production in sow granulosal cells by Per2, a crucial circadian regulator, remains unexplored. In this study, we have identified the presence of Per2 in ovarian granulosa cells and have observed its circadian expression pattern. Employing siRNA to interfere with Per2 expression, our investigation revealed that Per2 knockdown notably elevated progesterone (P4) levels along with increasing the expression of StAR but interference of Per2 did not alter the rhythm of clock-related gene (Bmal1, Clock, Per1 and Cry1) in granulosa cells. Subsequent mechanistic analysis showed that Per2 formed complexes with PPARγ and interference with Per2 promoted the formation of the PPARγ:RXRα heterodimer. Importantly, we uncovered that PPARγ:RXRα heterodimer could control the expression of StAR via direct peroxisome proliferator response element (PPRE) binding to its promoter to regulate its activity, and knockdown of Per2 promoted the transcription of StAR via increasing the binding of PPARγ:RXRα ligands. Altogether, these findings indicated a noncanonical role of Per2 in controlling PPARγ:RXRα binding to regulate transcription of StAR and progesterone synthesis, thus revealing potential avenues of pharmacological and therapeutic intervention.

Therapeutic effects of curculigoside on cyclophosphamide-induced premature ovarian failure in mice.

OBJECTIVE: The main purpose of this study was to elucidate the anti-apoptotic effects of curculigoside (CUR) on ovarian granulosa cells (GCs) in a mouse model of cyclophosphamide (CTX)-induced premature ovarian failure (POF). METHOD: Intraperitoneal injection of CTX (100 mg/kg body weight) induced POF in mice. Thirty-six female mice were divided into six groups: blank group; POF model group; low-dose CUR group; medium-dose CUR group; high-dose CUR group; and estradiol benzoate group. Mice were orally administered for 28 consecutive days. Twenty-four hours after the completion of treatment, mice were weighed and euthanized, and blood was collected from the eyeball under anesthesia. The ovaries were surgically separated and weighed, and the ovarian index was calculated. Hematoxylin-eosin (HE) staining was used to observe follicular development and corpus luteum morphology in the ovaries. Serum levels of follicle stimulating hormone (FSH), anti-Müllerian hormone (AMH) and estradiol (E2) were measured. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) content and malondialdehyde (MDA) levels in ovarian tissue were determined. The GC apoptosis level was measured. Western blotting was used to detect protein expression levels of Beclin-1, LC3, P62, AKT, p-AKT, mTOR and p-mTOR in the ovaries. RESULTS: The results showed that CUR can improve body weight and ovarian index; promote follicular development and reduce follicular atresia; improve FSH, AMH and E2 levels; downregulate MDA levels and restore antioxidant enzyme activity; inhibit the autophagy level; activate the AKT/mTOR signaling pathway; and alleviate GC apoptosis. CONCLUSION: CUR improves POF by activating the AKT/mTOR signaling pathway, inhibiting autophagy and alleviating GC apoptosis.

The role of miR-10a-5p in LPS-induced inhibition of progesterone synthesis in goose granulosa cells by down-regulating CYP11A1.

The poultry ovary is a preferred target for E. coli and Salmonella infection of tissues, and lipopolysaccharide (LPS) is a critical molecule in infecting the organism and interfering with cell function, invading the ovaries through the cloaca and interfering with progesterone (P4) secretion by follicular granulosa cells (GCs), seriously affecting the health of breeding geese. miRNAs are small, non-coding RNAs with a variety of important regulatory roles. To investigate the mechanism of miR-10a-5p mediated LPS inhibition of progesterone synthesis in goose granulosa cells, Yangzhou geese at peak laying period were selected as experimental animals to verify the expression levels of genes and transcription factors related to progesterone synthesis. In this study, bioinformatic predictions identified miR-10a-5p target gene CYP11A1, and genes and transcription factors related to the sex steroid hormone secretion pathway were screened. We detected that LPS inhibited CYP11A1 expression while increasing miR-10a-5p expression in vivo. Progesterone decreased significantly in goose granulosa cells treatment with 1 µg/mL LPS for 24 h, while progesterone-related genes and regulatory factors were also suppressed. We also determined that the downregulation of miR-10a-5p led to CYP11A1 expression. Overexpression of miR-10a-5p suppressed LPS-induced CYP11A1 expression, resulting in decreased progesterone secretion. Our findings indicated that miR-10a-5p was up-regulated by LPS and inhibited progesterone synthesis by down-regulating CYP11A1. This study provides insight into the molecular mechanisms regulating geese reproduction and ovulation.

