BPA and BPS Affect Connexin 37 in Bovine Cumulus Cells.
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ABSTRACT:
SUBMITTER: Sabry R
PROVIDER: S-EPMC7926832 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundExposure to endocrine-disrupting chemicals, such as Bisphenol A (BPA) and Bisphenol S (BPS), is widespread and has negative implications on embryonic development. Preliminary evidence revealed that in women undergoing IVF treatment, urinary BPA levels were associated with low serum anti-Mullerian hormone, however a definitive relationship between the two has not yet been characterized.MethodsThis study aimed to evaluate BPA and BPS effects on in vitro oocyte maturation and early preimplantation embryo development through i) analysis of anti-Mullerian hormone (AMH) and anti-Mullerian hormone receptor II (AMHRII), ii) investigation of developmental parameters, such as cleavage, blastocyst rates and developmental arrest, iii) detection of apoptosis and iv) assessment of possible sex ratio skew. An in vitro bovine model was used as a translational model for human early embryonic development. We first assessed AMH and AMHRII levels after bisphenol exposure during oocyte maturation. Zygotes were also analyzed during cleavage and blastocysts stages. Techniques used include in vitro fertilization, quantitative polymerase chain reaction (qPCR), western blotting, TUNEL and immunofluorescence.ResultsOur findings show that BPA significantly decreased cleavage (p < 0.001), blastocyst (p < 0.005) and overall developmental rates as well as significantly increased embryonic arrest at the 2-4 cell stage (p < 0.05). Additionally, both BPA and BPS significantly increased DNA fragmentation in 2-4 cells, 8-16 cells and blastocyst embryos (p < 0.05). Furthermore, BPA and BPS alter AMH and AMHRII at the mRNA and protein level in both oocytes and blastocysts. BPA, but not BPS, also significantly skews sex ratios towards female blastocysts (p < 0.05).ConclusionThis study shows that BPA affects AMH and AMHRII expression during oocyte maturation and that BPS exerts its effects to a greater extent after fertilization and therefore may not be a safer alternative to BPA. Our data lay the foundation for future functional studies, such as receptor kinetics, downstream effectors, and promoter activation/inhibition to prove a functional relationship between bisphenols and the AMH signalling system.
| S-EPMC8330045 | biostudies-literature
Project description:Bisphenol A (BPA) and its analogs, bisphenol S (BPS) and bisphenol F (BPF), might impact fertility by altering oxidative stress pathways. Here, we hypothesize that bisphenols-induced oxidative stress is responsible for decreased gamete quality. In both female (cumulus-oocyte-complexes-COCs) and male (spermatozoa), oxidative stress was measured by CM-H2DCFDA assay and key ROS scavengers (SOD1, SOD2, GPX1, GPX4, CAT) were quantified at the mRNA and protein levels using qPCR and Western blot (COCs)/immunofluorescence (sperm). Either gamete was treated in five groups: control, vehicle, and 0.05 mg/mL of BPA, BPS, or BPF. Our results show elevated ROS in BPA-treated COCs but decreased production in BPS- and BPF-treated spermatozoa. Additionally, both mRNA and protein expression of SOD2, GPX1, and GPX4 were decreased in BPA-treated COCs (p < 0.05). In sperm, motility (p < 0.03), but not morphology, was significantly altered by bisphenols. SOD1 mRNA expression was significantly increased, while GPX4 was significantly reduced. These results support BPA's ability to alter oxidative stress in oocytes and, to a lesser extent, in sperm. However, BPS and BPF likely act through different mechanisms.
| S-EPMC8774721 | biostudies-literature
Project description:Here, we used female adult rat adipose-derived stem cells (rASCs) and human adipose-derived stem cells (hASCs) to compare the toxicities and potencies of several widespread environmental toxins that may be endocrine-disrupting chemicals, including bisphenol A (BPA), and the newer BPA alternatives bisphenol S (BPS), bisphenol AF (BPAF), and tetramethyl bisphenol F (TMBPF). Adult stem cells were cultured to 80% confluency in vitro and then exposed to BPA (1 and 10 µM), 17β-estradiol (E2; 10 µM), BPS (1 and 10 µM), BPAF (3 × 10-3-30 µM), TMBPF (0.01-50 µM), or control media alone (with 0.01% ethanol) for varying time intervals from 20 min to 5 hrs. Using several cellular assays, the levels of cell death, apoptosis, caspase-6 activation, and potencies were compared across chemical treatments and vehicle-treated controls. There was significantly decreased cell viability and increased apoptosis in rat and human stem cells treated with each BPA analog, as early as 20 min of exposure, and at low doses. With higher magnification, higher resolution imaging it was evident that in many of the BPA analog-treated cells, the Apopxin Deep Red dye indicative of apoptosis was localized to the cytoplasmic compartments of cells, while the nuclear green DCS1 dye indicative of late-stage apoptosis and necrosis was localized to the nuclei of cells. Notably, BPAF and TMBPF showed cytotoxicity in a dose-dependent manner (BPAF LC50 = 0.014 µM (rASCs) and 0.009 µM (hASCs); TMBPF LC50 = 0.88 µM (rASCs) and 0.06 µM (hASCs); lethal concentration with 50% survival). The rank order of potency was BPAF>TMBPF>BPA>BPS. The majority of cell death was due to apoptosis as indicated by high levels of activated caspase-6 in the cytoplasm of almost 100% of cells treated with the BPA analogs. This data allows for further confirmation of caspase-6-mediated apoptosis using higher magnification imaging that definitively demonstrate the cytotoxic and apoptotic effects of these BPA analogs. For a complete description, interpretation, and discussion of the data refer to the article in press [1].
