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Background/Objectives: The avascular nature of the follicle creates a hypoxic microenvironment, establishing a niche where granulosa cells (GCs) rely on glycolysis to produce energy in the form of lactate (L-lactate). Autophagy, an evolutionarily conserved stress-response process, involves the formation of autophagosomes to encapsulate intracellular components, delivering them to lysosomes for degradation. This process plays a critical role in maintaining optimal follicular development. However, whether hypoxia regulates autophagy in GCs via lactate remains unclear. Methods: In this study, we investigated lactate-induced autophagy under hypoxia by utilizing glycolysis inhibitors or silencing related genes. Results: We observed a significant increase in autophagy in ovarian GCs under hypoxic conditions, indicated by elevated LC3II levels and reduced P62 levels. Suppressing lactate production through glycolytic inhibitors (2-DG and oxamate) or silencing lactate dehydrogenase (LDHA/LDHB) effectively reduced hypoxia-induced autophagy. Further investigation revealed that the HIF1-α/BNIP3/Beclin-1 axis is essential for lactate-induced autophagy under hypoxic conditions. Inhibiting HIF-1α activity using siRNAs or PX-478 downregulated BNIP3 expression and subsequently suppressed autophagy. Similarly, BNIP3 silencing with siRNAs repressed lactate-induced autophagy in hypoxic conditions. Mechanistically, immunoprecipitation experiments showed that BNIP3 disrupted pre-existing Bcl-2/Beclin-1 complexes by competing with Bcl-2 to form Bcl-2/BNIP3 complexes. This interaction released Beclin-1, which subsequently triggered lactate-induced autophagy under hypoxic conditions. Conclusions: These findings unveil a novel mechanism by which hypoxia regulates GC autophagy through lactate production, highlighting its potential role in sustaining follicular development under hypoxic conditions. [ABSTRACT FROM AUTHOR]

Background: Granulosa cell (GC) apoptosis, ferroptosis, and other programmed cell death processes are markers of follicular aging. Quercetin has been shown to reduce ferroptosis, however, its effects on ferroptosis in poultry remains unexplored. Our preliminary study identified ferroptosis in aging ovaries. Therefore, in the present study, 540-day-old Mountain Plum-blossom chickens were fed with quercetin supplementation at varying doses (0.2, 0.4, and 0.6 g/kg), and examined its molecular effects on GC ferroptosis using an in vitro Erastin-induced model. Results: The results showed that quercetin supplementation significantly increased egg production, which confirmed its potential to alleviate ferroptosis in chicken ovarian tissue. The in vitro experiment revealed that quercetin and Fer-1 (positive control) mitigated Erastin-induced ferroptosis in GCs. Further, transcriptome analysis revealed that quercetin modulated key genes such as acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor (TFRC), involved in ferroptosis regulation. The results further showed that quercetin also reduced Erastin-induced apoptosis and inflammation by modulating the expression of genes and proteins related to apoptosis and inflammatory factors (NF-κB, TNF-α, IL-6, and IL-10). Conclusion: Taken together, the results showed that quercetin improves egg production performance in chickens and mitigates ovarian ferroptosis in aging hens, and inhibits Erastin-induced ferroptosis, inflammation, and apoptosis in GCs. These findings revealed the protective role of quercetin in poultry ovarian tissue and its cellular mechanisms against detrimental factors in poultry production. [ABSTRACT FROM AUTHOR]

Background: Telomeres maintain chromosome stability, while telomerase counteracts their progressive shortening. Telomere length varies between cell types, with leukocyte telomere length (LTL) decreasing with age. Reduced telomerase activity has been linked to reproductive issues in females, such as low pregnancy rates and premature ovarian failure, with recent studies indicating correlations between telomere length in granulosa cells and IVF outcomes. Objectives: The study aims to explore the relationship between telomere length, telomerase activity, and euploid blastocyst rate in infertile women undergoing IVF/ICSI PGT-A cycles. Methods: This prospective study involves 108 patients undergoing controlled ovarian stimulation and PGT-A. Telomere length and telomerase activity were measured in peripheral mononuclear cells and granulosa cells (GC), respectively. Results: The telomere repeat copy number to single gene copy number ratio (T/S) results respectively 0.6 ± 0.8 in leukocytes and 0.7 ± 0.9 in GC. An inverse relationship was found between LTL and the patient's age (p <.01). A higher aneuploid rate was noticed in patients with short LTL, with no differences in ovarian reserve markers (p =.15), number of oocytes retrieved (p =.33), and number of MII (p = 0.42). No significant association was noticed between telomere length in GC and patients' age (p = 0.95), in ovarian reserve markers (p = 0.32), number of oocytes retrieved (p =.58), number of MII (p =.74) and aneuploidy rate (p =.65). Conclusion: LTL shows a significant inverse correlation with patient age and higher aneuploidy rates. Telomere length in GCs does not correlate with patient age or reproductive outcomes, indicating differential telomere dynamics between leukocytes and granulosa cells. [ABSTRACT FROM AUTHOR]

Ovarian dysfunction caused by aging restricts female reproductive capacity and is accompanied by oxidative stress and impaired autophagy. Recent studies have shown that trehalose (Tre) can activate autophagy and have antioxidant effects. However, whether Tre can be used to attenuate ovarian aging remains unclear. Therefore, the anti-aging effects of Tre on the ovary were explored both in vivo and in vitro. D-galactose (D-gal) was administered i.p. daily (200 mg/kg body weight) for 8 weeks to establish the mouse ovarian aging model (n = 10). We found that Tre significantly reversed ovarian weight loss and reduced the number of TUNEL-positive granulosa cells caused by D-gal in mouse ovaries. Tre elevated the protein expression levels of LC3-II, Parkin, PINK1, Beclin1, and LAMP2 in ovaries. Mitochondrial-related proteins TOM20 and COX IV expression levels were increased by Tre administration. In vitro studies further supported these findings, showing that Tre treatment significantly reduced the number of SA-β-gal and PI-positive cells, and decreased ROS levels in cultured granulosa cells. Thus, Tre alleviates ovarian aging by activating mitophagy and reducing oxidative stress, suggesting its potential as an anti-aging agent for ovarian health. [ABSTRACT FROM AUTHOR]