Pigment epithelium-derived factor expression and role in follicular development.

RESEARCH QUESTION: What is the involvement of pigment epithelium-derived factor (PEDF), expressed in granulosa cells, in folliculogenesis? DESIGN: mRNA expression of PEDF and other key factors [Cyp19, anti-Müllerian hormone receptor (AMHR) and vascular endothelial growth factor (VEGF)] in mice follicles was examined in order to typify the expression of PEDF in growing follicles and in human primary granulosa cells (hpGC), and to follow the interplay between PEDF and the other main players in folliculogenesis: FSH and AMH. RESULTS: mRNA expression of PEDF increased through folliculogenesis, although the pattern differed from that of the other examined genes, affecting the follicular angiogenic and oxidative balance. In hpGC, prolonged exposure to FSH stimulated the up-regulation of PEDF mRNA. Furthermore, a negative correlation between AMH and PEDF was observed: AMH stimulation reduced the expression of PEDF mRNA and PEDF stimulation reduced the expression of AMHR mRNA. CONCLUSIONS: Folliculogenesis, an intricate process that requires close dialogue between the oocyte and its supporting granulosa cells, is mediated by various endocrine and paracrine factors. The current findings suggest that PEDF, expressed in granulosa cells, is a pro-folliculogenesis player that interacts with FSH and AMH in the process of follicular growth. However, the mechanism of this process is yet to be determined.

Effect of TSH on aromatase expression of ovarian granulosa cells in obese mice.

PURPOSE: Aromatase plays an important role in ovarian development, the normal progress of the menstrual cycle, and fertility status. Elevated aromatase activity is linked to obesity. There is a bidirectional relationship between obesity and thyroid function. Few studies have investigated the relationship between TSH and ovarian aromatase in obesity. Our aim was to investigate the effect of TSH on aromatase expression of ovarian granulosa cells in obese mice. METHODS: Female mice pups were divided into an obesity group and a control group. Obese parameters and the time of pubertal onset were recorded. At the age of 5 weeks, blood and tissues were obtained. Serum aromatase and hormone concentrations were measured using ELISA. The granulosa cells were isolated and exposed to variable concentrations (0 µM, 1 µM, 10 µM, 100 µM) of TSH. The expression of CYP19A1 mRNA and protein were assessed via RT-qPCR and western blot. RESULTS: In female mice, body weight, Lee's obesity index, and serum levels of E2, aromatase, and TSH were significantly higher in the obesity group compared to the control group, whereas the time of pubertal onset and serum T3 and T4 concentrations were significantly lower (all P < 0.001). In granulosa cells, the expression of CYP19A1 mRNA in the obesity group was lower than that in the control group at 1 µM and 100 µM concentrations of TSH (both P < 0.001). The expression of CYP19A1 protein in the obesity group was higher than that in the control group after TSH stimulation (P = 0.014, P < 0.001, and P = 0.004, respectively). With the increase of TSH concentrations, the expression of CYP19A1 mRNA and protein in the two groups significantly increased (all P < 0.001). CONCLUSION: Early puberty and elevated serum aromatase and TSH levels were found in obese female mice. In the granulosa cells of obese mice, TSH directly regulates aromatase expression in a dose-dependent manner.

Downregulation of CASC15 attenuates the symptoms of polycystic ovary syndrome by affecting granulosa cell proliferation and regulating ovarian follicular development.

Polycystic ovary syndrome (PCOS) is a type of follicular dysplasia with an unclear pathogenesis, posing certain challenges in its diagnosis and treatment. Cancer susceptibility candidate 15 (CASC15), a long non-coding RNA closely associated with tumour development, has been implicated in PCOS onset and development. Therefore, this study aimed to investigate the molecular mechanisms underlying PCOS by downregulating CASC15 expression in both in vitro and in vivo models. We explored the potential regulatory relationship between CASC15 expression and PCOS by examining cell proliferation, cell cycle dynamics, cell autophagy, steroid hormone secretion capacity, and overall ovarian function in mice. We found that CASC15 expression in granulosa cells derived from patients with PCOS was significantly higher than those of the normal group (P < 0.001). In vitro experiments revealed that downregulating CASC15 significantly inhibited cell proliferation, promoted apoptosis, induced G1-phase cell cycle arrest, and influenced cellular autophagy levels. Moreover, downregulating CASC15 affected the follicular development process in newborn mouse ovaries. In vivo studies in mice demonstrated that disrupting CASC15 expression improved PCOS-related symptoms such as polycystic changes and hyperandrogenism, and significantly affected ovulation induction and embryo implantation in pregnant mice. Overall, CASC15 was highly expressed in granulosa cells of patients with PCOS and its downregulation improved PCOS-related symptoms by influencing granulosa cell function and follicular development in mice.

