Granulosa Cell Function Research Articles

Steroidogenic activity of liposomal methylated resveratrol analog 3,4,5,4′-tetramethoxystilbene (DMU-212) in human luteinized granulosa cells in a primary three-dimensional in vitro model

PurposeSteroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model.MethodsDMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels.ResultslipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity.ConclusionsThis is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.

Effect of IGF1 and FSH on the function of granulosa cells from prehierarchal follicles in chickens†.

Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3-5 and 6-8mm prehierarchal follicles were cultured with IGF1 (0, 10, 100ng/mL) in the presence or absence of FSH (0, 10ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6-8mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6-8mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6-8mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6-8mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3-5mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

Evaluation of the p53 pathway in polycystic ovarian syndrome pathogenesis and apoptosis enhancement in human granulosa cells through transcriptome data analysis

The polycystic ovarian syndrome (PCOS) is closely associated with enhanced apoptosis of granulosa cells, which have a vital role in maturation of oocytes. p53 plays a critical role in the regulation of apoptosis and cell cycle arrest, metabolism and insulin resistance. The aim of this study was to investigate the impact of p53 pathway in enhancing apoptosis and abnormal function of granulosa cells. In this study, microarray analysis and RNA sequencing were downloaded from the GEO and used as datasets. Principal Component Analysis (PCA) and online SSizer tool were applied to evaluate the experiment quality control and sample sufficiency, respectively. Bioinformatics’ analyses were performed on the selected datasets, and validated by qRT-PCR and western blot analyses. Three datasets out of five ones were chosen for re-analyzing based on the PCA outcomes. 21 deregulated genes were identified via filters including p < 0.05 and |log2FC|≥ 1. Functional enrichment analysis confirmed the relevance of cell cycle regulation and apoptosis as common biological hallmarks in PCOS. Results have shown differentially expressed p53 target genes involved in apoptosis (BAX, FAS, PMAIP1, and CASP8), cell cycle (Cyclins, Cyclin dependent kinases), glucose metabolism and insulin resistance (THBS1), and p53 regulation (MDM2). Subsequently, the relative mRNA expression of FAS, PMAIP1 and MDM2 genes, and protein levels of p53 and MDM2 were confirmed using granulosa cells collected from 20 PCOS women and 18 control individuals by qRT-PCR and western blot, respectively. Results of this study represent the possible role of p53 pathway in pathogenesis of PCOS particularly, through the enhancement of apoptosis in granulosa cells.

Effect of human granulosa cells vitrification on the expression of oocyte secreted factors

Background: Oocyte vitrification is a helpful fertility preservation technique for women at risk of losing their ovarian functions. Meanwhile, its application extends with the emerging trend of delayed childbearing, postponed marriage, and further diverse ethical, medical, legal, and social implications. Whereas its effect on oocytes has been widely elucidated, the potential impact of this procedure on the biological functions of granulosa cells remains poorly understood. The present study aimed to assess vitrification’s effect on the granulosa cells (GCs). Methods: 35 women with polycystic ovary syndromes who underwent in vitro fertilization (IVF) in Morula IVF Jakarta Clinic were recruited. The expression of prominent oocytes secreted factors (OSFs), including GDF-9 and BMP-15, were measured at messenger RNA (mRNA) levels. GCs from mature and immature oocytes were collected and calculated separately. Relative expression of GDF-9 and BMP-15 was quantified by a real-time quantitative PCR (RT-qPCR). All of the data was analyzed by using SPSS. Bivariate analysis used the Mann-Whitney test at a 95% confidence level. A significant p-value was ≤ 0.05. Results: The mean age of subjects was 32,39 ± 3,33 years old, with the mean duration of infertility being six years. The mean BMI, anti-Mullerian hormone (AMH) level, and antral follicle count (AFC) were 24.04 kg/m2, 3.43 ng/mL, and 15 follicles, respectively. The basal FSH, LH, estradiol, and progesterone were at the normal level. The expression of GDF-9 decreased significantly (0.5-fold, p &lt; 0.01) in the immature vitrified GC group but was not in the mature-vitrified GC group. Meanwhile, the expression of BMP-15 was stable in all examined groups (P&gt;0.05). Conclusion: Our results suggest that vitrification may alter oocyte maturation, as demonstrated by reduced GDF-9 expression in the immature vitrified GC group.