| S-EPMC8187815 | biostudies-literature
Project description:Cumulus cells are essential for nutrition of oocytes during maturation. In the absence of cumulus cells during maturation, oocyte developmental competence is severely compromised. In this study, we matured bovine cumulus-oocyte-complexes (COCs) for 8 h, the cumulus cells were removed and denuded oocytes were further matured for 15 h in either the medium conditioned by the initial 8 h culture, or in fresh unconditioned medium. Denuded oocytes that completed maturation in COC-conditioned medium demonstrated better developmental potential than denuded oocytes that completed maturation in standard maturation medium. An inventory was made of the metabolites secreted by COCs into the maturation medium during the first 8 h, from 8 to 23 h, and during an entire 23 h maturation protocol; the metabolomic changes in the cumulus cells during maturation were also investigated. In maturation medium, 173 biochemical components were detected compared to 369 different metabolites in cumulus cells. Significant changes in metabolomic components were evident in maturation medium and in cumulus cells during maturation, with most of the changes related to amino acid, carbohydrate, and lipid metabolism. The importance of two detected biochemicals, creatine and carnitine, for oocyte maturation was further investigated. The presence of carnitine, but not creatine during oocyte in vitro maturation improved the developmental competence of denuded oocytes.
| S-EPMC6013446 | biostudies-literature
Project description:Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.
| S-EPMC4556939 | biostudies-literature
Project description:microRNAs (miRNAs) are susceptible to environmental factors that might affect cellular function and impose negative effects on female reproduction. miR-21 is the most abundant miRNA in bovine granulosa cells and is widely reported as affected by Bisphenol A (BPA) exposure, yet the cause and consequences are not entirely elucidated. BPA is a synthetic endocrine disruptor associated with poor fertility. miR-21 function in bovine granulosa cells is investigated utilizing locked nucleic acid (LNA) oligonucleotides to suppress miR-21. Before measuring apoptosis and quantifying miR-21 apoptotic targets PDCD4 and PTEN, transfection was optimized and validated. BPA was introduced to see how it affects miR-21 regulation and which BPA-mediated effects are influenced by miR-21. miR-21 knockdown and specificity against additional miRNAs were confirmed. miR-21 was found to have antiapoptotic effects, which could be explained by its effect on the proapoptotic target PDCD4, but not PTEN. Previous findings of miR-21 overexpression were validated using BPA treatments, and the temporal influence of BPA on miR-21 levels was addressed. Finally, BPA effects on upstream regulators, such as VMP1 and STAT3, explain the BPA-dependent upregulation of miR-21 expression. Overall, this research enhances our understanding of miR-21 function in granulosa cells and the mechanisms of BPA-induced reproductive impairment.
| S-EPMC9368835 | biostudies-literature
Project description:Cumulus cells play an essential role during oocyte maturation and the acquisition of fertilizability and developmental competence. Micro(mi)RNAs can post-transcriptionally regulate mRNA expression, and we hypothesized that miRNA profiles in cumulus cells could serve as an indicator of oocyte quality. Cumulus cell biopsies from cumulus-oocyte-complexes that either yielded a blastocyst or failed to cleave after exposure to sperm cells were analyzed for miRNA expression. On average, 332 miRNA species with more than 10 reads and 240 miRNA species with more than 50 reads were identified in cumulus cells; this included nine previously undescribed microRNAs. The most highly expressed miRNAs in cumulus cells were miR-21, members of the let-7 family and miR-155. However, no repeatable differences in miRNA expression between the cumulus cells from oocytes that became blastocysts versus those from non-cleaved oocytes were identified. Further examination of individual cumulus cell samples showed a wide variability in miRNA expression level. We therefore conclude that miRNA expression in cumulus cells cannot be used as an oocyte quality marker.