YTHDF2 regulates MSS51 expression contributing to mitochondria dysfunction of granulosa cells in polycystic ovarian syndrome patients.

RESEARCH QUESTION: Granulosa cells (GCs) dysfunction plays a crucial role in the pathogenesis of polycystic ovary syndrome (PCOS). It is reported that YTH domain-containing family protein 2 (YTHDF2) is upregulated in mural GCs of PCOS patients. What effect does the differential expression of YTHDF2 have in PCOS patients? DESIGN: Mural GCs and cumulus GCs from 15 patients with PCOS and 15 ovulatory controls and 4 cases of pathological sections in each group were collected. Real-time PCR, Western Blot, immunohistochemistry, and immunofluorescence experiments were conducted to detect gene and protein expression. RNA immunoprecipitation assay was performed to evaluate the binding relationship between YTHDF2 and MSS51. Mitochondrial morphology, cellular ATP and ROS levels and glycolysis-related gene expression were detected after YTHDF2 overexpression or MSS51 inhibition. RESULTS: In the present study, we found that YTHDF2 was upregulated in GCs of PCOS patients while MSS51 was downregulated. YTHDF2 protein can bind to MSS51 mRNA and affect MSS51 expression. The reduction of MSS51 expression or the increase in YTHDF2 expression can lead to mitochondrial damage, reduced ATP levels, increased ROS levels and reduced expression of LDHA, PFKP and PKM. CONCLUSIONS: YTHDF2 may regulate the expression of MSS51, affecting the structure and function of mitochondria in GCs and interfering with cellular glycolysis, which may disturb the normal biological processes of GCs and follicle development in PCOS patients.

Toxicity mechanism of acrolein on energy metabolism disorder and apoptosis in human ovarian granulosa cells.

Acrolein (ACR), an unsaturated, highly reactive aldehyde, is a widespread environmental toxin. ACR exerts permanent and irreversible side effects on ovarian functions. Granulosa cells play a crucial role in supporting ovarian function. Thus, in this study, we investigated the toxicity effects of granulosa cells induced by ACR. Following treatment with varying ACR concentrations (0, 12.5, 25, 50, and 100µM), we observed that ACR exposure induced reactive oxygen species accumulation, mitochondrial energy metabolism disorder, and apoptosis in KGN cells (a human ovarian granulosa cell line) in a dose-dependent manner. In addition, mitochondrial biogenesis in KGN cells displayed biphasic changes after ACR exposure, with activation at a low ACR dose (12.5µM), but inhibition at higher ACR doses (≥50µM). SIRT1/PGC-1α-mediated mitochondrial biogenesis is crucial for maintaining intracellular mitochondrial homeostasis and cellular function. The inhibition/activation of the SIRT1/PGC-1α pathway in KGN cells validated its role in ACR-induced damage. The results indicated that the inhibition of the SIRT1/PGC-1α pathway aggravated ACR-induced cell damage, whereas its activation partially counteracted ACR-induced cell damage. This study attempted to uncover a novel mechanism of ACR-induced ovarian toxicity so as to provide an effective treatment option for safeguarding female reproductive health from the adverse effects of ACR.

METTL3 deficiency leads to ovarian insufficiency due to IL-1ß overexpression in theca cells.

