ZnT‐1 expression in the preimplantation mouse embryo and its effect on calcium influx

Abstract

ZnT-1 expression in the pre-implantation mouse embryo and its effect on calcium influx Pre-implantation embryos develop into 9 stages over the first 5 days post-fertilisation. Calcium influx from the external environment via calcium channels, including the L-type and T-type calcium channels, is critical for embryonic gene activation and cell proliferation. In cardiomyocytes these channels are regulated by the ubiquitously expressed zinc-transporter protein ZnT-1. When plasma membrane bound, ZnT-1 facilitates zinc-efflux. Free cellular zinc regulates ZnT-1 expression, with an increase in zinc inducing transcription. In this study, ZnT-1 mRNA and protein expression were investigated in pre-implantation embryo stages using qPCR and immunofluorescence. Embryos were cultured in vitro in zinc-supplemented media and compared to embryos cultured in the absence of zinc and to in vivo developed embryos. ZnT-1 mRNA was expressed at all stages and the presence of zinc increased mRNA expression a the late 2-cell stage only. There was no difference in expression between in vivo developed and cultured embryos. ZnT-1 protein was expressed from the early 2-cell stage onwards; not affected by zinc culture and localized to the plasma membrane at the late 2-cell stage only. Calcium imaging was performed to examine whether ZnT-1 membrane localization altered calcium influx. Experiments on early and late 2-cell embryos showed that there was no difference in calcium influx when ZnT-1 was localized to the plasma membrane. In summary ZnT-1 transcription was induced by zinc at the late 2-cell stage. Protein expression was not affected by zinc culture but was developmentally regulated, localizing to the plasma-membrane at the late 2-cell stage without effect on calcium influx.