Simple Summary: The common carp (Cyprinus carpio) shows pronounced sexual dimorphism in growth, and females grow faster than males. Studying the molecular mechanisms of ovarian development and oogenesis is vital for improving productivity and breeding all-female populations. Although many transcriptome analyses of bulk ovaries have been conducted, they have not been able to uncover cell type-specific differences in gene expression. In this study, we presented the transcriptional profile of common carp ovaries at the single-cell level using single-nucleus RNA sequencing. Our analysis revealed valuable information about regulating oogenesis and the development of granulosa cells. Our research provides a helpful resource that complements previously published bulk ovary transcriptomic analyses in the common carp for further investigating the molecular and cellular mechanisms underlying ovarian development. The common carp (Cyprinus carpio) is a crucial freshwater species cultivated worldwide for food consumption. Female carp have better growth performance than males, which fascinates scholars to uncover the mechanism of gonadal differentiation and produce mono-sex populations. However, the mechanism of ovarian development at single-cell resolution is limited. Here, we conducted single-nucleus RNA sequencing in adolescent common carp ovaries. Our study obtained transcriptional profiles of 13,155 nuclei and revealed 13 distinct cell clusters in the ovaries, including three subtypes of germ cells and four subtypes of granulosa cells. We subsequently performed pseudotime trajectory analysis to delineate potential mechanisms underlying the development of germ cells and granulosa cells. We identified 1250 dynamic expression genes in germ cells and 1815 in granulosa cells (q-value < 0.01), including zp3, eif4a2 and aspm in germ cells and fshr and esr1 in granulosa cells. The functional annotation showed that dynamic expression genes in germ cells were involved in sperm–egg recognition and some terms related to meiosis, such as sister chromatid segregation and homologous recombination. Genes expressed dynamically in granulosa cells were related to the TGF-β signaling pathway, response to gonadotropin, and development of primary female sexual characteristics. In addition, the dynamic genes expressed in granulosa cells might relate to the complex communication between different cell types. In summary, our study provided a transcriptome profile of common carp ovaries at single-nucleus resolution, and we further revealed the potential cell type-specific mechanisms underlying oogenesis and the differentiation of granulosa cells, which will facilitate breeding all-female common carp populations. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of baicalin (BAI) on the proliferation and apoptosis of ovarian granulosa cells in polycystic ovary syndrome (PCOS) and its mechanisms. Methods: Forty-eight female SD rats were randomly divided into the control group, PCOS group, BAI low, middle and high dose groups and positive control group, with 8 rats in each group. PCOS model was established by subcutaneous injection of DHEA, and rats in the low, medium, and high dose groups of BAI were given baicalin (12.5, 25, and 50 mg/kg) by gavage. The Positive control group was given intraperitoneal injection (10 mg/kg) of Vitipofen; The control group and PCOS group were given equal volume 0.9% NaCl solution instead. Once a day for 4 weeks. The serum sex hormone levels were detected by enzyme-linked immunosorbant assay (ELISA). The pathological changes of ovary were observed by HE staining. After successful modeling, ovarian granulosa cells were extracted, and their proliferation was detected by MTT assay. Apoptosis was detected by Annex- inV-FITC/PI double staining and TUNEL staining. The relative expressions of BCL2 and BAX, P13K/AKT pathway related proteins P13K, AKT, p-P13K and p-AKT were detected by western blot. Results: The extracted ovarian granulosa cells are fibroblast-like and adhered well, with positive expression of specific proteins FSHR. Compared with the control group, the ovarian structure of rats in the PCOS group is disrupted, with an increase in bleeding, congestion, and the number of cystic follicles, as well as thickening of the white membrane. The levels of serum testosterone (T) and luteinizing hormone (LH), the apoptosis rate of ovarian granulosa cells and the expression of Bax protein are significantly increased (P<0.05), while the levels of Estradiolm (E2) and follicle stimulating hormone (FSH), the viability of ovarian granulosa cells, the expression of Bcl-2, p-P13K, and p-AKT protein are significantly decreased (P<0.05). Compared with PCOS group, BAI groups have obvious recovery of polycystic features, significant reduction in cysts, and complete arrangement of ovarian structure and granular cell layer. The levels of E2, and FSH in serum, the viability of ovarian granulosa cells, the expressions of Bcl-2, p-P13K and p-AKT protein are significantly increased in a dose-dependent manner (P<0.05), while the levels of T and LH in serum, the apoptosis rate of ovarian granulosa cells and the expression of Bax protein are significantly decreased in a dose-dependent manner (P<0.05). Conclusion: Baicalin may promote the proliferation and inhibit the apoptosis of ovarian granulosa cell, which may alleviate PCOS by activating the P13K/AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Problem: There were not only granulosa cells (GCs) but also immune cells in preovulatory follicular fluid. The objective of this study was to explore the interactions between macrophages and GCs via adhesion molecules in preovulatory follicles and the regulatory mechanisms of the interactions. Method of Study: Flow cytometry and immunofluorescence were used to detect the expression of ITGB1 in macrophages and fibronectin (FN)1 in GCs in preovulatory follicles from 12 patients. The synthesis of FN1 protein in human ovarian GCs line (KGN) was detected by western blot. An adhesion experiment was performed to observe the changes of KGN cells adhesion to macrophages. Results: The progesterone levels 12 h after human chorionic gonadotropin (HCG) administration in the high proportion immune cells (high immune [HI]) group were significantly higher than that in the low proportion immune cells (low immune [LI]) group (p < 0.0001). The expression of ITGB1 in macrophages in the HI group was higher than in the LI group. The expression of FN1 in GCs in HI group was higher than in LI group (p < 0.01). Progesterone increased the synthesis of FN1 in KGN cells (p < 0.0001) and was suppressed by the elimination of PGR. The adhesion effect of KGN cells on macrophages was enhanced by progesterone (p < 0.0001). Conclusion: After luteinizing hormone (LH)/HCG surge, progesterone promotes the expression of FN1 in GCs by acting on the receptor PGR, and then GCs enhance the adhesion of macrophages by FN1‐ITGB1 interaction, further leading to the result that macrophages perform diverse functional activities to maintain tissue homeostasis during ovulation. [ABSTRACT FROM AUTHOR]

