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

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

Metabonomics analysis of Zi goose follicular granulosa cells using ENO1 gene expression interference.

The Zi goose is native to North-east China and is noted for its high egg production. Alpha enolase (ENO1) is a glycolytic enzyme which functions as a plasminogen receptor in follicular granulosa cells (FGCs), with several studies showing that FGCs can support follicular development. By transfecting the ENO1 interfering plasmid (shRNA) into FGCs, ENO1 expression in these cells was downregulated, suggesting the successful knock-down of ENO1 in these cells. In this knock-down model, we detected 13 metabolites from FGCs using LC/MS. When compared with the non-coding shRNA (NC) group, the lower level metabolites were (R)-(+)-citronellic acid, altretamine, 3-hydroxycaproic acid, heptadecanoic acid, cholecalciferol vitamin D3, indole, benzoic acid, capric acid, caffeic acid, azelaic acid, 3,4-dihydroxyhydrocinnamic acid and cholic acid, while oleic acid was detected at high levels. To further examine the results of metabolomics, six key metabolites were verified by gas chromatography-mass spectrometry (GC-MS). We found that vitamin D3, indole, benzoic acid, capric acid and cholic acid were significantly downregulated in the shRNA group, while oleic acid was significantly upregulated. This observation was consistent with the metabolomics data. Through these studies, we found that decreased ENO1 levels altered certain metabolite levels in FGCs.

Presence of growth/differentiation factor-15 cytokine in human follicular fluid, granulosa cells, and oocytes.

PURPOSE: The purpose of the study was to determine whether the GDF-15 is present in follicular fluid; to evaluate if there is a relation between follicular and serum levels of GDF-15 and fertility status of study subjects; and to test whether granulosa cells, oocytes, or both produce GDF-15. METHODS: This study used follicular fluid (FF, serum, and oocytes obtained under informed consent from women undergoing oocyte retrieval for in vitro fertilization. It also used ovaries from deceased preterm newborns. Collection of FF and blood at the time of oocyte retrieval, ELISA and western blot were performed to determine levels and forms of GDF-15. Concentrations of GDF-15 in FF and serum, its expression in ovarian tissue, and secretion from granulosa cells were analyzed. RESULTS: GDF-15 concentration in FF ranged from 35 to 572 ng/ml, as determined by ELISA. Western blot analysis revealed the GDF-15 pro-dimer only in FF. Both normal healthy and cancerous granulosa cells secreted GDF-15 into culture media. Primary oocytes displayed cytoplasmic GDF-15 positivity in immunostained newborn ovaries, and its expression was also observed in fully grown human oocytes. CONCLUSIONS: To the best of our knowledge, this is the first documentation of cytokine GDF-15 presence in follicular fluid. Its concentration was not associated with donor/patient fertility status. Our data also show that GDF-15 is expressed and inducible in both normal healthy and cancerous granulosa cells, as well as in oocytes.

The dual role of ATG7: Regulation of autophagy and apoptosis in porcine ovarian follicular granulosa cells.

The regulation of mammalian ovarian development involves the coordinated processes of autophagy and apoptosis. The autophagy-related gene ATG7 plays a pivotal role in mediating crosstalk between these pathways. Despite its recognized importance, the specific function of ATG7 in ovarian follicular granulosa cells remains poorly understood. This study aimed to explore the effects of ATG7 overexpression on apoptosis and autophagy in porcine ovarian follicular granulosa cells and thereby provide insights into the interplay between these fundamental cellular mechanisms. An ATG7 overexpression vector was introduced into cells, followed by assessment of cell proliferation using the CCK-8 assay, quantification of related gene expression via real-time quantitative PCR and western blotting, and evaluation of apoptosis using TUNEL staining. ATG7 exhibited a predominant cytoplasmic localization and additional nuclear expression in porcine ovarian follicular granulosa cells. The transfection efficiency of the vector was initially verified, indicating that its overexpression notably increased expression of ATG7 protein. Further investigations confirmed that overexpression of ATG7 inhibited cell proliferation, stimulated autophagy, and promoted apoptosis in these cells. In summary, overexpression of ATG7 influences the viability of porcine ovarian follicular granulosa cells by regulating the interplay between autophagy and apoptosis. This study not only broadens the understanding of functional regulation of autophagy and apoptosis by ATG7, but also sheds light on the intricate mechanisms governing ovarian follicular atresia.

Notch2 Regulates the Function of Bovine Follicular Granulosa Cells via the Wnt2/ß-Catenin Signaling Pathway.

