Bisphenol-A exposure and gene expression in human luteinized membrana granulosa cells in vitro.
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ABSTRACT: Does bisphenol-A (BPA) affect gene expression in human membrana granulosa cells (MGC)?In vitro, short exposure to supra-physiological concentrations of BPA alters human MGC gene expression.Exposure to BPA may interfere with reproductive endocrine signaling. In vitro studies, mostly in animal models, have shown an inverse correlation between exposure to BPA and follicular growth, meiosis, and steroid hormone production in granulosa cells.Primary cultures of MGC obtained from 24 patients undergoing IVF (for PGD, male factor infertility or unexplained infertility) were exposed to various concentrations of BPA (0, 0.02, 0.2, 2 or 20 µg/ml) for 48 h.The study was conducted in a university-affiliated hospital. Microarray analysis was used to identify genes exhibiting expression changes following BPA exposure. Genes significantly altered were identified based on changes greater than 2-fold relative to the control group (not treated by BPA) and a Student's t-test P-value <0.05. Statistical significance was adjusted for multiple comparisons using the Benjamini-Hochberg method. Alterations in the expression of genes that are involved in the enriched functional annotations altered by BPA at the concentration of 20 µg/ml were confirmed by real-time PCR.A distinct pattern of gene expression was observed in primary cultures of MGC exposed to the highest BPA concentration compared with untreated cells. We identified 652 genes that exhibited at least 2-fold differences in expression after BPA exposure (all P < 0.05 versus untreated). These genes were significantly enriched for annotations related to cell cycle progression, segregation of chromosomes, steroid metabolism, apoptosis, lipid synthesis, oocyte maturation and chromosomal alignment. No significant changes in gene expression were found at the lower doses of BPA most relevant to human exposure.N/A.Human exposure to BPA in vivo occurs over long periods of time. In this in vitro model, cells were exposed to the chemical for 48 h only. Thus, the effects of BPA on the human follicle might be underestimated.As BPA exposure is ubiquitous, understanding the effects of the chemical on the ovary, specifically in women of reproductive age, has public health significance. The clinical evidence to date points to an association between BPA exposure and impaired IVF outcome, although not all studies have shown negative effects. Our study adds valuable mechanistic information showing that exposure to BPA alters granulosa cell gene expression at high and supra-physiological doses.This study was supported by grant number 1936/12 from the ISF. The authors have nothing to disclose.
<h4>Study question</h4>Does bisphenol-A (BPA) affect gene expression in human membrana granulosa cells (MGC)?<h4>Summary answer</h4>In vitro, short exposure to supra-physiological concentrations of BPA alters human MGC gene expression.<h4>What is known already</h4>Exposure to BPA may interfere with reproductive endocrine signaling. In vitro studies, mostly in animal models, have shown an inverse correlation between exposure to BPA and follicular growth, meiosis, and steroid hormone production in ...[more]
Project description:The corpus luteum (CL) forms after ovulation and acts as a temporary endocrine gland that produces progesterone (P4), a hormone that is essential for implantation and maintenance of pregnancy in mammals. In pregnant women, human chorionic gonadotropin (hCG) secreted by the conceptus prevents luteolysis. hCG also increases the survival of cultured human luteinized granulosa cells (hLGCs). To clarify the maintenance mechanism of the human CL, we investigated the effects of hCG and P4 receptor antagonists, onapristone (OP) and RU486, on the viability of hLGCs. With the patients' consent, hLGCs were isolated from follicular aspirates for in vitro fertilization. The cells were cultured with hCG (0.1, 1, 10, 100 IU/ml), OP (10, 25, 50, 100 ?M), RU486 (100 ?M), P4 (1, 10, 25, 50 ?M) or some combination of the four for 24 h. Cell viability was significantly increased by hCG (100 IU/ml) and significantly decreased by OP (100 ?M) compared with the control. Cells treated with hCG and OP together were significantly less viable than the control and OP-treated cells. The combined treatment also significantly increased CASP3 activity and cleaved CASP3 protein expression. Furthermore, P4 addition reversed the reduction in cell viability caused by the combination of hCG and OP treatment. The overall findings suggest that hCG cooperates with P4 to increase survival of hLGCs and to induce apoptosis when P4 action supported by hCG is attenuated in the human CL.