Lipopolysaccharide accelerates tryptophan degradation in the ovary and the derivative kynurenine disturbs hormone biosynthesis and reproductive performance

Lipopolysaccharide (LPS), also known as endotoxin, is a component of the outer membrane of gram-negative bacteria. LPS is released into the surrounding environment during bacterial death and lysis. Due to its chemical and thermal stability, LPS can be detected anywhere and easily exposed to humans and animals. Previous studies have shown that LPS causes hormonal imbalances, ovarian failure, and infertility in mammals. However, the potential mechanisms remain unclear. In this study, we investigated the effects and mechanisms of LPS on tryptophan degradation, both in vivo and in vitro. The effects of kynurenine, a tryptophan derivative, on granulosa cell function and reproductive performance were explored. Results showed that p38, NF-κB, and JNK signaling pathways were involved in LPS-induced Ido1 expressions and kynurenine accumulation. Furthermore, the kynurenine decreased estradiol production, but increased granulosa cell proliferation. In vivo, experiments showed that kynurenine decreased estradiol and FSH production and inhibited ovulation and corpus luteum formation. Additionally, pregnancy and offspring survival rates decreased considerably after kynurenine treatment. Our findings suggest that kynurenine accumulation disrupts hormone secretion, ovulation, corpus luteal formation, and reproductive performance in mammals.

P-735 Are there any differences in preimplantation blastocyst chromosomal abnormalities between polycystic ovary syndrome patients and controls? A multi-center retrospective cohort study

Abstract Study question Are there possible differences in the preimplantation blastocyst chromosome aberrations between polycystic ovary syndrome (PCOS) patients and controls? Summary answer PCOS lowers aneuploidy risk but increases mosaicism risk in preimplantation embryos, and PCOS-related insulin resistance should be investigated as a potential cause. What is known already PCOS is thought to alter granulosa cell functions and effects on oocyte development, potentially leading to chromosomal abnormalities and increasing the risk of pregnancy loss. However, few studies have compared the chromosomal status of preimplantation embryos from PCOS patients to those from healthy controls. Study design, size, duration This was a multi-center retrospective cohort study conducted at 3 IVF centers. A total of 707 blastocysts from 147 PCOS patients and 3006 blastocysts from 821 control women receiving preimplantation genetic testing (PGT) between 2015 and 2021. Participants/materials, setting, methods PGT was performed using next-generation sequencing (NGS). Differences in the chromosome aberrations were compared. Multivariate and subgroup analyses were conducted to control for confounders. Influences of insulin resistance and PCOS phenotype on the risks of specific chromosomal abnormalities were also examined. Main results and the role of chance Compared to controls, blastocysts from PCOS patients demonstrated lower aneuploidy rate (14.7% vs. 25.4%, P &amp;lt; 0.001) but greater mosaicism rate (16.5% vs. 8.7%, P &amp;lt; 0.001). Multivariable analysis adjusting for age, IVF center, body mass index (BMI), total gonadotropin (Gn) dose, and peak serum estradiol (E2) on trigger day, identified PCOS as an independent protective factor against embryonic aneuploidy (adjusted OR = 0.56, 95% CI, 0.42–0.76, P&amp;lt;0.001) but a risk factor for embryonic mosaicism (adjusted OR = 2.05, 95% CI 1.48–2.85, P&amp;lt;0.001). These differences in chromosomal spectrum were also observed in subgroups stratified by age, IVF center and BMI. Further multivariate analysis suggested that insulin resistance could be responsible for the increased risk of embryonic mosaicism among PCOS patients (OR = 1.89, 95% CI, 1.06–3.34, P=0.03). Limitations, reasons for caution The main limitation is the retrospective design. Besides, methodological uncertainties still exist in detecting mosaicism in current reproductive medicine. A longer-term, multiple-center prospective study with greater methodological standardization is warranted to confirm the present results. Wider implications of the findings These results suggest that PCOS pathology may increase the risk of mitosis errors but not the risk of meiosis errors during oocyte development, and PCOS-related insulin resistance should be investigated as a potential cause. Trial registration number 2020[325]

