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

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 (E-m) hyperpolarizes. However, the mechanisms that regulate sperm E-m are not well understood. Here we show that sperm hyperpolarize when external Na+ is replaced by N-methyl-glucamine. Readdition of external Na+ restores a more depolarized E-m 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 E-m. 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-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta 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.