Project description:BackgroundExposure to bisphenol A (BPA), a chemical widely used in consumer products, has been associated with in vitro Cyp19 gene expression.ObjectiveTo evaluate an in vivo human model of Cyp19 gene expression in granulosa cells.Study designA subset of an ongoing prospective cohort study of women undergoing in vitro fertilization (IVF) at Massachusetts General Hospital.MethodsMixed effect models were used to evaluate the association of urinary BPA concentrations with granulosa cell Cyp19 mRNA expression.ResultsIn 61 women undergoing 76 IVF cycles, adjusted changes in mean Cyp19 expression (? estimate (95% CI)) for quartiles 2, 3 and 4 as compared to the lowest quartile were: -0.97 (-2.22, 0.28); -0.97 (-2.18, 0.24) and -0.38 (-1.58, 0.82).ConclusionsAn in vivo model for evaluation of Cyp19 gene expression was developed for use in epidemiologic studies. In this pilot study, we found no statistically significant linear association between urinary BPA concentrations and Cyp19 expression.
Project description:Interferon-tau (IFNT), serves as a signal to maintain the corpus luteum (CL) during early pregnancy in domestic ruminants. We investigated here whether IFNT directly affects the function of luteinized bovine granulosa cells (LGCs), a model for large-luteal cells. Recombinant ovine IFNT (roIFNT) induced the IFN-stimulated genes (ISGs; MX2, ISG15, and OAS1Y). IFNT induced a rapid and transient (15-45?min) phosphorylation of STAT1, while total STAT1 protein was higher only after 24?h. IFNT treatment elevated viable LGCs numbers and decreased dead/apoptotic cell counts. Consistent with these effects on cell viability, IFNT upregulated cell survival proteins (MCL1, BCL-xL, and XIAP) and reduced the levels of gamma-H2AX, cleaved caspase-3, and thrombospondin-2 (THBS2) implicated in apoptosis. Notably, IFNT reversed the actions of THBS1 on cell viability, XIAP, and cleaved caspase-3. Furthermore, roIFNT stimulated proangiogenic genes, including FGF2, PDGFB, and PDGFAR. Corroborating the in vitro observations, CL collected from day 18 pregnant cows comprised higher ISGs together with elevated FGF2, PDGFB, and XIAP, compared with CL derived from day 18 cyclic cows. This study reveals that IFNT activates diverse pathways in LGCs, promoting survival and blood vessel stabilization while suppressing cell death signals. These mechanisms might contribute to CL maintenance during early pregnancy.
Project description:Runt-related transcription factor 1 (RUNX1), a transcription factor, is transiently induced by the LH surge and regulates gene expression in periovulatory granulosa cells. Potential binding sites for RUNX are present in the 5'-flanking region of the Ptgs2 (prostaglandin-endoperoxide synthase 2) gene. Periovulatory Ptgs2 expression is essential for ovulation. In the present study, we investigated the role of RUNX1 in mediating the LH-induced expression of Ptgs2 in periovulatory granulosa cells. We first determined whether the suppression of Runx1 expression or activity affects Ptgs2 expression using cultured preovulatory granulosa cells isolated from immature rat ovaries primed with pregnant mare serum gonadotropin for 48 h. Knockdown of human chorionic gonadotropin-induced Runx1 expression by small interfering RNA or inhibition of endogenous RUNX activities by dominant-negative RUNX decreased human chorionic gonadotropin or agonist-stimulated Ptgs2 expression and transcriptional activity of Ptgs2 promoter reporter constructs. Results from chromatin immunoprecipitation assays revealed in vivo binding of endogenous RUNX1 to the Ptgs2 promoter region in rat periovulatory granulosa cells. Direct binding of RUNX1 to two RUNX-binding motifs in the Ptgs2 promoter region was confirmed by EMSA. The mutation of these two binding motifs resulted in decreased transcriptional activity of Ptgs2 promoter reporter constructs in preovulatory granulosa cells. Taken together, these findings provide experimental evidence that the LH-dependent induction of Ptgs2 expression results, in part, from RUNX1-mediated transactivation of the Ptgs2 promoter. The results of the present study assign potential significance for LH-induced RUNX1 in the ovulatory process via regulating Ptgs2 gene expression.
Project description:Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone morphogenetic protein 6 (BMP6) is an important intraovarian modulator of follicular development. In this study, we demonstrated that BMP6 treatment significantly increased the expression of CTGF in both primary and immortalized human granulosa cells. Using both pharmacological inhibitors and Small interfering RNA-mediated knockdown approaches, we showed that ALK2 and ALK3 type I receptors are required for BMP6-induced cellular activities. Furthermore, this effect is most likely mediated by a Sma- and Mad-related protein (SMAD)-dependent pathway. Our studies provide novel insight into the molecular mechanisms by which an intraovarian growth factor affects the production of another factor via a paracrine effect in human granulosa cells.
