Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm

Abstract

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (Em) hyperpolarizes. However, the mechanisms that regulate sperm Em are not well understood. Here we show that sperm hyperpolarize when external Na+ is replaced by N-methyl-glucamine. Readdition of external Na+ restores amore depolarized Em by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na+ transporter, possibly involving an amiloride sensitive Na+ channel, may contribute to the sperm resting Em. Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na+ current whose characteristics match those of the epithelial Na+ channel (ENaC) family of epithelial Na+ channels. Indeed, ENaC-α and -δ mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-α and -δ proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-α to the flagellar midpiece and of ENaC-δ to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na +. These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.