Transcriptomic analysis of granulosa cells in patients with endometriosis-related infertility: identification of potential molecular mechanisms

Objective: To investigate the impact of endometriosis on granulosa cell function and elucidate the molecular mechanisms involved. Methods: RNA sequencing, differential expression analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, gene set enrichment analysis, protein-protein interaction analysis, and RT-qPCR were employed to assess the effects of endometriosis on granulosa cell function. Results: The results revealed significant differences in gene expression between the endometriosis and control groups, including genes related to immune regulatory functions and ferroptosis. Hub gene modules and hub genes were identified, including those related to cell cycle and immune and inflammatory pathways. RT-qPCR revealed significant up-regulation of the hub genes CCL3 and IL1B in granulosa cells of patients with endometriosis. Conclusion: The results of RNA sequencing demonstrated that endometriosis induces significant transcriptional alterations in granulosa cells of affected patients. These findings provide important insights into the diagnosis and treatment of endometriosis and highlight the importance of further investigation of CCL3 and IL1B as potential biomarkers for endometriosis.

Organophosphate Esters Disrupt Steroidogenesis in KGN Human Ovarian Granulosa Cells.

Organophosphate esters (OPEs) are used extensively as flame retardants and plasticizers and are found ubiquitously in the environment and human matrices. Previous studies suggested that exposure to some of these chemicals may disrupt the homeostasis of female sex hormones and have detrimental effects on female fertility. Here, we determined the effects of OPEs on the function of KGN ovarian granulosa cells. We hypothesized that OPEs alter the steroidogenic ability of these cells by dysregulating the expression of transcripts involved in steroid and cholesterol biosynthesis. KGN cells were exposed for 48 hours to 1 of 5 OPEs (1-50μM): triphenyl phosphate (TPHP), tris(methylphenyl) phosphate (TMPP), isopropylated triphenyl phosphate (IPPP), tert-butylphenyl diphenyl phosphate (BPDP), and tributoxyethyl phosphate (TBOEP), or to a polybrominated diphenyl ether flame retardant, 2,2',4,4' tetrabromodiphenyl ether (BDE-47), in the presence or absence of Bu2cAMP. OPEs increased the basal production of progesterone (P4) and 17β-estradiol (E2) and had either no effect or inhibited Bu2cAMP-stimulated P4 and E2 synthesis; exposure to BDE-47 had no effect. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that OPEs (≥5μM) increased the basal expression of critical genes (STAR, CYP11A1, CYP19A1, HSD3B2, and NR5A1) involved in steroidogenesis; upon stimulation, the expression of all genes tested was downregulated. An overall inhibition in cholesterol biosynthesis was induced by OPEs, characterized by a downregulation in HMGCR and SREBF2 expression. TBOEP consistently showed the least effect. Therefore, OPEs perturbed steroidogenesis in KGN granulosa cells by targeting the expression of steroidogenic enzymes and cholesterol transporters; these effects may have an adverse impact on female reproduction.

CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9

BackgroundClock circadian regulator (CLOCK) is a core factor of the mammalian biological clock system in regulating female fertility and ovarian physiology. However, CLOCK's specific function and molecular mechanism in porcine granulosa cells (GCs) remain unclear. In this study, we focused on CLOCK’s effects on GC proliferation.ResultsCLOCK significantly inhibited cell proliferation in porcine GCs. CLOCK decreased the expression of cell cycle-related genes, including CCNB1, CCNE1, and CDK4 at the mRNA and protein levels. CDKN1A levels were upregulated by CLOCK. ASB9 is a newly-identified target of CLOCK that inhibits GC proliferation; CLOCK binds to the E-box element in the ASB9 promoter.ConclusionsThese findings suggest that CLOCK inhibits the proliferation of porcine ovarian GCs by increasing ASB9 level.

Perfluorooctanoic acid (PFOA) affects steroidogenesis and antioxidant defence in granulosa cells from swine ovary

PFOA is mainly employed in products with water and oil repellent properties. Due to its persistence, bioaccumulation and critical effects on health, its use has been restricted in several countries. This research was intended to explore PFOA action on the main functions of swine ovarian granulosa cells, a valuable model for translational medicine. Moreover, since we previously demonstrated a disruptive effect on free radical generation we sought to explore PFOA effects on the main antioxidant enzymes. PFOA inhibited cell proliferation (p < 0.001), assessed by BrdU uptake. Steroidogenesis was disrupted: PFOA also stimulated 17β-estradiol production (p < 0.05), increased progesterone production (p < 0.05) at the lowest dose while it displayed an inhibitory effect at higher concentrations (p < 0.05). SOD (p < 0.001), catalase (p < 0.05) and peroxidase (p < 0.01) activities were stimulated. Therefore, our study supports a disruptive effect of PFOA in cultured swine granulosa cells.

MicroRNA miR-152 can support ovarian granulosa cell functions and modify apigenin actions

The study aimed to evaluate the involvement of apigenin, microRNA (miR)-152, and their interrelationships in the control of basic ovarian granulosa cell functions. The effects of apigenin (0, 10, and 100 µg/mL), miR-152 analogues or miR-152 inhibitor, and their combinations with apigenin on porcine granulosa cells were examined. Expression levels of miR-152, viability, proliferation, apoptosis, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analyzed. Apigenin increased the expression of miR-152, cell proliferation, and estradiol release and reduced apoptosis, progesterone, and IGF-I output. MicroRNA-152 analogues promoted cell viability and proliferation, as well as the release of progesterone, IGF-I, oxytocin, and prostaglandin E2; however, it inhibited apoptosis and estradiol output. miR-152 inhibitor had the opposite effect. Moreover, miR-152 analogues suppressed the effect of apigenin on cell apoptosis and estradiol release. These observations 1) confirm the involvement of apigenin in the control of basic ovarian cell functions; 2) are the first demonstration of importance of miR-152 in the control of these functions; 3) show the ability of apigenin to promote miR-152 expression and the ability of miR-152 to modify apigenin effects on ovarian cells.

Effects of cortisol on granulosa cell function during follicle maturation in the horse

Follicle maturation and acquisition of developmental competence by the oocyte require an interplay between granulosa cells and the oocyte, involving Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) mediated modulation of follicular steroidogenesis. In the horse, dynamic intrafollicular concentrations of cortisol were associated with follicle growth and oocyte maturation, suggesting a functional relationship between cortisol and oocyte-derived factors. The aim of this study was to analyze effects of cortisol on steroidogenic activity, apoptosis and proliferative capacity of equine granulosa cells, as well as the relationship with the oocyte-derived factor GDF9. For this purpose, granulosa cells were collected three times in four mares each during the breeding season by transvaginal aspiration in the absence of a dominant follicle. Cells collected on the same day were pooled and then seeded on 12-well plates to grow to 70-80% confluence before being stimulated with 100 ng/ml IGF1 for 24h, in the presence or absence of cortisol in increasing concentrations: 50, 100 and 150 ng/ml. These concentrations were previously observed in antral follicles before deviation (50 ng/ml) and after deviation (120 ng/ml). In blocking experiments, cells were preincubated with 20 uM H89 (protein kinase A inhibitor). All experiments were repeated three times. GraphPad software was used for statistical analysis with one-way analysis of variance (ANOVA), followed by non-parametric multiple comparison post-hoc test.Expression of Star was similar in all groups (P>0.05), independent of cortisol concentration and was decreased (P<0.05) after preincubation with H89. Transcriptional levels of HSD3B2 were higher (P<0.001) after adding IGF1 and cortisol simultaneously, but not separately. The rise in HSD3B2 levels was independent of cortisol dose used (P>0.05). Blocking of Star with H89 increased GDF9 expression (P<0.01), both in the presence and absence of cortisol. Interestingly, addition of cortisol alone in all analyzed dosages raised BCL2 levels (P<0.05) and this effect was inhibited by H89 (P<0.05). However, there was no effect on CASP3 levels (P>0.05), regardless of treatment used. A higher proliferating capability of equine granulosa cells, as assessed by PCNA levels, was observed in response to IGF1 treatment (P<0.05), but only in the presence of 150 ng/ml cortisol. Despite apparently elevated, the expression of Ki67 marker in response to IGF1 in the presence of cortisol proved to be not significant (P>0.05). Taken together, our results demonstrate enhanced steroidogenesis as well as reduced apoptosis in equine granulosa cells in response to cortisol. Moreover, reduced steroidogenic activity (i.e. decreased expression of genes involved in progesterone synthesis) of granulosa cells increased GDF9 expression, which has previously been shown to be detrimental for the acquisition of developmental competence by the oocyte.

Effects of feeding exogenous vitamins and microelements on follicular development and molecular function of granulosa cells in donkeys

In mammalian ovarian physiology, vitamins and microelements are essential for follicular development, ovulation, and the synthesis and secretion of hormones and growth factors. However. the molecular mechanisms by which they influence maturation and growth of follicles in donkeys are still unknown. Here, we investigated how vitamins and microelements affected the development of follicles and the proliferation of ovarian granulosa cells (GCs). For this experiment, 40 jennies (average body weight: 292.946±33.543 kg, age: 3∼4 years) were randomly assigned into 4 groups (n=10) and fed 50% (A), 75% (B), 100% (C), and 150% (D) of a mixture of vitamins A, D and E and microelements once a day (dose 5 ml) for two consecutive months. After that, ultrasonography was applied to record the change in follicular dynamics in three estrous cycles. Using a transvaginal ultrasound-guided aspiration device to obtain follicular fluid (containing GCs) from the follicles (Ф=30mm-35mm), multi-omics analysis revealed its molecular mechanisms and physiological functions. The results showed that the time required for growing follicles to reach ovulation (Ф>40mm) was shortened in the vitamin D group compared to the three other groups(P<0.05). Of note, the follicles (Ф=30mm-35mm) in the vitamin C group had less discrete diameters and developed more evenly and had significantly more twin ovulations than the other three groups (P<0.05). The omics analysis showed that945 metabolites, including organic acids and derivatives (24.44%), lipids and lipid-like molecules (24.34%), organic heterocyclic compounds (13.44%), and benzenoids (12.06%) were identified in follicular fluid. The results also revealed 232, 207, 210 and 202 differential cumulative metabolites (DAMs) in group A vs. group B, group B vs. group C, group C vs. group D and group A vs. group C, respectively. The DAMs were most significantly enriched in protein digestion and absorption, ABC transporters, amino acid biosynthesis, mineral absorption, aspartate-arginine biosynthesis, and ovarian steroid synthesis process. Meanwhile, nine DEGs (INSR, CYP19A1, ALOX5, PLA2G4A, ADCY6, CYP1B1, PRKACB, CYP17A1, and PLA2G4F) associated with the secretory function of GCs were involved in the ovarian steroidogenesis pathway, and they were significantly correlated with estradiol (E2), cholesterol, progesterone (P4), and testosterone (T)(P<0.05). Furthermore, STEAP2, CYBRD1, ATP1B3, ATP1A1, SLC26A6, SLC5A1, SLC6A19, SLC40A1, and TRPV6 were closely associated with the mineral absorption of GCs, and they had a positive correlation with glycine and L-methionine (P<0.05). In conclusion, exogenous supplementation of jennies with vitamins A, D and E and micronutrients can contribute to follicular growth and ovulation and this was associated with the interaction and expression of key genes related to -metabolites in ovarian steroidogenesis and mineral absorption pathways.