The zinc oxide nanoparticles (ZnO NPs) is one of the most extensively utilized metal oxide nanoparticles in biomedicine, human food, cosmetics and livestock farming. However, growing evidence suggests that there is a potential risk for humans and animals because of the accumulation of ZnO NPs in cells, which leads to cell death through several different pathways. Nevertheless, the effects of ZnO NPs on porcine granulosa cells (PGCs) and how ZnO NPs regulate the follicular cells are unknown. In this study, we aimed to elucidate the role of ZnO NPs in the porcine ovary by using PGCs. Firstly, we identified the characterization of ZnO NPs used in this study and the results showed that the size of ZnO NPs was 29.0 nm. The results also demonstrated that ZnO NPs impaired cell viability and decreased steroid hormone secretion in PGCs. In addition, ZnO NPs induced reactive oxygen species (ROS) production, leading to oxidative stress of PGCs. Meanwhile, ZnO NPs also triggered autophagy in PGCs by increasing the ratio of LC3-II/LC3-I, along with the expression of SQSTM1 and ATG7. Finally, the results from N-acetylcysteine (NAC) addition suggested that ZnO NPs promoted autophagy through the enhancement of ROS production. In summary, this study demonstrates that ZnO NPs impair the viability and function of PGCs through autophagy, which is regulated by ROS production. [ABSTRACT FROM AUTHOR]

Abstract Background In-depth understanding of dynamic expression profiles of human granulosa cells (GCs) during follicular development will contribute to the diagnostic and targeted interventions for female infertility. However, genome-scale analysis of long non-coding ribonucleic acid (lncRNA) in GCs across diverse developmental stages is challenging. Meanwhile, further research is needed to determine how aberrant lncRNA expression participates in ovarian diseases. Methods Granulosa cell-related lncRNAs data spanning five follicular development stages were retrieved and filtered from the NCBI dataset (GSE107746). Stage-specific lncRNA expression patterns and mRNA-lncRNA co-expression networks were identified with bioinformatic approaches. Subsequently, the expression pattern of SNHG18 was detected in GCs during ovarian aging. And SNHG18 siRNA or overexpression plasmids were transfected to SVOG cells in examining the regulatory roles of SNHG18 in GC proliferation and apoptosis. Moreover, whether PKCɛ/SNHG18 signaling take part in GC glycolysis via ENO1 were verified in SVOG cells. Results We demonstrated that GC-related lncRNAs were specifically expressed across different developmental stages, and coordinated crucial biological functions like mitotic cell cycle and metabolic processes in the folliculogenesis. Thereafter, we noticed a strong correlation of PRKCE and SNHG18 expression in our analysis. With downregulated SNHG18 of GCs identified in the context of ovarian aging, SNHG18 knockdown could further induce cell apoptosis, retard cell proliferation and exacerbate DNA damage in SVOG cell. Moreover, downregulated PKCɛ/SNHG18 pathway interrupted the SVOG cell glycolysis by lowering the ENO1 expression. Conclusions Altogether, our results revealed that folliculogenesis-related lncRNA SNHG18 participated in the pathogenesis of ovarian aging, which may provide novel biomarkers for ovarian function and new insights for the infertility treatment.

Reactive oxygen species (ROS) are a by-product of the activity of cytochrome P450 steroidogenic enzymes. Antioxidant enzymes protect against ROS damage. To identify if any particular antioxidant enzyme is used to protect against ROS produced by granulosa cells as follicles enlarge and produce oestradiol, we measured in the bovine granulosa cells the expression of two steroidogenic enzymes (CYP11A1, CYP19A1), important for progesterone and oestradiol production. We also measured the expression of the members (FDXR, FDX1, POR) of their electron transport chains (ETC). We measured antioxidant enzymes (GPXs 1–8, CAT, SODs 1 and 2, PRDXs 1–6, GSR, TXN, TXNRDs 1–3). Since selenium is an active component of GPXs, the selenium-uptake receptors (LRPs 2 and 8) were measured. Only the selenium-dependent GPX1 showed the same increase in expression as the steroidogenic enzymes did with increasing follicle size. GPX4 and PRDX2/6 decreased with follicle size, whereas SOD1/2, CAT, GSR, and TXNRD3 were lowest at the intermediate sizes. The other antioxidant enzymes were unchanged or expressed at low levels. The expression of the selenium-uptake receptor LRP8 also increased significantly with follicle size. Correlation analysis revealed statistically significant and strongly positive correlations of the steroidogenic enzymes and their ETCs with both GPX1 and LRP8. These results demonstrate a relationship between the expression of genes involved in steroidogenesis and selenium-containing antioxidant defence mechanisms. They suggest that during the late stages of folliculogenesis, granulosa cells are dependent on sufficient expression of GPX1 and the selenium transporter LRP8 to counteract increasing ROS levels caused by the production of steroid hormones.

