title: Guanine-nucleotide exchange factors (RAPGEF3/RAPGEF4) induce sperm membrane depolarization and acrosomal exocytosis in capacitated stallion sperm.
abstract: Capacitation encompasses the molecular changes sperm undergo to fertilize an oocyte, some of which are postulated to occur via a cAMP-PRKACA (protein kinase A)-mediated pathway. Due to the recent discovery of cAMP-activated guanine nucleotide exchange factors RAPGEF3 and RAPGEF4, we sought to investigate the separate roles of PRKACA and RAPGEF3/RAPGEF4 in modulating capacitation and acrosomal exocytosis. Indirect immunofluorescence localized RAPGEF3 to the acrosome and subacrosomal ring and RAPGEF4 to the midpiece in equine sperm. Addition of the RAPGEF3/RAPGEF4-specific cAMP analogue 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8pCPT) to sperm incubated under both noncapacitating and capacitating conditions had no effect on protein tyrosine phosphorylation, thus supporting a PRKACA-mediated event. Conversely, activation of RAPGEF3/RAPGEF4 with 8pCPT induced acrosomal exocytosis in capacitated equine sperm at rates (34%) similar (P > 0.05) to those obtained in progesterone- and calcium ionophore-treated sperm. In the mouse, capacitation-dependent hyperpolarization of the sperm plasma membrane has been shown to recruit low voltage-activated T-type Ca(2+) channels, which later open in response to zona pellucida-induced membrane depolarization. We hypothesized that RAPGEF3 may be inducing acrosomal exocytosis via depolarization-dependent Ca(2+) influx, as RAPGEF3/RAPGEF4 have been demonstrated to play a role in the regulation of ion channels in somatic cells. We first compared the membrane potential (E(m)) of noncapacitated (-37.11 mV) and capacitated (-53.74 mV; P = 0.002) equine sperm. Interestingly, when sperm were incubated (6 h) under capacitating conditions in the presence of 8pCPT, E(m) remained depolarized (-32.06 mV). Altogether, these experiments support the hypothesis that RAPGEF3/RAPGEF4 activation regulates acrosomal exocytosis via its modulation of E(m), a novel role for RAPGEF3/RAPGEF4 in the series of events required to achieve fertilization.
authors.affiliation: Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: McPartlin
authors.forename: P E
authors.initials: PE
authors.lastname: Visconti
authors.forename: S J
authors.initials: SJ
authors.lastname: Bedford-Guaus
mesh_terms: D000177:Acrosome / Q000502:physiology*; D000818:Animals; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives; D005089:Exocytosis; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006736:Horses / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013075:Sperm Capacitation; D013873:Thionucleotides
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004004:Dichlororibofuranosylbenzimidazole; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits