Primary Cultures Of Granulosa Cells Research Articles

P–229 Oxidative stress and Metformin; an in-vitro study on serum and primary human granulosa cell cultures

Abstract Study question What is the effect of administration of Metformin on the oxidative stress (OS) levels in serum and primary human granulosa cell cultures of infertile females? Summary answer Metformin suppresses oxidative stress in serum and human granulosa cells and increases the expression of SIRT1 in OS induced environment. What is known already Oxidative stress (OS) is a resultant of mitochondrial dysfunction when it either fails to fight against the oxidants or the expression of the antioxidants is not sufficient. Cellular damage including DNA damage is a common resultant of oxidative stress. OS effects the oocyte maturation and moreover, the cleavage phase in the early embryonic stage. The raised levels of OS makers are hypothesized to compromise the nuclear maturation and the mitotic spindles of the maturing oocytes. Metformin seemed to decrease oxidative stress and improve insulin resistance, dyslipidaemia and endothelial dysfunction in PCOS patients Study design, size, duration This cross-sectional study was conducted from August 2017 – July 2019, at Aga Khan Hospital in collaboration with Australian Concept Infertility Medical Centre (ACIMC) on ten infertile patients undergoing egg retrieval after ethical approval from of Aga Khan Hospital (AKU-ERC–2018–0557–601). Participants/materials, setting, methods Serum samples were obtained and analysed. Follicular fluid of these subjects was collected for establishment of primary cell culture model of normal human granulosa cells (hGCs). Serum and hGC cultures were grouped as; a) control: treatment, b) Test1: H2O2 induced OS, and c) Test2: H2O2 induced OS treated with metformin. OS was estimated in all groups by Mishra method. The two Test groups were assessed for SIRT1 levels using quantitative PCR employing SIRT1 specific primers Main results and the role of chance With mean age of 32.04 ± 2.29 years the mean BMI was 27.61 ± 2.15 kg/m2. OS was induced and measured by an increase in optical density (OD) in hGC Test samples which showed 0.28 (0.16–0.40) OD when compared with control hGC samples 0.153 (0.09–0.23). There was a significant reduction in ODs after metformin treatment in the stress induced cells 0.182 (0.05–0.30). A similar pattern was observed in the serum samples in ODs; control: 0.105 (0.09–0.15), stress induced samples: 0.199 (0.19–0.20). and stress induced serum sample with metformin treatment: 0.1415 (0.06–0.18). The Ct values obtained to express the effect of metformin on SIRT1 levels, for OS induced (Test1) and OS induced metformin treated (Test2) cells were found to be 29.12 and 26.42, respectively. We also observed a significant (85%) difference in the fold change of SIRT1 expression between metformin treated and untreated cells. Limitations, reasons for caution Small sample size is the limitation of this study. The impact of metformin on cell cultures due to different causes of infertility could not be ascertained Wider implications of the findings: Metformin suppresses oxidative stress in serum and human granulosa cells and increases the expression of SIRT1 in OS induced environment, therefore, metformin may be considered as a treatment of oxidative stress in infertile patients. Randomized control trial with large sample size is recommended to confirm the cause and effect relationship. Trial registration number Not applicable

Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis.

Estrogen receptor beta (ERβ) plays a critical role in granulosa cell (GC) functions. The existence of four human ERβ splice isoforms in the ovary suggests their differential implication in 17β-estradiol (E2) actions on GC apoptosis causing follicular atresia. In this study, we investigated whether E2 can regulate ERβ isoforms expression to fine tune its apoptotic activities in human GC. For this purpose, we measured by RT-qPCR the expression of ERβ isoforms in primary culture of human granulosa cells (hGCs) collected from patients undergoing in vitro fertilization, before and after E2 exposure. Besides, we assessed the potential role of ERβ isoforms on cell growth and apoptosis after their overexpression in a human GC line (HGrC1 cells). We confirmed that ERβ1, ERβ2, ERβ4, and ERβ5 isoform mRNAs were predominant over that of ERα in hGCs, and found that E2 selectively regulates mRNA levels of ERβ4 and ERβ5 isoforms in these cells. In addition, we demonstrated that overexpression of ERβ1 and ERβ4 in HGrC1 cells increased cell apoptosis by 225% while ERβ5 or ERβ2 had no effect. Altogether, our study revealed that E2 may influence GC fate by specifically regulating the relative abundance of ERβ isoforms mRNA to modulate the balance between pro-apoptotic and non-apoptotic ERβ isoforms.

Involvement of BMP-15 in Glucocorticoid Actions on Ovarian Steroidogenesis by Rat Granulosa Cells

Glucocorticoid receptor (GR) are known to be expressed in the ovary and glucocorticoids are shown to exert direct effects on granulosa cell functions. In the clinical setting, menstrual abnormality, amenorrhea and hypermenorrhea can be shown in patients with glucocorticoid excess. On the other hand, glucocorticoids can also be used for the treatment of PCOS with hyperandrogenism. However, the effects of glucocorticoids on the reproductive system have not been fully elucidated. In the present study, we investigated the influence of glucocorticoids on follicular steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic proteins (BMPs) acting as a luteinizing inhibitor. Granulosa cells isolated from female immature rats were treated with follicle-stimulating hormone (FSH) in the presence of dexamethasone (Dex) in serum-free conditions. After treatment with Dex for 48 h, the changes of estradiol (E2) and progesterone (P4) production and cAMP synthesis induced by FSH treatments were measured by ELISA. Total RNAs of granulosa cells treated with FSH, Dex and BMPs were extracted and mRNA levels of steroidogenetic factors and enzymes, BMP receptors and Id-1 were quantified by real-time RT-PCR. Phosphorylation of Smad1/5/9 induced by BMPs was evaluated by Western blotting using cell lysates in the presence or absence of Dex. As a result, it was revealed that Dex treatment decreased FSH-induced E2 production by granulosa cells. In accordance with the steroid results, Dex suppressed FSH-induced P450arom mRNA expression as well as FSH-induced cAMP synthesis by granulosa cells. By contrast, Dex treatment augmented FSH-induced P4 production by granulosa cells in a concentration-dependent manner. Dex treatment was found to enhance basal and FSH-induced mRNA levels of P4-synthetic enzymes including P450scc and 3βHSD. Of note, Dex treatment activated the BMP target gene Id-1 transcription and Smad1/5/9 phosphorylation, in particular, induced by BMP-15 among various BMP ligands including BMP-2, -4, -6, -7, -9 and -15. It was also revealed that Dex treatment increased mRNA levels of ALK-6, a type-I receptor for BMP-15, and that BMP-15 treatment in turn upregulated GR mRNA levels expressed by granulosa cells. Given that BMP-15 acts as an inhibitor for P4 production by suppressing FSH-receptor actions, it was suggested that glucocorticoid is functionally linked to the enhancement of endogenous BMP-15, leading to the negative feedback toward the P4 overproduction induced by FSH and Dex in granulosa cells. Collectively, it was revealed that glucocorticoids elicit differential effects on the ovarian steroidogenesis of E2 and P4, in which GR and BMP-15 actions are mutually enhanced in granulosa cells.

Involvement of BMP-15 in glucocorticoid actions on ovarian steroidogenesis by rat granulosa cells

To elucidate the impact of glucocorticoids on ovarian steroidogenesis and its molecular mechanism by focusing on bone morphogenetic proteins (BMPs), we examined the effect of dexamethasone (Dex) on estradiol and progesterone synthesis by using primary culture of rat granulosa cells. It was revealed that Dex treatment dose-dependently decreased estradiol production but increased progesterone production induced by follicle-stimulating hormone (FSH) by granulosa cells. In accordance with the effects of Dex on estradiol synthesis, Dex suppressed P450arom mRNA expression and cAMP synthesis induced by FSH. Dex treatment in turn enhanced basal as well as FSH-induced levels of mRNAs encoding the enzymes for progesterone synthesis including P450scc and 3βHSD but not StAR and 20αHSD. Of note, Dex treatment significantly upregulated transcription of the BMP target gene Id-1 and Smad1/5/9 phosphorylation in the presence of BMP-15 among the key ovarian BMP ligands. It was also found that Dex treatment increased the expression level of BMP type-I receptor ALK-6 among the type-I and –II receptors for BMP-15. Inhibitory Smad6/7 expression was not affected by Dex treatment. On the other hand, BMP-15 treatment upregulated glucocorticoid receptor (GR) expression in granulosa cells. Collectively, it was revealed that glucocorticoids elicit differential effects on ovarian steroidogenesis, in which GR and BMP-15 actions are mutually enhanced in granulosa cells.