Ovarian cysts are linked to hormone imbalances and altered gene expressions, but the connection between cysts and ion channel expression is understudied. This study explored the role of TWIK-related acid-sensitive K+ (TASK) channels in bovine ovarian cyst formation. The ovarian follicles were split into small (5 to 10 mm in diameter) and large (>25 mm in diameter) groups. Among the measured K+, Na+, and Cl− concentrations in follicular fluid (FF) obtained from small-sized follicles (SFs) and large-sized follicles (LFs), the K+ concentration was significantly lower in LFFF. Quantitative PCR, Western blot, and immunocytochemistry data revealed that TASK-3 expression levels significantly decreased by approximately 50% in LFs and granulosa cells obtained from LFs (LFGCs) compared to the corresponding controls. The TASK-3 protein was localized to the plasma membranes of GCs. The diameters of LFGCs were larger than those of SFGCs. The cell swelling response to exposure to a hypotonic solution (200 mOsm/L) was highly reduced in TASK-3-overexpressing cells compared to vector-transfected cells. TASK-3-knockdown cells showed arrested growth. Senescence markers were detected in LFGCs and TASK-3-knockdown cells. These findings suggest that reduced TASK-3 expression in LFs is associated with the inhibition of GC growth, leading to senescence and cyst formation. [ABSTRACT FROM AUTHOR]

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics. • Ovine granulosa cells undergo autophagy during follicular atresia at antral stage • The autophagic marker level is higher in atretic than in healthy granulosa cells • Granulosa cells of atretic follicles undergo apoptosis as well • Autophagy and apoptosis could regulate granulosa cell death • Both autophagy and apoptosis could be involved in antral follicle atresia in sheep [ABSTRACT FROM AUTHOR]

Background: In-depth understanding of dynamic expression profiles of human granulosa cells (GCs) during follicular development will contribute to the diagnostic and targeted interventions for female infertility. However, genome-scale analysis of long non-coding ribonucleic acid (lncRNA) in GCs across diverse developmental stages is challenging. Meanwhile, further research is needed to determine how aberrant lncRNA expression participates in ovarian diseases. Methods: Granulosa cell-related lncRNAs data spanning five follicular development stages were retrieved and filtered from the NCBI dataset (GSE107746). Stage-specific lncRNA expression patterns and mRNA-lncRNA co-expression networks were identified with bioinformatic approaches. Subsequently, the expression pattern of SNHG18 was detected in GCs during ovarian aging. And SNHG18 siRNA or overexpression plasmids were transfected to SVOG cells in examining the regulatory roles of SNHG18 in GC proliferation and apoptosis. Moreover, whether PKCɛ/SNHG18 signaling take part in GC glycolysis via ENO1 were verified in SVOG cells. Results: We demonstrated that GC-related lncRNAs were specifically expressed across different developmental stages, and coordinated crucial biological functions like mitotic cell cycle and metabolic processes in the folliculogenesis. Thereafter, we noticed a strong correlation of PRKCE and SNHG18 expression in our analysis. With downregulated SNHG18 of GCs identified in the context of ovarian aging, SNHG18 knockdown could further induce cell apoptosis, retard cell proliferation and exacerbate DNA damage in SVOG cell. Moreover, downregulated PKCɛ/SNHG18 pathway interrupted the SVOG cell glycolysis by lowering the ENO1 expression. Conclusions: Altogether, our results revealed that folliculogenesis-related lncRNA SNHG18 participated in the pathogenesis of ovarian aging, which may provide novel biomarkers for ovarian function and new insights for the infertility treatment. [ABSTRACT FROM AUTHOR]

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post‐ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC‐layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post‐ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)‐cAMP‐PKA‐CREB signaling cascade, is necessary for mGC‐layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC‐layer stiffening", shedding light on the mechanism that prevents mGC escape from the post‐ovulatory follicle. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) have attracted significant attention due to their environmental toxicity. However, the detrimental impact of PFAS on the development of the female reproductive system remains controversial. In this study, we investigated the effects of three specific PFAS compounds perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) on ovarian development. Among these compounds, PFDA demonstrated the most pronounced cytotoxic effect on ovarian granulosa cells. The results showed that a 200 μM concentration of PFDA induced cell apoptosis via the intrinsic pathway by elevating reactive oxygen species (ROS) levels and activating Caspase-9 and Caspase-3. Furthermore, 200 μM PFDA triggered necroptosis, a form of regulated cell death (RCD), through the receptor-interacting serine/threonine kinase 1 (RIPK1), receptor interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like protein (MLKL) axis, mediated by inhibition of the canonical apoptosis proteolytic enzyme Caspase-8. In vivo experiments confirmed that mice exposed to PFDA displayed a significantly reduced ovarian index compared to the control group, accompanied by evident follicular atresia. Ovarian tissues from the PFDA-exposed group showed upregulated necroptosis markers, which were effectively mitigated by inhibiting the phosphorylation of RIPK1 at Ser166. Importantly, this study provides the first evidence that PFDA disrupts ovarian development through a novel mechanism involving the RIPK1-mediated necroptosis pathway, alongside the detection of the intrinsic apoptosis pathway. This greatly expands our insight into the effects of PFDA on cell death. This finding highlights the potential public health hazards associated with PFDA exposure and emphasizes the need for further research to fully understand its broader implications.

