P-221 Bisphenol A affects Ca2+ homeostasis in mouse oocytes through GPR30 receptor and MAPK/ERK signalling pathway

Abstract

Abstract Study question Does bisphenol A (BPA), xenoestrogen present in plastics, affect Ca2+ homeostasis in mouse oocytes? If yes, what is the mechanism of BPA action? Summary answer BPA (2 ng/ml, concentration reported for human follicular fluid) alternates the Ca2+ pattern generated in fertilized oocytes acting through GPR30 receptor and MAPK/ERK signaling pathway. What is known already BPA is a monomeric organic compound widely used in the production of resins, polycarbonates, and plastics. It is a xenoestrogen, i.e. a synthetic compound exerting an estrogen-like effect on cells. It can therefore disrupt the functioning of animal (including human) organisms. Recent literature data indicate that BPA may affect fertility in mammals by disturbing several processes in oocytes and embryos, including epigenetic modifications, energy metabolism, and spindle assembly. However, its effect on Ca2+ homeostasis in oocytes, key for activation of embryonic development upon fertilization, has not been yet examined. Study design, size, duration The study was carried out on mouse (a C57Bl6/Tar x CBA/Tar cross) oocytes. Each experimental variant was repeated at least 3 times, the number of oocytes analyzed was 40-75 per group, depending on the experimental variant. Participants/materials, setting, methods BPA was added to M16 medium, in which mouse oocytes matured in vitro. Oocytes that reached metaphase II were labeled with Oregon Green 488 BAPTA-1 AM, a fluorescent Ca2+ indicator, fertilized in vitro, and subjected to time-lapse imaging. The fluorescence intensity was analyzed to assess the pattern of Ca2+ oscillations triggered in oocytes upon fertilization. In some experiments, specific inhibitors were used to analyze the molecular pathway mediating the BPA action in oocytes. Main results and the role of chance 2ng/ml BPA added to oocytes during their in vitro maturation decreased the maturation rate (from 77% in control to 52%). BPA shortened the total duration of Ca2+ oscillations induced in metaphase II oocytes by fertilization (183 ± 80.4 min in control vs. 118.2 ± 84.2 min in BPA group) and the mean interval between the subsequent Ca2+ transients (14.3 ± 4.9 min vs. 9.7 ± 4.5 min). As GPR30, G protein-coupled estrogen receptor, can bind BPA and is expressed in oocytes, we investigated whether it mediates BPA action. G1, a GPR30 activator, added to the oocyte maturation medium instead of BPA, mimicked the BPA effect only partially: it shortened the total duration of Ca2+ oscillations (175.6 ± 71.5 in control vs. 134.5 ± 90.8 min in G1 group), but increased the inter-transient interval (11.6 ± 5.2 min vs. 15.2 ± 4.7 min). On the other hand, G15, a GPR30 inhibitor, alleviated the BPA-induced alternations (total duration of Ca2+ oscillations: 192.7 ± 77.5 min, the interval between Ca2+ transients: 11.4 ± 4.6 min). Moreover, PD0325901, a MAPK/ERK pathway inhibitor, rescued BPA-induced changes in the duration and frequency of Ca2+ oscillations (195.9 ± 95 min and 18.2 ± 4.8 min, respectively). Limitations, reasons for caution The susceptibility of oocytes to BPA, and, in consequence, its effect on Ca2+ homeostasis, may be different for various mammalian species. Wider implications of the findings BPA disturbs the pattern of Ca2+ oscillations in fertilized mouse oocytes through GRP30 and MAP/ERK pathway, but it is possible that BPA acts also through other estrogen receptors. Ca2+ oscillations are crucial for activation of embryonic development, so our observations provide a novel mechanism of BPA action on mammalian fertility. Trial registration number n/a