title: Acetylcholine causes an increase of intracellular calcium in human sperm.
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 microM 3-quinuclidinyl benzilate (QNB) or 50 nM strychnine failed to inhibit the ACh-initiated AR, demonstrating that muscarinic AChRs and nAChRs containing alpha9 subunits do not mediate this event. Choline (2.5, 5 and 10 mM), a highly specific but low potency agonist of the alpha7 nAChR initiated AR, with its effect blocked by the nAChR antagonist methyllycaconitine (MLA). ACh (50-400 microM) stimulated a small transient rise in the intracellular Ca2+ in sperm populations loaded with FURA-2, with 200 microM 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 alpha7 or alpha9 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+ ( approximately 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.
authors.affiliation: Department of Cell Biology and Human Anatomy, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA, USA. cmbray@ucdavis.edu
authors.forename: C
authors.initials: C
authors.lastname: Bray
authors.forename: J-H
authors.initials: JH
authors.lastname: Son
authors.forename: S
authors.initials: S
authors.lastname: Meizel
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D020101:Acrosome Reaction / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008297:Male; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000502:physiology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
chemical_list: D011978:Receptors, Nicotinic; D012964:Sodium; D000109:Acetylcholine; D002118:Calcium