| S-EPMC5832000 | biostudies-literature
Project description:ObjectiveRecently, the decellularization technique is introduced as one of the tissue engineering procedures for the treatment of various deficiencies. Here, we aimed to assess the dynamic activity of CCs and HUVECs within decellularized bovine ovarian tissue transplanted subcutaneously in rats. Ovarian tissue was decellularized using a cocktail consisting of different chemicals, and the efficiency of decellularization was assessed using hematoxylin-eosin and DAPI staining. The cell survival was evaluated using an LDH leakage assay. Thereafter, decellularized samples were recellularized using HUVECs and CCs, encapsulated inside alginate (1.2%)-gelatin, (1%) hydrogel, and transplanted subcutaneously to rats. The existence of CD31- and estrogen-positive cells was assessed using immunohistochemistry staining.ResultsBright-field imaging and DAPI staining revealed the lack of nuclei with naive matrix structure in ovarian tissue subjected to decellularization protocol. SEM imaging revealed a normal matrix in decellularized ovaries. LDH assay showed a lack of cytotoxicity for CCs after 7-days compared to the control group. Immunohistochemistry staining showed both CD31- and estrogen-positive cells in CCs + HUVECs compared to the CCs group. CD31 cells appeared with flattened morphology aligned with matrix fibers. The existence of estrogen and CD31 positive cells showed the efficiency of decellularized ovarian tissue to restore cellular function and activity.
| S-EPMC9644448 | biostudies-literature
Project description:The connexin 37 (Cx37) channel is clustered at gap junctions between cells in the renal vasculature or the renal tubule where it is abundant in basolateral cell interdigitations and infoldings of epithelial cells in the proximal tubule, thick ascending limb, distal convoluted tubule and collecting duct; however, physiological data regarding its role are limited. In this study, we investigated the role of Cx37 in fluid homeostasis using mice with a global deletion of Cx37 (Cx37-/- mice). Under baseline conditions, Cx37-/- had ~40% higher fluid intake associated with ~40% lower urine osmolality compared to wild-type (WT) mice. No differences were observed between genotypes in urinary adenosine triphosphate or prostaglandin E2, paracrine factors that alter renal water handling. After 18-hours of water deprivation, plasma aldosterone and urine osmolality increased significantly in Cx37-/- and WT mice; however, the latter remained ~375 mmol/kg lower in Cx37-/- mice, an effect associated with a more pronounced body weight loss despite higher urinary AVP/creatinine ratios compared to WT mice. Consistent with this, fluid intake in the first 3 hours after water deprivation was 37% greater in Cx37-/- vs WT mice. Cx37-/- mice showed significantly lower renal AQP2 abundance and AQP2 phosphorylation at serine 256 than WT mice in response to vehicle or dDAVP, suggesting a partial contribution of the kidney to the lower urine osmolality. The abundance and responses of the vasopressin V2 receptor, AQP3, NHE3, NKCC2, NCC, H+-ATPase, αENaC, γENaC or Na+/K+-ATPase were not significantly different between genotypes. In summary, these results demonstrate that Cx37 is important for body water handling.
| S-EPMC7746157 | biostudies-literature
Project description:This study aims to investigate the effects of CLAUDIN-6 (CLDN6) on cell apoptosis and proliferation of bovine cumulus cells (CCs). Immunofluorescence staining was used to localize CLDN6 protein in CCs. Three pairs of siRNA targeting CLDN6 and one pair of siRNA universal negative sequence as control were transfected into bovine CCs. Then, the effective siRNA was screened by real-time quantitative PCR (RT-qPCR) and Western blotting. The mRNA expression levels of apoptosis related genes (CASPASE-3, BAX and BCL-2) and proliferation related genes (PCNA, CDC42 and CCND2) were evaluated by RT-qPCR in CCs with CLDN6 knockdown. Cell proliferation, apoptosis and cell cycle were detected by flow cytometry with CCK-8 staining, Annexin V-FITC staining and propidium iodide staining, respectively. Results showed that the CLDN6 gene was expressed in bovine CCs and the protein was localized in cell membranes and cytoplasms. After CLDN6 was knocked down in CCs, the cell apoptosis rate significantly decreased and the pro-apoptotic genes BAX and CASPASE-3 were down-regulated significantly, whereas the anti-apoptotic gene BCL-2 was markedly up-regulated (p < 0.05). Additionally, CLDN6 knockdown significantly enhanced cell proliferation of CCs at 72 h after siRNA transfection. The mRNA levels of proliferation-related genes PCNA, CCND2 and CDC42 increased obviously in CCs with CLDN6 knockdown (p < 0.05). After CLDN6 was down-regulated, the percentage of CCs at S phase was significantly increased (p < 0.05). However, there was no remarkable difference in the percentages of cells at the G0/G1 phase and G2/M phase between CCs with or without CLDN6 knockdown (p > 0.05). Therefore, the expression of CLDN6 and its effects on cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. CLDN6 low expression inhibited cell apoptosis, induced cell proliferation and cell cycle arrest of bovine CCs.
| S-EPMC9656489 | biostudies-literature