ABSTRACT: The development and maturation of follicles are intricately linked to egg production and reproductive performance of chickens. Granulosa cells death directly affects the development and maturation of follicles, thereby impacting the reproductive performance of hens. Ferroptosis is a new type of cell death, it is unknown how it affects the growth and development of chicken follicles. In this study, RNA-seq analysis revealed significant differences in the expression of ferroptosis-related genes between normal follicles and atretic follicles, suggesting a potential role for ferroptosis in follicle growth and development. In addition, we found that ubiquitin-specific protease 13 (USP13) was significantly upregulated in atrophic follicles. Overexpression of USP13 results in depletion of glutathione (GSH), peroxidation of lipids, accumulation of iron, and activation of ferroptosis in chicken granulosa cells. In contrast, USP13 knockdown significantly inhibited ferroptosis events. Mechanistically, USP13 prevents the degradation of autophagy related 7 (ATG7) by deubiquitinating it, thereby enhancing the stability of ATG7 protein and ultimately promoting ferroptosis. In conclusion, this study elucidates the crucial role of the USP13-ATG7 axis in regulating ferroptosis in chicken follicle granulosa cells, thereby presenting a novel avenue for molecular breeding research in chickens.

Abstract Background Adiposity profoundly impacts reproductive health in both humans and animals. However, the precise subpopulations contributing to infertility under obese conditions remain elusive. Results In this study, we established an obese mouse model through an eighteen-week high-fat diet regimen in adult female mice. Employing single-cell RNA sequencing (scRNA-seq), we constructed a comprehensive single-cell atlas of ovarian tissues from these mice to scrutinize the impact of obesity on the ovarian microenvironment. ScRNA-seq revealed notable alterations in the microenvironment of ovarian tissues in obese mice. Granulosa cells, stromal cells, T cells, and macrophages exhibited functional imbalances compared to the control group. We observed heightened interaction strength in the SPP1-CD44 pairing within lgfbp7+ granulosa cell subtypes and Il1bhigh monocyte subtypes in the ovarian tissues of obese mice. Moreover, the interaction strength between Il1bhigh monocyte subtypes and Pdgfrb+ stromal cell subtypes in the form of TNF − TNFrsf1α interaction was also enhanced subsequently to obesity, potentially contributing to ovarian fibrosis pathogenesis. Conclusions We propose a model wherein granulosa cells secrete SPP1 to activate monocytes, subsequently triggering TNF-α secretion by monocytes, thereby activating stromal cells and ultimately leading to the development of ovarian fibrosis. Intervening in this process may represent a promising avenue for improving clinical outcomes in fertility treatments for obese women.

Previous research identified the expression of EDN3 in granulosa cells of preovulatory follicles in chickens. Notably, the expression level of EDN3 in Silky Fowl with low egg-laying performance was significantly higher than that in high-yield laying breed White Leghorn. Given the crucial role of granulosa cells in follicular development and maturation, it is very important to study the effect of EDN3 on the biological function of granular cells. In this study, an EDN3 overexpression plasmid was constructed and transfected into granular cells. The viability of these cells was detected using quantiative (qPCR), Cell Counting Kit-8 (CCK8), and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Gonadal hormone synthesis was detected using enzyme-linked immunosorbent assay (ELISA) techniques. Finally, transcriptome sequencing was employed to identify differentially expressed genes. Result showed thatoverexpression of EDN3 was observed to promote cell viability. In addition, it significantly inhibits the expressions of LHR and cAMP-PKA signaling pathways. Cell transcriptome sequencing data displayed that EDN3 can upregulate energy metabolism and immune-related signaling pathways, whereas follicle maturation and the GnRH signaling pathway were downregulated. In conclusion, this study demonstrates that EDN3 can enhance granulosa cell viability and inhibit the expression of LHCGR, a process likely mediated through the cAMP-PKA signaling pathway. However, further evidence is required to substantiate the regulatory relationship between EDN3 and the cAMP-PKA signaling pathway.

Background: The follicular fluid (FF) of mature oocytes contains a high concentration of growth factors and cytokinesthat have the potential to influence implantation in either a paracrine or autocrine manner. During the physiologicalprocesses of ovulation, FF enters the fallopian tubes in conjunction with the oocyte. The purpose of this studyis to evaluate implantation and clinical pregnancy rates following uterine flushing with FF and granulosa cells ininfertile women with moderate male factor infertility after ovum retrieval for intracytoplasmic sperm injection (ICSI).Materials and Methods: This phase III randomised clinical trial enrolled 140 women with moderate male factorinfertility who intended to undergo ICSI at Royan Infertility Clinic (Tehran, Iran). A computer-generated program andopaque sealed envelopes were used to randomly allocate patients to either an intervention group (n=70) or a controlgroup (n=70). Participants in the intervention group received 2 ml of clear FF (without blood contamination) from 2to 3 dominant follicles after oocyte retrieval. The control group only underwent uterine cavity catheterisation.Results: The intervention group had a clinical pregnancy rate of 38.5% (25/65) compared to the control group [42.9%(27/63); P=0.719] and an implantation rate of 24.1% compared to the control group (27%; P=0.408). These rates did notdiffer between the groups. There were no statistically significant differences between the intervention and control groupsin terms of pregnancy-related complications-ectopic pregnancy, blighted ovum or anembryonic pregnancy, and abortion.Conclusion: Uterine cavity flushing with FF from mature follicles following oocyte retrieval had no effect, eitherpositively or negatively, on clinical pregnancy or implantation rates in women with moderate male factor infertility(registration number: NCT04077970).

Abstract 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.

