244 INVOLVEMENT OF Na+/H+ ANTIPORTER 1 IN EMBRYONIC DEVELOPMENT OF RAT OOCYTES ACTIVATED BY ETHANOL

Abstract

In mammalian oocytes, artificial activation is essential for further embryonic development of somatic cell nuclear transferred or round spermatid-injected oocytes. Unlike the protocol for mice, the optimal activation protocol for rat oocytes is not well established. Recently, we showed for the first time that a combined treatment with ethanol and 6-dimethylaminopurine (6-DMAP) efficiently induced activation of rat oocytes, resulted in a higher proportion of blastocyst formation as compared with other combined treatments or with the 6-DMAP treatment alone (Sano et al. 2009 Zygote 17, 29–36). Although the ethanol treatment has been used for oocyte activation not only in rats, but also in other species to date, it is unclear why the ethanol treatment is effective, especially in activating rat oocytes. Because it has been reported that alcohol alters intracellular pH via exchangers, transporters, or both on the membrane surface in the myocardium, it leads us to the possibility that the ethanol treatment regulates such exchangers, transporters, or both in rat oocytes. The aim of the present study was to clarify the role of Na+/H+ exchanges (NHE) in activating rat oocytes treated with ethanol. First, we examined which types of NHE are expressed in rat ovaries, oocytes, and parthenogenetic embryos. Expression of the NHE1 protein was also confirmed in both oocytes and parthenogenetic embryos, but not in NHE2 and NHE3. Treatment with 5-(N-ethyl-N-isopropyl)amiloride hydrochloride, an NHE inhibitor, did not affect the development of ethanol-activated oocytes to the 2-cell stage; however, it decreased the rate of blastocyst formation in a dose-dependent manner. The 5-(N-ethyl-N-isopropyl)amiloride hydrochloride treatment did not decrease development to either the 2-cell or the blastocyst stage of oocytes activated by 6-DMAP alone. Taken together, we conclude that ethanol treatment was involved in NHE1 activation, resulting in high developmental ability of rat oocytes. This work was supported in part by a Grant-in-Aid for Scientific Research from JSPS (KAKENHI; 21780253) to J. I. The work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities, to J. I. and N. K.