Project description:Bisphenol A (BPA) is a widely used chemical that has been detected in follicular fluid and associated with adverse reproductive effects. Granulosa cells have an important role in follicular growth and oocyte maturation, however, little is known about the biological mechanisms of BPA toxicity on human granulosa cells. In this study, we exposed primary granulosa cells to different concentrations of BPA (0, 20, 200, 2000, and 20 000?ng/ml) and used quantitative polymerase chain reaction to measure the expression levels of miRNAs enriched in extracellular vesicles (EV-enriched miRNAs), and cellular levels of selected target genes of differentially expressed EV-enriched miRNAs. We found that exposure to 20 000?ng/ml BPA was associated with decreased levels of EV-miR-27b-3p (FC?=?0.58, p?=?.04) and increased levels of its biologically relevant target genes FADD (FC?=?1.22, p?=?.01), IGF1 (FC?=?1.59, p?=?.06), and PPARG (FC?=?1.73, p?=?.001) as compared with the control. In addition, we observed that under the same exposure conditions, the expression levels of miR-27b-3p in granulosa cells were also downregulated (FC?=?0.65, p?=?.03) as compared with the control. Our findings suggest that both cellular and extracellular changes in gene expression may mediate BPA toxicity in granulosa cells.
Project description:Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3', 5'-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1-500 μg/ml; dimethylsulfoxide = vehicle control) for 0.5-36 h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500 μg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500 μg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women.
Project description:It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.
Project description:BackgroundBisphenol A (BPA), a synthetic endocrine-disrupting chemical, is a reproductive toxicant. Granulosa cells have significant roles in follicle development, and KIT ligand (KITL) and Anti-Müllerian hormone (AMH) are essential biomolecules produced by them during folliculogenesis.ObjectiveDue to the widespread use of BPA and its potential epigenetic effects, this study examined the impact of BPA on promoter methylation of amh and kitl genes in mouse granulosa cells.Materials and methodsPreantral follicles were isolated from ovaries of immature mice and cultured for eight days. Then, follicles were treated with 50 and 100 ?M of BPA, and 0.01% (v/v) ethanol for 24 and 72 hr. Growth and degeneration of follicles and antrum formation were analyzed. The granulosa cells were isolated mechanically, and their extracted DNA was treated with sodium bisulfite. The promoter regions of the amh and kitl were analyzed with PCR and sequencing.ResultsBPA did not change follicle survival and antrum formation significantly (p = 0.41). However, the culture in the presence of 100 ?M BPA had an inhibitory effect on growth. Before BPA treatment, the CpG of the kitl and amh promoters were unmethylated and partially methylated, respectively. While the percent of 5mC in the amh promoter reduced at 100 ?M of BPA, it did not alter the kitl promoter methylation.ConclusionBPA at higher concentrations has an inhibitory effect on follicle growth. Moreover, it seems that the epigenetic impact of BPA restricts to the demethylation of CpG sites.
Project description:Zearalenone (ZEA), a metabolite of Fusarium fungi, is commonly found on moldy grains. Because it can competitively combine to estrogen receptor to disrupt estrogenic signaling, it has been reported to have serious adverse effects on animal reproduction systems. In order to explore the genotoxic effects of ZEA exposure on ovarian somatic cells, porcine granulosa cells were exposed to 10 μM and 30 μM ZEA for 24 or 72 h in vitro. The results showed that ZEA exposure for 24 h remarkably reduced the proliferation of porcine granulosa cells in a dose-dependent manner as determined by MTT analysis and flow cytometry. Furthermore, exposure to ZEA for 72 h induced apoptosis, and RNA sequence analysis also revealed that the expression of apoptosis related genes were altered. RT-qPCR, immunofluorescence and western blot analysis further confirmed the expression of DNA damage and repair related genes (γ-H2AX, BRCA1, RAD51 and PRKDC) were increased in ZEA exposed granulosa cells. When the estrogen antagonist, tamoxifen, was added with ZEA in the culture medium, the DNA damage and repairment by ZEA returned to normal level. Collectively, these results illustrate that ZEA disrupts genome stability and inhibits growth of porcine granulosa cells via the estrogen receptors which may promote granulosa cell apoptosis when the DNA repair system is not enough to rescue this serious damage.