BACKGROUND: Oxidative modification of high-density lipoprotein occurs in patients with polycystic ovary syndrome. However, the relationship between oxidized high-density lipoprotein and ovulation dysfunction and its mechanism are unknown. OBJECTIVE: To investigate the effect and potential mechanism of oxidized high-density lipoprotein on ovarian granulosa cell apoptosis. METHODS: Polycystic ovary syndrome rat model was established, then the high-density lipoprotein was harvested from the rat serum of heart blood. The degree of oxidation of the high-density lipoprotein was detected by high-density lipoprotein inflammation index assay, malondialdehyde assay and lipoprotein agarose gel electrophoresis assay. The normal rat ovarian granulosa cells were isolated and treated with high-density lipoprotein and oxidized high-density lipoprotein isolated from the model rat serum. Cell viability was detected by CCK-8 assay. Cell apoptosis was detected by flow cytometry. The generation of reactive oxygen species was detected by H2DCF-DA staining. The p38 signaling activity was detected by western blot assay. Ovarian granulosa cells were pretreated with reactive oxygen species inhibitors N-acetylcysteine, tetramethylpiperidine (Tempol) and p38 inhibitor SB203580, and then treated with oxidized high-density lipoprotein. Finally, cell apoptosis, reactive oxygen species production and p38 signaling activity were detected. RESULTS AND CONCLUSION: A portion of the high-density lipoprotein from the serum of polycystic ovary syndrome model rats affected oxidative modification. High-density lipoprotein and oxidized high-density lipoprotein isolated from the model rat serum inhibited granulosa cell viability and promoted apoptosis (all P < 0.05). They promoted rat granulosa cell reactive oxygen species production and p38 activation (all P < 0.05). N-acetylcysteine, Tempol and SB203580 reversed oxidized high-density lipoprotein induced granulosa cell apoptosis (all P < 0.05). N-acetylcysteine and Tempol suppressed oxidized high-density lipoprotein-induced p38 activation (all P < 0.05). SB203580 did not have a regulatory effect on reactive oxygen species production (P > 0.05). In summary, polycystic ovary syndrome can promote partial oxidative modification of high-density lipoprotein. The oxidized high-density lipoprotein promotes rat granulosa cell apoptosis by the activation of the reactive oxygen species-initiated p38 signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Adiposity profoundly impacts reproductive health in both humans and animals. However, the precise subpopulations contributing to infertility under obese conditions remain elusive. Results: In this study, we established an obese mouse model through an eighteen-week high-fat diet regimen in adult female mice. Employing single-cell RNA sequencing (scRNA-seq), we constructed a comprehensive single-cell atlas of ovarian tissues from these mice to scrutinize the impact of obesity on the ovarian microenvironment. ScRNA-seq revealed notable alterations in the microenvironment of ovarian tissues in obese mice. Granulosa cells, stromal cells, T cells, and macrophages exhibited functional imbalances compared to the control group. We observed heightened interaction strength in the SPP1-CD44 pairing within lgfbp7+ granulosa cell subtypes and Il1bhigh monocyte subtypes in the ovarian tissues of obese mice. Moreover, the interaction strength between Il1bhigh monocyte subtypes and Pdgfrb+ stromal cell subtypes in the form of TNF − TNFrsf1α interaction was also enhanced subsequently to obesity, potentially contributing to ovarian fibrosis pathogenesis. Conclusions: We propose a model wherein granulosa cells secrete SPP1 to activate monocytes, subsequently triggering TNF-α secretion by monocytes, thereby activating stromal cells and ultimately leading to the development of ovarian fibrosis. Intervening in this process may represent a promising avenue for improving clinical outcomes in fertility treatments for obese women. [ABSTRACT FROM AUTHOR]

Background: The follicular fluid (FF) of mature oocytes contains a high concentration of growth factors and cytokines that have the potential to influence implantation in either a paracrine or autocrine manner. During the physiological processes of ovulation, FF enters the fallopian tubes in conjunction with the oocyte. The purpose of this study is to evaluate implantation and clinical pregnancy rates following uterine flushing with FF and granulosa cells in infertile women with moderate male factor infertility after ovum retrieval for intracytoplasmic sperm injection (ICSI). Materials and Methods: This phase III randomised clinical trial enrolled 140 women with moderate male factor infertility who intended to undergo ICSI at Royan Infertility Clinic (Tehran, Iran). A computer-generated program and opaque sealed envelopes were used to randomly allocate patients to either an intervention group (n=70) or a control group (n=70). Participants in the intervention group received 2 ml of clear FF (without blood contamination) from 2 to 3 dominant follicles after oocyte retrieval. The control group only underwent uterine cavity catheterisation. Results: The intervention group had a clinical pregnancy rate of 38.5% (25/65) compared to the control group [42.9% (27/63); P=0.719] and an implantation rate of 24.1% compared to the control group (27%; P=0.408). These rates did not differ between the groups. There were no statistically significant differences between the intervention and control groups in terms of pregnancy-related complications-ectopic pregnancy, blighted ovum or anembryonic pregnancy, and abortion. Conclusion: Uterine cavity flushing with FF from mature follicles following oocyte retrieval had no effect, either positively or negatively, on clinical pregnancy or implantation rates in women with moderate male factor infertility (registration number: NCT04077970). [ABSTRACT FROM AUTHOR]

Background: The impaired functions of granulosa cells (GCs) in the delayed development and immaturity of oocytes have been reported in polycystic ovary syndrome (PCOs). Even with ovarian stimulation, a large number of oocytes in these patients are still in the stage germinal vesicle (GV). Objective: The levels of Smad2/3, phosphorylated Smad2/3 (P-Smad2/3), the expression of SARA, Smad4, and SMURF2 genes in the GCs surrounding metaphase II (MII) or GV oocytes in PCOs women were investigated. Materials and Methods: GCs of MII and GV oocytes were isolated from 38 women with PCOs and the expression levels of SARA, Smad4, and SMURF2 in surrounding GCs of MII and GV oocytes were determined using reverse-transcription polymerase chain reaction. Also, Smad2/3 and P-Smad2/3 proteins were determined using western blotting. Results: The expression level of SMURF2 was significantly higher in GCs surrounding GV oocytes compared with that of GCs encompassing MII oocytes (p < 0.001). At the same time, no significant differences were observed in SARA and Smad4 expression levels in GCs surrounding GV and MII oocytes. A lower level of P-Smad2/3 was also found in GCs GV oocytes compared with GCs of MII oocytes (p < 0.001). Conclusion: It seems that P-Smad2/3 plays a role in oocyte development, and the downregulation of this protein is associated with a defect in the maturation of GV oocytes. On the other hand, the upregulation of the SMURF2 gene also affects the growth process of GCs and the maturation of GV oocytes. [ABSTRACT FROM AUTHOR]