Curcumin in Combination with Aerobic Exercise Improves Follicular Dysfunction via Inhibition of the Hyperandrogen‐Induced IRE1α/XBP1 Endoplasmic Reticulum Stress Pathway in PCOS‐Like Rats

Combining diet with exercise can improve health and performance. Exercise can reduce androgen excess and insulin resistance (IR) in polycystic ovary syndrome (PCOS) patients. Curcumin is also presumed to improve the follicle development disorder. Here, we investigated the effects of a combination therapy of oral intake of curcumin and exercise on hyperandrogen-induced endoplasmic reticulum (ER) stress and ovarian granulosa cell (GC) apoptosis in rats with PCOS. We generated a PCOS model via continuous dehydroepiandrosterone subcutaneous injection into the necks of Sprague Dawley rats for 35 days. PCOS-like rats then received curcumin treatment combined with aerobic (treadmill) exercise for 8 weeks. We found that compared to control rats, the ovarian tissue and ovarian GCs of hyperandrogen-induced PCOS rats showed increased levels of ER stress-related genes and proteins. Hyperandrogen-induced ovarian GC apoptosis, which was mediated by excessive ER stress and unfolded protein response (UPR) activation, could cause follicle development disorders. Both curcumin gavage and aerobic exercise improved ovarian function via inhibiting the hyperandrogen-activated ER stress IRE1α-XBP1 pathway. Dihydrotestosterone- (DHT-) induced ER stress was mitigated by curcumin/irisin or 4μ8C (an ER stress inhibitor) in primary GC culture. In this in vitro model, the strongly expressed follicular development-related genes Ar, Cyp11α1, and Cyp19α1 were also downregulated.

Daily exposure to phthalates and alkylphenols alters miR biogenesis and expression in mice ovaries.

Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and Western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicer1 and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.

β-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

The beta-nerve growth factor (β-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama β-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with β-NGF, β-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of β-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3β-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. β-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that β-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that β-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Aldosterone enhances progesterone biosynthesis regulated by bone morphogenetic protein in rat granulosa cells

Aldosterone (Aldo) is involved in various cardiovascular diseases such as hypertension and heart failure. Aldo levels are known to be increased in patients with polycystic ovary syndrome, and expression of the mineralocorticoid receptor (MR) has also been detected in the ovary. However, the effect of Aldo on reproductive function has yet to be elucidated. Here, we examined the effects of Aldo on follicular steroidogenesis using primary culture of rat granulosa cells by focusing on the ovarian bone morphogenetic protein (BMP) system acting as a luteinizing inhibitor. We found that Aldo treatment increased FSH-induced progesterone production in a concentration-responsive manner. Consistent with the effects on steroidogenesis, Aldo increased mRNA levels of progesterogenic factor and enzymes including StAR and P450scc, whereas Aldo failed to change FSH-induced estradiol and cAMP synthesis or P450arom expression by granulosa cells. Progesterone production and StAR expression induced by FSH and Aldo were reversed by co-treatment with spironolactone, suggesting the involvement of geonomic MR action. Aldo treatment attenuated Smad1/5/9 phosphorylation and Id1 transcription induced by BMP-6. Furthermore, Aldo enhanced the expression of inhibitory Smad6 in the presence of BMP-6. In addition, BMP-6 downregulated MR expression, while Aldo modulated the mRNA levels of endogenous BMP-6 and BMP type-II receptors, indicating the existence of a feedback loop between the BMP system and MR in granulosa cells. Collectively, the results indicated that Aldo predominantly enhances FSH-induced progesterone production by inhibiting BMP-Smad signaling, suggesting a novel role of Aldo in ovarian steroidogenesis and a functional link between MR and BMP pathways in granulosa cells.