Premature ovarian insufficiency (POI) is a clinical syndrome characterised by a decline in ovarian function in women before 40 years of age and is associated with oestradiol deficiency and a complex pathogenesis. However, the aetiology of POI is still unclear and effective preventative and treatment strategies are still lacking. Methyltransferase like 3 (METTL3) is an RNA methyltransferase that is involved in spermatogenesis, oocyte development and maturation, early embryonic development, and embryonic stem cell differentiation and formation, but its role in POI is unknown. In the present study, METTL3 deficiency in follicular theca cells was found to lead to reduced fertility in female mice, with a POI-like phenotype, and METTL3 knockout promoted ovarian inflammation. Further, a reduction in METTL3 in follicular theca cells led to a decrease in the m6A modification of pri-miR-21, which further reduced pri-miR-21 recognition and binding by DGCR8 proteins, leading to a decrease in the synthesis of mature miR-21-5p. Decrease of miR-21-5p promoted the secretion of interleukin-1ß (IL-1ß) from follicular theca cells. Acting in a paracrine manner, IL-1ß inhibited the cAMP-PKA pathway and activated the NF-κB pathway in follicular granulosa cells. This activation increased the levels of reactive oxygen species in granulosa cells, causing disturbances in the intracellular Ca2+ balance and mitochondrial damage. These cellular events ultimately led to granulosa cell apoptosis and a decrease in oestradiol synthesis, resulting in POI development. Collectively, these findings reveal how METTL3 deficiency promotes the expression and secretion of IL-1ß in theca cells, which regulates ovarian functions, and proposes a new theory for the development of POI disease.

UCHL1 promotes the proliferation of porcine granulosa cells by stabilizing CCNB1.

BACKGROUND: The proliferation of porcine ovarian granulosa cells (GCs) is essential to follicular development and the ubiquitin-proteasome system is necessary for maintaining cell cycle homeostasis. Previous studies found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) regulates female reproduction, especially in ovarian development. However, the mechanism by which UCHL1 regulates porcine GC proliferation remains unclear. RESULTS: UCHL1 overexpression promoted GC proliferation, and knockdown had the opposite effect. UCHL1 is directly bound to cyclin B1 (CCNB1), prolonging the half-life of CCNB1 and inhibiting its degradation, thereby promoting GC proliferation. What's more, a flavonoid compound-isovitexin improved the enzyme activity of UCHL1 and promoted the proliferation of porcine GCs. CONCLUSIONS: UCHL1 promoted the proliferation of porcine GCs by stabilizing CCNB1, and isovitexin enhanced the enzyme activity of UCHL1. These findings reveal the role of UCHL1 and the potential of isovitexin in regulating proliferation and provide insights into identifying molecular markers and nutrients that affect follicle development.

The Effect of Human Umbilical Cord Mesenchymal Stem Cell on Premature Ovarian Cell Senilism Through miR-10a.

Objective: To investigate the potential protective impact of miR-10a-modified HUMSCs-derived exosomes on both premature ovarian failure and the functionality of ovarian granulosa cells in a POF model. Methods: KGN cells were co-cultured with cisplatin-diaminedichloroplatinum (II) (10 µM) for 24 h to establish an in vitro POF model. The cells were distributed into three distinct groups: the control group, the POF group, and the POF + HUCMSC group. The plasmid sh-NC, sh-miR-10 a and miR-10 a mimic were transfected into KGN cells. After co-cultured with HUCMSC-EVs for 48 h, they were divided into HUCMSC group, sh-miR-10 a-HUMSCs-exosomes group and miR-10 a-HUMSCs-exosomes group. Flow cytometry was adopted to assess the impact of HUMSCs surface immune antigens and miR-10a-HUCMSCs-exosomes on KGN cell apoptosis. Additionally, the evaluation of cell proliferation was carried out through CCK-8 and EDU assays. Western blot analysis was utilized to detect the Caspase-3, Bax, and Bcl-2 proteins levels. Furthermore, the levels of TNF-α, IL-6, IL-10, MDA, SOD, and CAT were quantified using ELISA. Results: Compared with the Control group, the POF group inhibited the growth of ovarian granulosa cells (P<0.01), reduced the number of EDU cells (P<0.01), and increased the protein expression of Caspase-3 (P<0.05) and Bax (P<0.01). HUMSCs treatment significantly down-regulated the expression of IL-6, TNF-α and MDA, while up-regulating the expression of IL-10, SOD and CAT (P<0.01); the overexpression of miR-10a promoted cell growth, besides, the introduction of miR-10a-HUMSCs-derived exosomes led to an elevation in the proliferation rate of OGCs affected by POF and concurrently suppressed the apoptosis rate. Conclusion: HUMSCs-derived exosomes modified by miR-10a have protective effects on premature ovarian failure and ovarian granulosa cell function in POF model.

Human Amniotic Epithelial Stem Cells Alleviate Autoimmune Premature Ovarian Insufficiency in Mice by Targeting Granulosa Cells via AKT/ERK Pathways.