1. The ferritin heavy chain (FHC) has a vital impact on follicular development in geese, due to its ability to regulate apoptosis of granulosa cells (GCs) and follicular atresia. However, its specific regulatory mechanisms remain unclear. The present study characterised how FHC regulates oxidative stress, cell proliferation and apoptosis in goose GCs by interfering with and overexpressing the FHC gene. 2. After 72 h of interference with FHC expression, the activity of GCs decreased remarkably (p < 0.05), reactive oxygen species (ROS) levels and the expression levels of antioxidant enzyme genes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) increased significantly (p < 0.05). The overexpression of FHC for 72 h was found to significantly reduce the expression of CAT and SOD genes (p < 0.05). 3. Interfering with FHC expression revealed that the expression levels of the cell proliferation gene Aurora kinase A (AURORA-A) were significantly decreased (p < 0.05), while the expression levels of the apoptosis genes B-cell lymphoma-2 (BCL-2) and cysteine aspartate-specific protease 8 (CASPASE 8) increased (p < 0.05). Further research has shown that, when interfering with FHC expression for 72 h, apoptosis rate increased by 1.19-fold (p < 0.05), but the current data showed a lower apoptosis rate after FHC overexpression by 59.41%, 63.39%, and 52.31% at three different treatment times (p < 0.05). 4. In conclusion, FHC improved the antioxidant capacity of GCs, promotes GCs proliferation, and inhibits GCs apoptosis of ovarian follicles in Sichuan white geese. [ABSTRACT FROM AUTHOR]

Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment. [ABSTRACT FROM AUTHOR]

Geese are susceptible to oxidative stress during reproduction, which can lead to follicular atresia and impact egg production. Follicular atresia is directly triggered by the apoptosis and autophagy of granulosa cells (GCs). Adiponectin (ADPN), which is secreted by adipose tissue, has good antioxidant and anti-apoptotic capacity, but its role in regulating the apoptosis of GCs in geese is unclear. To investigate this, this study examined the levels of oxidative stress, apoptosis, and autophagy in follicular tissues and GCs using RT-qPCR, Western blotting, immunofluorescence, flow cytometry, transcriptomics and other methods. Atretic follicles exhibited high levels of oxidative stress and apoptosis, and autophagic flux was obstructed. Stimulating GCs with H2O2 produced results similar to those of atretic follicles. The effects of ADPN overexpression and knockdown on oxidative stress, apoptosis and autophagy in GCs were investigated. ADPN was found to modulate autophagy and reduced oxidative stress and apoptosis in GCs, in addition to protecting them from H2O2-induced damage. These results may provide a reasonable reference for improving egg-laying performance of geese. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The yak, a unique species of cattle found exclusively on the western plateau of China, is a valuable source of livelihood for local residents. However, their low fecundity restricts the expansion of yak farming, whereas regional factors limit studies on yak breeding. Granulosa cells (GCs), which provide essential steroid hormones and growth factors for oocytes, have been the focus of many studies on the mechanisms of follicular growth and atresia. This study aimed to establish an immortalized cell line model that could serve as a tool for future studies on the mechanisms of ovarian follicle development in yaks. First, we isolated primary yak granulosa cells (yGCs) and evaluated their replicative senescence after continuous in vitro subculturing. Subsequently, an immortalized culture method for primary yGC was explored, and a new cell line model was established to study the mechanism of follicular development in vitro. We used a mammalian gene expression lentivirus vector to transfer the simian virus 40 large T antigen (SV40T) into primary yGC to obtain an immortalized cell line. The immortalized yGCs were morphologically identical to the primary yGCs, and cell proliferation and growth were normal within a limited number of generations. Folliclestimulating hormone receptor (FSHR), a specific marker for GCs, was positively expressed in immortalized yGCs. Furthermore, the immortalized yGCs retained the ability of GCs to synthesize estradiol and progesterone and expressed genes related to steroid synthesis. The establishment of immortalized yGC opens up a myriad of possibilities for advancing our understanding of yak reproductive biology and improving yak breeding strategies. [ABSTRACT FROM AUTHOR]