Ovarian follicular GCs are strongly implicated in the growth, development, and atresia of ovarian follicles. The Wnt/ß-catenin and Notch signaling pathways participate in GC proliferation, differentiation, apoptosis, and steroid hormone production during follicular development. However, the crosstalk between Wnt and Notch signaling in GCs remains unclear. This study investigated this crosstalk and the roles of these pathways in apoptosis, cell cycle progression, cell proliferation, and steroid hormone secretion in bovine follicular GCs. The interaction between ß-catenin and Notch2 in GCs was assessed by overexpressing CTNNB1, which encodes ß-catenin. The results showed that inhibiting the Notch pathway by Notch2 silencing in GCs arrested the cell cycle, promoted apoptosis, reduced progesterone (P4) production, and inhibited the Wnt2-mediated Wnt/ß-catenin pathway in GCs. IWR-1 inhibited Wnt2/ß-catenin and Notch signaling, reduced GC proliferation, stimulated apoptosis, induced G1 cell cycle arrest, and reduced P4 production. CTNNB1 overexpression had the opposite effect and increased 17ß-estradiol (E2) production and Notch2 protein expression. Co-immunoprecipitation assays revealed that Notch2 interacted with ß-catenin. These results elucidate the crosstalk between the Wnt/ß-catenin and Notch pathways and the role of these pathways in bovine follicular GC development.

Selenium Protects Follicular Granulosa Cells from Apoptosis Induced by Mercury Through Inhibition of ATF6/CHOP Pathway in Laying Hens.

The purpose of this research was to explore the effect of selenium on mercury-mediated apoptosis of follicular granulosa cells in laying hens. Moreover, the ATF6/CHOP pathway was investigated to explore the mechanism in this progress. Hg, Se, and 4-phenyl butyric acid were used alone or in combination to treat the cells. Our results showed that the nuclear in cells became condensate after Hg exposure, while Se addition significantly alleviated this change. Hg exposure significantly induced the apoptosis and the reduction of mitochondrial membrane potential in cells (P < 0.05). Nevertheless, co-treatment of Se significantly inhibited these effects (P < 0.05). Additionally, Hg exposure dramatically elevated the gene expressions of Bax/Bcl-2 (P < 0.05), caspase-3 (P < 0.05), caspase-9 (P < 0.05), protein kinase RNA-like endoplasmic reticulum kinase (P < 0.05), activating transcription factor 6 (P < 0.05), C/EBP homologous protein (CHOP; P < 0.05), inositol-requiring enzyme 1α (P < 0.05), tumor necrosis factor-associated factor 2 (P < 0.05), activating transcription factor 6 (ATF6; P < 0.05), and apoptosis signal-regulating kinase 1 (P < 0.05) in cells, whereas Se addition avoided these changes. The exposure to Hg considerably boosted the expression of ATF6 and CHOP protein (P < 0.05), while Se addition significantly alleviated the above-mentioned enhancements (P < 0.05). In summary, Hg exposure induced apoptosis, which was considerably reduced alleviated by Se addition, which was linked to the ATF6/CHOP pathway in follicular granulosa cells in laying hens.

Proteomic Analysis Identifies Distinct Protein Patterns for High Ovulation in FecB Mutant Small Tail Han Sheep Granulosa Cells.

The Booroola fecundity (FecB) mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene increases ovulation in sheep. However, its effect on follicular maturation is not fully understood. Therefore, we collected granulosa cells (GCs) at a critical stage of follicle maturation from nine wild-type (WW), nine heterozygous FecB mutant (WB), and nine homozygous FecB mutant (BB) Small Tail Han sheep. The GCs of three ewes were selected at random from each genotype and consolidated into a single group, yielding a total of nine groups (three groups per genotype) for proteomic analysis. The tandem mass tag technique was utilized to ascertain the specific proteins linked to multiple ovulation in the various FecB genotypes. Using a general linear model, we identified 199 proteins significantly affected by the FecB mutation with the LIMMA package (p < 0.05). The differential abundance of proteins was enriched in pathways related to cholesterol metabolism, carbohydrate metabolism, amino acid biosynthesis, and glutathione metabolism. These pathways are involved in important processes for GC-regulated 'conservation' of oocyte maturation. Further, the sparse partial least-squares discriminant analysis and the Fuzzy-C-mean clustering method were combined to estimate weights and cluster differential abundance proteins according to ovulation to screen important ovulation-related proteins. Among them, ZP2 and ZP3 were found to be enriched in the cellular component catalog term "egg coat", as well as some apolipoproteins, such as APOA1, APOA2, and APOA4, enriched in several Gene Ontology terms related to cholesterol metabolism and lipoprotein transport. A higher abundance of these essential proteins for oocyte maturation was observed in BB and WB genotypes compared with WW ewes. These proteins had a high weight in the model for discriminating sheep with different FecB genotypes. These findings provide new insight that the FecB mutant in GCs improves nutrient metabolism, leading to better oocyte maturation by altering the abundance of important proteins (ZP2, ZP3, and APOA1) in favor of increased ovulation or better oocyte quality.

