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Mol. Hum. Reprod. Advance Access published online on January 18, 2006
Molecular Human Reproduction, doi:10.1093/molehr/gah245
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received June 7, 2005
Accepted November 11, 2005

Article

Acetylcholine causes an increase of intracellular calcium in human sperm

C. Bray 1 *, J.-H. Son 1, and S. Meizel 1

1 Department of Cell Biology and Human Anatomy, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA, USA

* To whom correspondence should be addressed.
C. Bray, E-mail: cmbray{at}ucdavis.edu


  Abstract

Sperm nicotinic acetylcholine receptors (nAChRs) can influence motility and the initiation of acrosome reaction (AR). We report that AR initiation by acetylcholine (ACh) in capacitated human sperm requires both Na+ and Ca2+ in the external medium. Pre-incubation with 50 µM 3-quinuclidinyl benzilate (QNB) or 50 nM strychnine failed to inhibit the ACh-initiated AR, demonstrating that muscarinic AChRs and nAChRs containing {alpha}9 subunits do not mediate this event. Choline (2.5, 5 and 10 mM), a highly specific but low potency agonist of the {alpha}7 nAChR initiated AR, with its effect blocked by the nAChR antagonist methyllycaconi-tine (MLA). ACh (50-400 µM) stimulated a small transient rise in the intracellular Ca2+ in sperm populations loaded with FURA-2, with 200 µM ACh being maximal (146 nM ± 23 SEM). The nAChR antagonists, {alpha}-bungarotoxin ({alpha}-BTX) and MLA, reduced the ACh-initiated Ca2+ rise by 75 and 78%, respectively, demonstrating the majority of the rise is mediated through nAChRs containing {alpha}7 or {alpha}9 subunits. Single cell imaging studies using FLUO-3 resolved two patterns of ACh-stimulated Ca2+ increase in the sperm head: 94% of responding sperm displayed a rise (59.6% ± 5.7 SEM increase from resting fluorescence intensity), returning to resting levels over a period of 2-3 min. The remaining sperm (6%) displayed a sharp spike of Ca2+ (~1 min; 86% ± 4.3 SEM change in fluorescence intensity), followed by abrupt loss of fluorescence, a pattern suggestive of AR. A Ca2+ influx in the sperm midpiece appeared to accompany the Ca2+ influx seen in the head. These observations confirm an ionotropic role for nAChRs in sperm function.

Keywords: calcium/nicotinic acetylcholine receptors/sperm.
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