Objective: To define the clinical, histopathological features and the prognostic factors affecting survival in patients with adult granulosa cell tumors of the ovary (AGCT). Methods: A 322 patients whose final pathologic outcome was AGCT treated at nine tertiary oncology centers between 1988 and 2021 participated in the study. Results: The mean age of the patients was 51.3 ± 11.8 years and ranged from 21 to 82 years. According to the International Federation of Gynecology and Obstetrics 2014, 250 (77.6%) patients were stage I, 24 (7.5%) patients were stage II, 20 (6.2%) patients were stage III, and 3 (7.8%) were stage IV. Lymphadenectomy was added to the surgical procedure in 210 (65.2%) patients. Lymph node involvement was noted in seven (3.3%) patients. Peritoneal cytology was positive in 19 (5.9%) patients, and 13 (4%) had metastases in the omentum. Of 285 patients who underwent hysterectomy, 19 (6.7%) had complex hyperplasia with atypia/endometrial intraepithelial neoplasia, and 8 (2.8%) had grade 1 endometrioid endometrial carcinoma. It was found that 93 (28.9%) patients in the study group received adjuvant treatment. Bleomycin, etoposide, cisplatin was the most commonly used chemotherapy protocol. The median follow-up time of the study group was 41 months (range, 1-276 months). It was noted that 34 (10.6%) patients relapsed during this period, and 9 (2.8%) patients died because of the disease. The entire cohort had a 5-year disease-free survival (DFS) of 86% and a 5-year disease-specific survival of 98%. Recurrences were observed only in the pelvis in 13 patients and the extra-abdominal region in 7 patients. The recurrence rate increased 6.168-fold in patients with positive peritoneal cytology (95% confidence interval [CI]=1.914-19.878; p=0.002), 3.755-fold in stage II-IV (95% CI=1.275-11.063; p=0.016), and 2.517-fold in postmenopausal women (95% CI=1.017-6.233; p=0.046) increased. Conclusion: In this study, lymph node involvement was detected in 3.3% of patients with AGCT. Therefore, it was concluded that lymphadenectomy can be avoided in primary surgical treatment. Positive peritoneal cytology, stage, and menopausal status were independent prognostic predictors of DFS. [ABSTRACT FROM AUTHOR]

ABSTRACT: Red light (RL) can enhance egg production in poultry. CircRNAs play a crucial role by serving as transcriptional regulators. However, their role in influencing follicle development in White King pigeons remains unexplored. In this study, 54 paired White King pigeons were chosen and divided into RL and white light (WL) groups, each with 3 subgroups. The egg production of paired pigeons in each replicate was recorded for 45 d, and the characteristics of follicle development were monitored during the laying interval (LI). The granulosa cell layer from follicles of the second-largest follicle (F2) was collected, and high-throughput sequencing was performed to elucidate the molecular mechanism of follicle development in pigeons. The study confirmed that RL enhances egg production in pigeons. Additionally, under RL, the F2 follicle was selected, while under WL, small follicles were kept on the third day (LI3). A total of 5,510 circRNAs were identified across all samples, revealing differentially expressed circRNAs (DECs) in various comparisons: 627 in RF1 vs. WF1, 900 in RF2 vs. WF2, 606 in RF1 vs. RF2, and 937 in WF1 vs. WF2. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that host genes of DECs were enriched in pathways like steroid hormone biosynthesis, oocyte meiosis, GnRH signaling pathway, and apoptosis pathway. Moreover, circRNA_5497, circRNA_2016, and circRNA_3328 were common DECs across 4 groups, sharing miRNA binding sites with follicle selection-associated genes. In conclusion, our findings suggest that RL promotes egg production by stimulating follicle selection during LI, offering insights into the regulatory mechanisms of circRNAs in follicle selection under RL. This knowledge can help enhance the reproductive performance of pigeons.

ABSTRACT: Follicle selection in chicken refers to the process of selecting a follicle to enter hierarchy from a cohort of small yellow follicles (SY) with a diameter of 6 to 8 mm. The follicle being selected will develop rapidly and ovulate. Follicle selection is a key stage affecting chicken egg-laying performance. Our previous study showed that the phosphorylation level of lysine (K)-specific demethylase 1A (LSD1) at serine 54 (LSD1Ser54p) was significantly increased in F6 follicles compared to prehierarchal SY follicles, but its function was unclear. Here, the mechanism of this modification, the effect of LSD1Ser54p dephosphorylation on gene expression profile of chicken hierarchal granulosa cells and the function of fibroblast growth factor 9 (FGF9) that is regulated by LSD1Ser54p were further investigated. The modification of LSD1Ser54p was predicted to be mediated by cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). Treatment of chicken hierarchal granulosa cells with CDK5 inhibitor significantly decreased LSD1Ser54p level (P < 0.05) and LSD1Ser54p interacted with CDK5, suggesting that, in the granulosa cells of chicken hierarchal follicles, LSD1Ser54p modification was carried out by CDK5. When the LSD1Ser54p level decreased in the granulosa cells of chicken hierarchal follicles, both the mRNA expression of FGF9 and α-actinin 2 (ACTN2) and the H3K4me2 level in their promoter regions significantly increased (P < 0.05), indicating that this phosphorylation modification enhanced the demethylation activity of LSD1. Moreover, in chicken hierarchal granulosa cells, overexpression of chicken FGF9 stimulated their proliferation and increased the mRNA expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b) and steroidogenic acute regulatory protein (StAR). This study collectively revealed that phosphorylation of LSD1 at serine 54 by CDK5 enhanced its demethylation activity in chicken ovarian granulosa cells and regulated genes including FGF9 that is engaged in chicken follicle selection.

Abstract Polycystic ovary syndrome (PCOS) is a complex reproductive endocrinological disorder influenced by a combination of genetic and environmental factors. Linoleic acid (LA) is a widely consumed ω-6 polyunsaturated fatty acid, accounting for approximately 80% of daily fatty acid intake. Building upon the prior investigations of our team, which established a connection between LA levels in the follicular fluid and PCOS, this study deeply examined the specific impact of LA using a granulosa cell line. Our findings revealed that LA exerts its influence on granulosa cells (GCs) by binding to the estrogen receptor (ER). Activated ER triggers the transcription of the FOXO1 gene. Reactive oxygen species (ROS)-related oxidative stress (OS) and inflammation occur downstream of LA-induced FOXO1 activation. Increased OS and inflammation ultimately culminate in GC apoptosis. In summary, LA modulates the apoptosis and inflammation phenotypes of GCs through the ER-FOXO1-ROS-NF-κB pathway. Our study provides additional experimental evidence to comprehend the pathophysiology of PCOS and provides novel insights into the dietary management of individuals with PCOS.