Recombinant Anti-Müllerian Hormone (rAMH) for Stalling In Vitro Granulosa Cell Replication.

To investigate whether recombinant AMH (rAMH) is able to decrease cellular proliferation/apoptosis in luteinized granulosa cells (GCs) through hormonal regulation, a primary culture of GCs was established from GCs obtained at time of oocyte retrieval from follicular fluid of 3 patients. Cells were seeded in well cell culture plates at a density of 100,000 cells/well in medium and treated with rAMH 20ng/ml (rAMH group), or phosphate-buffered saline (PBS-control group), for 24h. Total RNA was extracted from all cells, followed by cDNA synthesis and real-time RT-PCR to quantify the expression levels of AMH, AMH-R2, FSH-R, inhibin B, cell proliferation (Ki67), and apoptosis (Caspase 3). We used independent sample t test (SPSS v25) and a p < 0.05 significance. Cellular expressions of AMH, AMH-R2, FSH-R, and inhibin B were reduced greater than 50% in the rAMH group, compared with that of the the control group (p ≤ 0.005 for all). Ki67 and Caspase3 were also reduced greater than 30% in the rAMH group (p ≤ 0.001 for both). Our findings show a direct inhibitory effect of AMH on luteinized GCs' expression of the major regulatory hormones, in addition to a significant decrease in markers of cell proliferation and apoptosis. These results confirm the inhibitory effects of AMH on follicular development.

Non-Esterified Fatty Acid-Induced Reactive Oxygen Species Mediated Granulosa Cells Apoptosis Is Regulated by Nrf2/p53 Signaling Pathway.

Negative energy balance (NEB) during the perinatal period can affect dairy cow follicular development and reduce the fecundity. Non-esterified fatty acid (NEFA) concentration is elevated during NEB, and is known to be toxic for multiple cell types. In the ovary, NEB increased NEFA, and may influences follicular growth and development. However, the effect and mechanism of NEFA on granulosa cells (GCs) in vitro remains unknown. In this study, we found that NEFA dose-dependently induced apoptosis in primary cultured granulosa cells. Mechanistically, our data showed that NEFA significantly increased reactive oxygen species (ROS) levels, resulting in the activation of endoplasmic reticulum stress (ERS) and eventually cell apoptosis in GCs. Moreover, NEFA also increased the phosphorylation levels of ERK1/2 and p38MAPK pathways, upregulated the expression of p53 and potentially promoted its translocation to the nuclear, thus transcriptionally activated Bax, a downstream gene of this pathway. NEFA also promoted nuclear factor E2 (Nrf2) expression and its level in the nuclear. To elucidate the mechanism of NEFA action, N-acetyl-l-cysteine (NAC), a ROS scavenger was used to verify the role of ROS in NEFA induced apoptosis of GCs. NAC pretreatment reversed the NEFA-induced ERS-related protein and apoptosis-related protein levels. Meanwhile, NAC pretreatment also blocked the phosphorylation of ERK1/2 and p38 induced by NEFA, and the nucleation of Nrf2 and p53, suggesting that ROS plays a crucial role in regulating the NEFA-induced apoptosis of GCs. Together, these findings provide an improved understanding of the mechanisms underlying GCs apoptosis, which could potentially be useful for improving ovarian function.

ERO1α inhibits cell apoptosis and regulates steroidogenesis in mouse granulosa cells