Mechanisms of and Potential Medications for Oxidative Stress in Ovarian Granulosa Cells: A Review.

Granulosa cells are essential for follicle initiation and development, and their abnormal function or apoptosis is a crucial factor leading to follicular atresia. A state of oxidative stress occurs when the balance between the production of reactive oxygen species and the regulation of the antioxidant system is disturbed. Oxidative stress is one of the most important causes of the abnormal function and apoptosis of granulosa cells. Oxidative stress in granulosa cells causes female reproductive system diseases, such as polycystic ovary syndrome and premature ovarian failure. In recent years, studies have confirmed that the mechanism of oxidative stress in granulosa cells is closely linked to the PI3K-AKT signaling pathway, MAPK signaling pathway, FOXO axis, Nrf2 pathway, NF-κB signaling pathway, and mitophagy. It has been found that drugs such as sulforaphane, Periplaneta americana peptide, and resveratrol can mitigate the functional damage caused by oxidative stress on granulosa cells. This paper reviews some of the mechanisms involved in oxidative stress in granulosa cells and describes the mechanisms underlying the pharmacological treatment of oxidative stress in granulosa cells.

Effects of vascular endothelial growth factor (VEGF) on the viability, apoptosis and steroidogenesis of yak (Bos grunniens) granulosa cells

Vascular endothelial growth factor (VEGF) is crucial for follicle development through the regulation of granulosa cell (GC) function in some mammals, but its mechanism is unclear in yak (Bos grunniens). Therefore, the objectives of this study were to investigate the effects of VEGF on the viability, apoptosis and steroidogenesis of yak GCs. First, we investigated the localization of VEGF and its receptor (VEGFR2) in yak ovaries by immunohistochemistry analysis and evaluated the effect of culture medium containing different VEGF concentrations and culture times on the viability of yak GCs by Cell Counting Kit-8. Then, optimal treatment with 20 ng/mL VEGF for 24 h was selected to analyze the effects of this compound on intracellular reactive oxygen species levels by DCFH-DA kit, cell cycle and apoptosis by flow cytometry, steroidogenesis by ELISA kit and the expression of the related genes by RT‒qPCR. The results showed that VEGF and VEGFR2 were highly coexpressed in GCs and theca cells. GCs cultured in medium containing 20 ng/mL VEGF for 24 h significantly improved cell viability, decreased ROS production, promoted the transition from G1 phase to S phase (P < 0.05), increased the expression of the CCND1 (P < 0.05), CCNE1, CDK2, CDK4, and PCNA genes (P < 0.01) and decreased the expression of the P53 gene (P < 0.05). This treatment significantly reduced GC apoptosis (P < 0.05) by promoting the expression of BCL2 and GDF9 (P < 0.01) and inhibiting the expression of BAX and CASPASE3 (P < 0.05). VEGF promoted progesterone secretion (P < 0.05) accompanied by increased expression of HSD3B, StAR and CYP11A1 (P < 0.05). Taken together, our findings highlight the beneficial influence exerted by VEGF in improving GC viability and reducing ROS production and the apoptosis rate through the modulation of related gene expression.

MiR-128-3p regulates chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways

MicroRNAs (miRNAs) are class of 22 nt short RNA sequences which inhibit protein translation through binding to the 3’UTR of its target genes. The continuous ovulatory property of chicken follicle makes it a perfect model for studying granulosa cell (GC) functions. In this study, we found that large number of miRNAs including miR-128-3p, were differentially expressed in the GCs of F1 and F5 follicles of chicken. Subsequently, the results revealed that miR-128-3p inhibited proliferation, the formation of lipid droplets, and hormone secretion in chicken primary GCs through directly targeting YWHAB and PPAR-γ genes. To determine the effects of 14-3-3β (encoded by YWHAB) protein on GCs functions, we overexpressed or inhibited the expression of YWHAB, and the results showed that YWHAB inhibited the function of FoxO proteins. Collectively, we found that miR-128-3p was highly expressed in the chicken F1 follicles compared to the F5 follicles. In addition, the results indicated that miR-128-3p promoted GC apoptosis through 14-3-3β/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen. Taken together, the results showed that miR-128-3p plays a regulatory role in chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways.