Expression of genes regulating cell division in porcine follicular granulosa cells.

BACKGROUND: Cell cycle regulation influences the proliferation of granulosa cells and affects many processes related to ovarian folliclular growth and ovulation. Abnormal regulation of the cell cycle can lead to many diseases within the ovary. The aim of this study was to describe the expression profile of genes within granulosa cells, which are related to the formation of the cytoskeleton, organization of cell organelles inside the cell, and regulation of cell division. Established in vitro primary cultures from porcine ovarian follicle granulosa cells were maintained for 48, 96, 144 h and evaluated via microarray expression analysis. RESULTS: Analyzed genes were assigned to 12 gene ontology groups "actin cytoskeleton organization", "actin filament organization", "actin filament-based process", "cell-matrix adhesion", "cell-substrate adhesion", "chromosome segregation", "chromosome separation", "cytoskeleton organization", "DNA integrity checkpoint", "DNA replication initiation", "organelle fision", "organelle organization". Among the genes with significantly changed expression, those whose role in processes within the ovary are selected for consideration. Genes with increased expression include (ITGA11, CNN1, CCl2, TPM2, ACTN1, VCAM-1, COL3A1, GSN, FRMD6, PLK2). Genes with reduced expression inlcude (KIF14, TACC3, ESPL1, CDC45, TTK, CDC20, CDK1, FBXO5, NEK2-NIMA, CCNE2). For the results obtained by microarray expressions, quantitative validation by RT-qPCR was performed. CONCLUSIONS: The results indicated expression profile of genes, which can be considered as new molecular markers of cellular processes involved in signaling, cell structure organization. The expression profile of selected genes brings new insight into regulation of physiological processes in porcine follicular granulosa cells during primary in vitro culture.

CYP19A1 May Influence Lambing Traits in Goats by Regulating the Biological Function of Granulosa Cells.

Abnormal expression of CYP19A1, a gene related to steroid hormone synthesis, causes steroid hormone disruption and leads to abnormal ovulation in granulosa cells. However, the exact mechanism of CYP19A1 regulation is unclear. In this study, we confirmed the localization of CYP19A1 in goat ovarian tissues using immunohistochemistry. Subsequently, we investigated the effects of CYP19A1 on granulosa cell proliferation, steroid hormone secretion, and expression of candidate genes for multiparous traits by overexpressing and silencing CYP19A1 in goat granulosa cells (GCs). The immunohistochemistry results showed that CYP19A1 was expressed in all types of follicular, luteal, and granulosa cells, with subcellular localization results revealing that CYP19A1 protein was mainly localized in the cytoplasm and nucleus. Overexpression of CYP19A1 significantly increased the mRNA levels of CYP19A1, FSHR, and INHBA, which are candidate genes for multiple birth traits in goats. It also promoted cell proliferation, PCNA and Cyclin E mRNA levels in granulosa cells, and secretion of estrogen and progesterone. However, it inhibited the mRNA levels of STAR, CYP11A1, and 3ßSHD, which are genes related to steroid synthesis. Silencing CYP19A1 expression significantly reduced CYP19A1, FSHR, and INHBA mRNA levels in granulosa cells and inhibited granulosa cell proliferation and PCNA and Cyclin E mRNA levels. It also reduced estrogen and progesterone secretion but enhanced the mRNA levels of STAR, CYP11A1, and 3ßSHD. CYP19A1 potentially influenced the lambing traits in goats by affecting granulosa cell proliferation, hormone secretion, and expression of candidate genes associated with traits for multiple births.

[Effects of bisphenol A on apoptosis of ovarian preantral follicular granulosa cells and ovarian development in mice].