Autoimmune factors play an important role in premature ovarian insufficiency (POI). Human amniotic epithelial stem cells (hAESCs) have recently shown promising treatment effects on chemotherapy-induced POI. However, the therapeutic efficacy and underlying mechanisms of hAESCs in autoimmune POI remain to be investigated. In this study, we showed for the first time that intravenous transplantation of hAESCs could reside in the ovary of zona pellucida 3 peptide (pZP3) induced autoimmune POI mice model for at least 4 weeks. hAESCs could improve ovarian function and fertility, alleviate inflammation and reduce apoptosis of granulosa cells (GCs) in autoimmune POI mice. The transcriptome analysis of mice ovaries and in vitro co-cultivation experiments suggest that activation of the AKT and ERK pathways may be the key mechanism in the therapeutic effect of hAESCs. Our work provides the theoretical and experimental foundation for optimizing the administration of hAESCs, as well as the clinical application of hAESCs in autoimmune POI patients.

FOXL2 regulates RhoA expression to change actin cytoskeleton rearrangement in granulosa cells of chicken pre-ovulatory follicles.

Forkhead box L2 (FOXL2) is an indispensable key regulator of female follicular development, and it plays important roles in the morphogenesis, proliferation, and differentiation of follicle granulosa cells (GCs), such as establishing normal estradiol signaling and regulating steroid hormone synthesis. Nevertheless, the effects of FOXL2 on GC morphology and the underlying mechanism remain unknown. Using FOXL2 ChIP-seq analysis, we found that FOXL2 target genes significantly enriched in the actin cytoskeleton-related pathways. We confirmed that FOXL2 inhibited the expression of RhoA, a key gene for actin cytoskeleton rearrangement, by binding to TCATCCATCTCT in RhoA promoter region. In addition, the overexpression of FOXL2 in GCs induced the depolymerization of F-actin and the disordered of the actin filaments, resulting in a slowdown in the expansion of GCs, while silencing FOXL2 inhibited F-actin depolymerization and stabilized the actin filaments, thereby accelerating GC expansion. RhoA/ROCK pathway inhibitor Y-27632 exhibited similar effects to FOXL2 overexpression, even reversed the actin polymerization in FOXL2 silencing GCs. This study revealed for the first time that FOXL2 regulated GC actin cytoskeleton by RhoA/ROCK pathway, thus affecting GC expansion. Our findings provide new insights for constructing the regulatory network of FOXL2 and propose a potential mechanism for facilitating rapid follicle expansion, thereby laying a foundation for further understanding follicular development.

Progesterone and 17-hydroxy-progesterone concentrations in follicular fluid and serum reflect their production in granulosa and theca cells.

RESEARCH QUESTION: How is the production of progesterone (P4) and 17-hydroxy-P4 (17-OH-P4) regulated between theca cells and granulosa cells during the follicular phase, during ovulation and after transformation into a corpus luteum? DESIGN: Three cohorts were examined: (i) 31 women undergoing natural and stimulated cycles, with serum hormone measurements taken every 3 days; (ii) 50 women undergoing ovarian stimulation, with hormone concentrations in serum and follicular fluid assessed at five time points during final follicle maturation; and (iii) 12 women undergoing fertility preservation, with hormone concentrations evaluated via the follicular fluid of small antral follicles. RESULTS: In the early follicular phase, theca cells primarily synthesized 17-OH-P4 while granulosa cells produced limited P4, maintaining the P4:17-OH-P4 ratio <1. As follicles reached follicle selection at a diameter of approximately 10 mm, P4 synthesis in granulosa cells was up-regulated, but P4 was mainly accumulated in follicular fluid. During final maturation, enhanced activity of the enzyme HSD3B2 in granulosa cells enhanced P4 production, with the P4:17-OH-P4 ratio increasing to >1. The concentration of 17-OH-P4 in the luteal phase was similar to that in the follicular phase, but P4 production increased in the luteal phase, yielding a P4:17-OH-P4 ratio significantly >1. CONCLUSIONS: The P4:17-OH-P4 ratio reflects the activity of granulosa cells and theca cells during the follicular phase and following luteinization in the corpus luteum. Managing the function of granulosa cells is key for reducing the concentration of P4 during ovarian stimulation, but the concerted action of FSH and LH on granulosa cells during the second half of the follicular phase makes this complex.