Inhibiting oocyte spontaneous activation (SA) is essential for successful rat cloning by nuclear transfer (NT). This study tested the hypothesis that activities of the Na /Ca2 exchanger (NCX) would decrease with oocyte aging and that SA of rat oocytes could be inhibited if the intraoocyte Ca2 rises were prevented by activating the NCX through increasing Na concentrations in the culture medium. Elevating Na levels in culture medium by supplementing NaCl inhibited SA of rat oocytes, while maintaining a constant level of maturation-promoting factor and mitogen-activated protein kinase activities. Experiments using the NCX inhibitor bepridil, the Na /K -ATPase inhibitor ouabain, and an assay for intraoocyte Ca2 concentrations showed that extracellular Na inhibited rat oocyte SA by enhancing NCX activity and preventing intracellular Ca2 rises. Immunohistochemical quantification indicated that the density of NCX1 decreased significantly in aged oocytes that were prone to SA compared with that in freshly ovulated oocytes whose SA rates were low during in vitro culture. Cumulus cell NT showed that sham enucleation caused marked SA in freshly ovulated rat oocytes and that Na supplementation prevented the manipulation-induced SA and improved the in vitro and in vivo development of rat somatic cell NT embryos. Taken together, the results have confirmed our hypothesis that the NCX is active in rat oocytes and its activity decreases with oocyte aging and that activating the NCX by increasing extracellular Na inhibits SA of rat oocytes and improves the development of rat somatic cell NT embryos. These data are also important for understanding the mechanisms of oocyte aging.
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Vol. 88 • No. 6