ER oxidoreduclin 1α (ERO1α), an oxidase that exists in the ER, participates in protein folding and secretion and inhibiting apoptosis, and regulates tumor progression, which is a novel factor of poor cancer prognosis. However, the other physiological functions of ERO1α remain undiscovered. Although our preliminary results of this study indicated that ERO1α revealed the robust expression in ovary, especially in granulosa cells, the role of ERO1α in follicular development is not well known. Therefore, the aims of the present study were to explore the role of ERO1α and the possible mechanisms in regulating cell apoptosis and steroidogenesis in ovarian granulosa cells. ERO1α was mainly localized in granulosa cells and oocytes in the adult ovary by immunohistochemistry. Western blot analysis showed that the expression of ERO1α was highest at oestrous stage during the estrous cycle. The effect of ERO1α on cell apoptosis and steroidogenesis was detected by transduction of ERO1α overexpression and knockdown lentiviruses into primary cultured granulosa cells. Flow cytometry analysis showed that ERO1α decreased granulosa cells apoptosis. Western bolt and RT-qPCR analysis found that ERO1α increased the ratio of BCL-2/BAX, and decreased BAD and Caspase-3 expression. ELISA analysis showed that ERO1α enhanced estrogen (E2) secretion. Western bolt and RT-qPCR analysis found that ERO1α increased StAR, CYP11A1, 3β-HSD, CYP17A1, and CYP19A1 expression, and decreased CYP1B1 expression. Furthermore, Western bolt analysis found that ERO1αincreased PDI and PRDX 4 expression, and activated the PI3K/AKT/mTOR signaling pathway through increasing the phosphorylation of AKT and P70 S6 kinase. In summary, these results suggested that ERO1α might play an anti-apoptotic role and regulate steroidogenesis in granulosa cells, at least partly, via activation of the PI3K/AKT/mTOR signaling pathway.

PDGFRα/β-PI3K-Akt pathway response to the interplay of mitochondrial dysfunction and DNA damage in Aroclor 1254-exposed porcine granulosa cells

Metabolic dysfunction and genomic instability are known to affect female fertility. Aroclor 1254 (A1254) is an endocrine disruptor that affects mitochondrial function following ingestion, inhalation, or dermal exposure. Numerous studies to date have addressed associations between A1254 toxicity and chronic neurological disorders, while A1254 exposure is little known to have a toxic effect on the female reproductive system. Furthermore, interactive mechanisms between metabolic dysfunction and the repair of DNA damage deserve further investigation. In this paper, an in vitro porcine primary granulosa cell (GC) culture model was used to investigate the mechanisms of exposure and effects of the exogenous chemical carcinogen A1254 on reproductive toxicology. High-throughput RNA sequencing obtained 2329 differentially expressed genes (DEGs) to be analyzed using COG classification, GO, and KEGG. When combined with immunofluorescence, Western blot analysis, and real-time RT-PCR analysis, this data showed that the mitochondrial-ROS-driven feed-forward loop increased phospho-PDGFRα/β, which stimulates apoptosis by suppressing the PI3K-Akt pathway. We also noticed that inhibition of the Akt-PDP1-PDK1 axis attenuated mitochondrial function. In contrast, following iPath analysis, partial metabolic pathways were enhanced. Importantly, we found that A1254 activated a DNA damage response, the major regulators of which belong to the PI3K-related protein kinases (PIKKs) and oncogenes, which led to the “Warburg effect”. It is not easy to restore the damage that A1254 causes to metabolism through dysregulation and the Warburg effect, owing to the fact that oncogenes can regulate cytoplasmic metabolism. Therefore, we suspect that the PDGFR-PI3K-Akt pathway may be a latent interaction between mitochondrial dysfunction and the response of DNA damage.

Paraben concentrations found in human body fluids do not exert steroidogenic effects in human granulosa primary cell cultures

In cosmetics and food products, parabens are widely used as antimicrobial agents. Reports have suggested that parabens may be linked to infertility, owing to their effects on basal steroidogenesis properties or their capacity to inflict mitochondrial damage. Despite growing concerns about parabens as endocrine disruptors, it is unclear whether they affect any of these actions in humans, particularly at environmentally relevant concentrations. In this work, an in vitro primary culture of human granulosa cells was used to evaluate steroidogenesis, based on the assessment of progesterone production and regulation of critical steroidogenic genes: CYP11A1, HSD3B1, CYP19A1, and HSD17B1. The effects of two commercially relevant parabens, methylparaben (MPB) and butylparaben (BPB), were screened. Cells were exposed to multiple concentrations ranging from relatively low (typical environmental exposure) to relatively high. The effect was assessed by the parabens’ ability to modify steroidogenic genes, progesterone or estradiol production, and on mitochondrial health, by evaluating mitochondrial activity as well as mtDNA content. Neither MPB nor BPB showed any effect over progesterone production or the expression of genes controlling steroid production. Only BPB affected the mitochondria, decreasing mtDNA content at supraphysiological concentrations (1000 nM). Prolonged exposure to these compounds produced no effects in neither of these parameters. In conclusion, neither MPB nor BPB significantly affected basal steroidogenesis in granulosa cells. Although evidence supporting paraben toxicity is prevalent, here we put forth evidence that suggests that parabens do not affect basal steroidogenesis in human granulosa cells.