OBJECTIVE: To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism. METHODS: Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured in vitro. The cultured cells were treated with 0, 1, 10, 50, 100, 150, 200 and 500 µmol/L BPA, and the changes in cell proliferation, cell cycle, apoptosis and mitochondrial membrane potential were analyzed with CCK-8 method and flow cytometry. The protein expressions of Bcl-2, Bax, p53 and cyclin D1 in the treated cells were determined with Western blotting. Pregnant ICR mice were treated for a week with BPA at the concentration that produced significant effects on the preantral follicular granulosa cells, and the weight changes of the pregnant mice were recorded. The ovarian tissues of the offspring female mice were weighed at PND 10, 17, 21 and 42 followed by histological observation with HE staining and examination of Bcl-2 mRNA expression level with RT-qPCR. RESULTS: Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 µmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (P < 0.05). Exposure to a higher BPA concentration at 200 µmol/L obviously enhanced cell apoptosis by reducing the mitochondrial membrane potential and repressed the cell proliferation (P < 0.05). BPA exposure at 50 µmol/L and 200 µmol/L produced opposite effects on the protein expressions of Bcl-2 (P < 0.01), Bax (P < 0.05) and p53 (P < 0.05) in mouse ovarian preantral follicular granulosa cells. BPA exposure at the doses of 10 and 35 mg/kg caused rapid weight increment of the pregnant mice and changes in ovarian index of the offspring female mice. In the offspring female mice, the changes in Bcl-2 mRNA expression in the ovarian tissue showed a similar pattern to that of ovarian index. Exposure of the pregnant mice to a high BPA concentration at 35 mg/kg resulted in accelerated follicular development into antral follicular stage in PND 21 offspring female mice. CONCLUSIONS: BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

EGFR promotes the proliferation of quail follicular granulosa cells through the MAPK/extracellular signal-regulated kinase (ERK) signaling pathway.

Follicles develop into preovulatory follicles during folliculogenesis and the majority of small yellow follicles become atretic and gets reabsorbed. In this study, based the RNA-seq results of duck ovary, epidermal growth factor receptor (EGFR) was selected as a candidate gene in follicular development and the role was explored. The results demonstrated that EGFR-P8 was the quail EGFR core promoter. It had an E2F4 binding site within EGFR core promoter. E2F4 overexpression significantly increased EGFR expression in quail granulosa cells (GCs). However, the effect was abolished when the GCs were treated with corynoxeine, an inhibitor of the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) signaling pathway. Moreover, luciferase reporter assay and chromatin immunoprecipitation experiments showed that E2F4 upregulated the expression of EGFR expression, which increased E2 and P4 production. In addition, EGFR regulated GCs proliferation and affected follicular development. Taken together, our findings suggested that EGFR, which was regulated by E2F4, enhanced the expression of MAPK/ERK pathway components and follicular development. These results provided an important basis for an improved understanding of the MAPK/ERK pathway and new insight into the development of quail follicles.

Effects of bisphenol A on apoptosis of ovarian preantral follicular granulosa cells and ovarian development in mice / 南方医科大学学报

OBJECTIVE@#To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism.@*METHODS@#Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured @*RESULTS@#Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (@*CONCLUSIONS@#BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

Antagonism of resveratrol on oxidative stress injury of follicular granulosa cells / 解剖学报

Objctive To investigate the protective effect of resveratrol on oxidative stress damage of follicular granulosa cells induced by hydrogen peroxide. Methods Granulosa cells were collected from the follicular fluid of in vitro fertilization(IVF) patients after oocyte retrieval and cultured. The cultured granulosa cells were divided into four groups: control group, injury model group, 10 μmol / L resveratrol group and 50 μmol / L resveratrol group. Cell viability was determined by CCK-8 assay, malondialdehyde(MDA) content by thiobarbituric(TBA) assay, superoxide dismutase(SOD) level by water soluble tetrazdium-1(WST-1) assay, apoptosis by AnnexinV-FITC / PI double-staining flow cytometry, Bcl-2 and Caspase-3 protein expression by Western blotting, and progesterone secretion by competitive ELISA. Resutls Compared with the control group, the cell viability, SOD level, Bcl-2 protein expression and progesterone secretion were significantly decreased in the injury model group, while MDA content, apoptosis rate and Caspase-3 apoptotic protein expression were significantly increased (P<0. 05) . Compared with the injury model group, the 10 μmol / L resveratrol group showed no statistically significant differences in various parameters; however, the cell viability, SOD level, progesterone secretion, and Bcl-2 and silent information regulator factor 2 related enzyme 1(SIRT1) protein expression were significantly increased, and the MDA content, apoptosis rate, and Caspase-3 apoptotic protein expression were significantly decreased in the 50 μmol / L resveratrol group (P < 0. 05) . Conclusion 50 μmol / L resveratrol can increase the activity of SIRT1, enhance the anti-oxidation and anti-apoptosis ability of granulosa cells and improve the function of granulosa cells.