Effect of Mitochondrial Function of Ovarian Granulosa Cells on In Vitro Fertilization and Embryo Transfer Outcomes in Obese Polycystic Ovary Syndrome Patients

Effect of Mitochondrial Function of Ovarian Granulosa Cells on In Vitro Fertilization and Embryo Transfer Outcomes in Obese Polycystic Ovary Syndrome Patients

MiR-29c-5p regulates the function of buffalo granulosa cells to induce follicular atresia by targeting INHBA

MicroRNAs (miRNAs) are involved in many physiological processes such as signal transduction, cell proliferation and apoptosis. Many studies have shown that miRNAs can regulate the process of follicular development. Our previous studies found that the expression of miR-29c-5p in buffalo atretic follicles was much higher than that in healthy follicles, suggesting that this miRNA may participate in the process of buffalo follicular atresia. In this study, we aim to explore to the role and molecular mechanisms of miR-29c-5p on the functions of buffalo granulosa cells (GCs). GCs cultured in vitro were transfected with miR-29c-5p mimics and its inhibitor, respectively, and it was found that the mimics significantly increased the apoptotic rate of GCs. They also inhibited the proliferation of GCs and the secretion of steroid hormones. The effect of the inhibitor was opposite to that of the mimics. MiR-29c-5p was subsequently shown to target the inhibin subunit beta A, (INHBA). Overexpression of INHBA could promote the production of activin A and inhibin A, and then reverse the effect of miR-29c-5p on buffalo GCs. In conclusion, these results suggest that miR-29c-5p promotes apoptosis and inhibits proliferation and steroidogenesis by targeting INHBA in buffalo GCs. This may ultimately promote atresia in buffalo follicles.

MiR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27

Ovarian follicle development is associated with the physiological functions of granulosa cells (GCs), including proliferation and apoptosis. The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows. However, the functions of miR-24-3p on GCs are unclear. In this study, using flow cytometry, 5-ethynyl-2´-de-oxyuridine (EdU) staining, and cell count, we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes, moreover, miR-24-3p inhibited GC apoptosis. Mechanistically, on-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B (P27/CDKN1B). Furthermore, the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression. These findings suggest that miR-24-3p regulates the physiological functions of GCs.

SDNOR, a Novel Antioxidative lncRNA, Is Essential for Maintaining the Normal State and Function of Porcine Follicular Granulosa Cells.

Increasing evidence shows that lncRNAs, an important kind of endogenous regulator, are involved in the regulation of follicular development and female fertility, but the mechanism remain largely unknown. In this study, we found that SDNOR, a recently identified antiapoptotic lncRNA, is a potential multifunctional regulator in porcine follicular granulosa cells (GCs) through RNA-seq and multi-dimension analyses. SDNOR-mediated regulatory networks were established and identified that SOX9, a transcription factor inhibited by SDNOR, mediates SDNOR's regulation of the transcription of downstream targets. Functional analyses showed that loss of SDNOR significantly impairs GC morphology, inhibits cell proliferation and viability, reduces E2/P4 index, and suppresses the expression of crucial markers, including PCNA, Ki67, CDK2, CYP11A1, CYP19A1, and StAR. Additionally, after the detection of ROS, SOD, GSH-Px, and MDA, we found that SDNOR elevates the resistance of GCs to oxidative stress (OS) and also inhibits OS-induced apoptosis. Notably, GCs with high SDNOR levels are insensitive to oxidative stress, leading to lower apoptosis rates and higher environmental adaptability. In summary, our findings reveal the regulation of porcine GCs in response to oxidative stress from the perspective of lncRNA and demonstrate that SDNOR is an essential antioxidative lncRNA for maintaining the normal state and function of GCs.