Effects of α-enolase Gene Silencing on Reproductive-related Hormone Receptor Expression and Steroid Hormone Synthesis of Primary Granulosa Cells from Goose F1 Follicles.

IntroductionEnolases are enzymes in the glycolytic pathway, which catalyse the reversible conversion of D-2-phosphoglycerate into phosphoenol pyruvate in the second half of the pathway. In this research, the effects of α-enolase (ENO1) on steroid reproductive-related hormone receptor expression and on hormone synthesis of primary granulosa cells from goose F1 follicles were studied.Material and MethodsPrimary granulosa cells from the F1 follicles of eight healthy 8-month-old Zi geese were separated and cultured. An ENO1 interference expression vector was designed, constructed and transfected into primary cultured granulosa cells. The mRNA expression levels of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LHR), oestrogen receptor α (ER α), oestrogen receptor β (ER β), growth hormone receptor (GHR) and insulin-like growth factor binding protein-1 (IGFBP-1) in the cells were evaluated as were the secretion levels of oestradiol, activin, progesterone, testosterone, inhibin and follistatin in cell supernatant.Resultsα-enolase gene silencing reduced the expression of FSHR, LHR, ERα, ERβ, GHR, and IGFBP-1 mRNA, potentiated the secretion of oestrogen, progesterone, testosterone, and follistatin of granulosa cells, and hampered the production of activin and inhibin.ConclusionENO1 can regulate the reactivity of granulosa cells to reproductive hormones and regulate cell growth and development by adjusting their hormone secretion and reproductive hormone receptor expression. The study provided a better understanding of the functional action of ENO1 in the processes of goose ovary development and egg laying.

Lipopolysaccharide-induced mechanisms of ovarian dysfunction in cows with uterine inflammatory diseases.

Uterine inflammatory diseases commonly occur in postpartum dairy cows, resulting in reduced reproductive performance due to aberrant uterine and ovarian activity. Infection of the uterus with gram-negative bacteria results in the detection of lipopolysaccharide (LPS) in the plasma and follicular fluid of cows along with uterine inflammation. LPS acts on follicular components such as theca cells, granulosa cells, and follicle-enclosed oocytes, leading to impaired follicular activity. Follicles with a high LPS environment exhibit reduced follicular steroidogenesis due to the inhibition of steroidogenic enzyme transcription. Primary cell cultures of bovine granulosa and theca cells have shown that LPS acts on follicular cells to impair steroid production, which may disturb follicle growth and/or reduce their ability to ovulate. Even if ovulation occurs, cows with uterine inflammation are less likely to conceive because in addition to uterine damage, LPS also impairs the developmental competence of oocytes. LPS perturbs the nuclear and cytoplasmic maturation of bovine oocytes. Moreover, oocytes matured using LPS treatment are less likely to develop into the blastocyst stage. Such oocytes also have a reduced number of trophoblast cells in blastocysts. Therefore, the detrimental effects of LPS on ovarian activity may be partly responsible for infertility in cows with uterine inflammation. Novel treatment and prevention strategies for uterine inflammatory diseases can be developed by advancing our knowledge of the pathophysiology underlying ovarian dysfunction, and this can only be achieved by further research. The present review outlines the molecular pathogenesis of LPS-induced ovarian dysfunction.

Thermotolerance of camel (Camelus dromedarius) somatic cells affected by the cell type and the dissociation method.

Researchers dealing with heat stress experiments use different cell kinds and use trypsin that has been reported to affect the cellular proteins of cultured cells. Therefore, we compared the effects of acute and chronic exposures to high temperature (45 °C) on camel skin fibroblast and granulosa cells. Primary culture of fibroblasts and granulosa cells tolerated the acute heat shock for 2 h; however, granulosa cells cultured for long duration (20 h) showed thermotolerance when compared with the fibroblasts. Moreover, the effect of cell dispersion method (trypsin and mechanical dissociation) on the thermotolerance of sub-cultured cells was examined. Trypsin altered the morphology of fibroblasts and granulosa cells exposed to 45 °C for 4 h. Moreover, trypsin significantly reduced the fibroblast and granulosa cell migration in the wound healing assay. The current results demonstrate that cell passaging and cell type can affect the thermotolerance of the cells; it also revealed that trypsin could alter the cellular response to the heat shock. We raise the demand for another alternative method for cell dispersion in experiments dealing with cellular responses to the heat shock.

HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress.

Heat stress negatively affects reproduction in cattle by disrupting the normal function of ovarian granulosa cells (GCs), ultimately leading to oxidative damage and cell death via apoptosis. Heme oxygenase-1(HO-1) is a member of the heat shock protein family, which are associated with cellular antioxidant defenses and anti-apoptotic functions. Recent studies demonstrated that HO-1 is upregulated in heat-stressed cells. In the present study, we investigated the expression of HO-1 in bovine GCs transiently exposed to heat stress and characterized the expression and activity of key oxidative stress enzymes and molecules. We show that heat stress induced oxidative stress and apoptosis, and enhanced Nrf2 and HO-1 expression in primary GC cultures. Knocking down HO-1 expression using siRNA exacerbated both oxidative stress and apoptosis, whereas pre-treating GCs with hemin, which induces HO-1 expression, partially prevented these effects. These findings demonstrate that HO-1 attenuates heat stress-induced apoptosis in bovine GCs by decreasing production of reactive oxygen species and activating the antioxidant response.

Dysregulated miR-142, -33b and -423 in granulosa cells target TGFBR1 and SMAD7: a possible role in polycystic ovary syndrome

It is well established that microRNA (miRNA) expression profiles are altered in patients with polycystic ovary syndrome (PCOS). In addition, abnormal transforming growth factor beta (TGFB) signaling in granulosa cells is related to the pathological conditions of PCOS. However, the function of dysregulated miRNAs in PCOS is still unclear. In this study, we aimed to elucidate the roles of specific miRNAs in PCOS. We collected follicular fluid from 46 patients with PCOS and 32 healthy controls. Granulosa cells (GCs) were separated and the levels of six candidate miRNAs were determined by quantitative RT-PCR. The direct targets of three dysregulated miRNAs were predicted using bioinformatic tools and confirmed using a dual luciferase assay and immunoblotting. The biological function of three dysregulated miRNAs in primary GCs was determined using a cell proliferation assay and flow cytometry. We found that miR-423 expression was downregulated (P = 0.038), and the levels of miR-33b (P = 0.032) and miR-142 (P = 0.021) were upregulated in GCs from patients with PCOS, compared to controls. miR-423 directly repressed SMAD family member 7 (SMAD7) expression, while transforming growth factor beta receptor 1 (TGFBR1) was a direct target of both miR-33b and miR-142. An RNA oligonucleotide mixture containing miR-423 inhibitor, miR-33b mimic, and miR-142 mimic repressed TGFB signaling, promoted cell proliferation (P = 0.0098), repressed apoptosis (P = 0.027), and increased S phase cell numbers (P = 0.0036) in primary cultures of GCs, compared to the cells treated with a sequence scrambled control RNA oligonucleotide. This study unveiled the possible roles of three miRNAs in PCOS and might provide candidate biomarkers for PCOS diagnosis while in vivo functional studies, using transgenic or knockout mouse models, are expected to confirm the roles of dysregulated miRNAs in the pathogenesis of PCOS.

SUN-193 Effects of Aldosterone on Ovarian Steroidogenesis and Involvement of BMP Action in Rat Granulosa Cells

Many local factors expressed in the ovary are involved in folliculogenesis. Among these, the ovarian renin-angiotensin system is critical for normal processes of follicle development, steroidogenesis and ovulation. In various clinical conditions such as secondary hypertension, heart failure, and liver and/or renal damages, hyperaldosteronism can be detected. In addition, Aldo levels are known to be high in patients with PCOS and/or OHSS. However, the effect of high concentration of Aldo on reproductive function has not been fully elucidated. In this study, we examined the influence of Aldo on follicular steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system acting as a luteinizing inhibitor. Granulosa cells isolated from female rats were treated with follicle stimulating hormone (FSH) in the presence of low to high concentrations of Aldo (1-300 nM) and cultured in serum-free conditions. After incubation with Aldo for 48 hours, changes in estradiol (E2) and progesterone (P4) productivity induced by FSH stimulation were measured by CLIA, and cAMP synthesis was evaluated by EIA. Total RNA was extracted and mRNA level of steroidogenetic enzymes induced by FSH and that of BMP-receptors and Id-1 induced by BMP-6 were quantified by real-time RT-PCR. Phosphorylation of Smad1/5/9 was further evaluated by Western blotting using cell lysate after the co-treatments with Aldo and BMP-6. As a result, treatment with Aldo increased FSH-induced P4 production by granulosa cells in a concentration-dependent manner at the maximum effects by 100-300 nM of Aldo. Aldo treatment increased mRNA levels of P4-synthetic enzymes expression including StAR, P450scc and 3βHSD, and Aldo also upregulated FSH-induced cAMP synthesis. On the contrary, Aldo failed to change E2 production stimulated by FSH. Moreover, Aldo treatment attenuated Id-1 transcription and phosphorylation of Smad1/5/9 induced by BMP-6 stimulation. These data indicated that Aldo elicits a specific effect on the enhancement of P4 production by granulosa cells. On the other hand, BMP-6 treatment downregulated MR expression in granulosa cell, suggesting the existence of a negative feedback loop between BMP-receptor and MR signaling. Taken it into consider that BMP ligands act as specific suppressor for P4 production as a luteinizing inhibitor, it is possible that Aldo is functionally linked to the suppression of endogenous BMP-receptor signaling in granulosa cells, leading to the deterioration of follicular growth and ovulation process. Collectively, it is revealed that high concentration of Aldo specifically enhances FSH-induced P4 production by inhibiting BMP signal in granulosa cells. This effect may be associated with impaired fertility and/or ovulatory failure in the patients with hyperaldosteronism.

Maternal short and medium chain fatty acids supply during early pregnancy improves embryo survival through enhancing progesterone synthesis in rats

Exploring strategies to prevent miscarriage in women or early pregnancy loss in mammals is of great importance. Manipulating maternal lipid metabolism to maintain sufficient progesterone level is an effective way. To investigated the embryo loss and progesterone synthesis impacts of short and medium chain fatty acids on the lipid metabolism, pregnancy outcome and embryo implantation were investigated in rats fed the pregnancy diets supplemented without or with 0.1% sodium butyrate (SB), 0.1% sodium hexanoate (SH), or 0.1% sodium caprylate (SC) during the entire pregnancy and early pregnancy, respectively, followed with evaluation of potential mechanisms. Maternal SB, SH, or SC supply significantly improved live litter size and embryo implantation in rats. Serum progesterone, arachidonic acid, and phospholipid metabolites levels were significantly increased in response to maternal SB, SH, and SC supply. The expression of key genes involved in ovarian steroidogenesis and granulosa cell luteinization were elevated in ovaries and primary cultured granulosa cells, including cluster of differentiation 36 (CD36), steroidogenic acute regulatory protein (StAR), and cholesterol side-chain cleavage enzyme (CYP11A1). Additionally, the expression of lysophosphatidic acid receptor 3 (LPA3) and cyclooxygenase-2 (COX2) related with phospholipid metabolism were enhanced in uterus in vivo and in in vitro cultured uterine tissue. In conclusion, maternal SB, SH and SC supply reduced early pregnancy loss through modulating maternal phospholipid metabolism and ovarian progesterone synthesis in rats. Our results have important implications that short or medium chain fatty acids have the potential to prevent miscarriage in women or early pregnancy loss in mammals.