Guanine-Nucleotide Exchange Factors (RAPGEF3/RAPGEF4) Induce Sperm Membrane Depolarization and Acrosomal Exocytosis in Capacitated Stallion Sperm1

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 Ca2+ channels, which later open in response to zona pellucida-induced membrane depolarization. We hypothesized that RAPGEF3 may be inducing acrosomal exocytosis via depolarization-dependent Ca2+ 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 (Em) 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, Em remained depolarized (−32.06 mV). Altogether, these experiments support the hypothesis that RAPGEF3/RAPGEF4 activation regulates acrosomal exocytosis via its modulation of Em, a novel role for RAPGEF3/RAPGEF4 in the series of events required to achieve fertilization

The 5,10-methylenetetrahydrofolate reductase C677T polymorphism interacts with smoking to increase homocysteine.

Elevated homocysteine is a risk marker for several human pathologies. Risk factors for elevated homocysteine include low folate and homozygosity for the T allele of the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Because nitric oxide may inhibit folate catabolism and endothelial nitric oxide synthase activity is reduced in smokers, we postulated that smoking status might modify the impact of the MTHFR C677T polymorphism on homocysteine (tHcy) concentrations. We tested this hypothesis in a healthy young adult population for which MTHFR C677T genotypes and tHcy concentrations were previously reported. The MTHFR 677TT genotype was significantly associated with elevated tHcy concentrations in smokers (P = 0.001) but not in non-smokers (P = 0.36). Among smokers, the MTHFR 677TT genotype was significantly associated with high tHcy in heavy smokers (P = 0.003) but not light smokers (P = 0.09), in men (P = 0.003) but not women (P = 0.11), and in subjects from the lowest serum folate quartile (P = 0.49) but not from folate quartiles 2-4 (P = 0.49). After adjustment for nutritional variables, interactions between MTHFR C677T genotype and NOS3 G894T genotype, and between MTHFR genotype, smoking status and gender were statistically significant. We propose that hyperhomocysteinemia in MTHFR 677TT homozygote smokers is the consequence of mild intracellular folate deficiency caused by a smoking-related reduction of NOS3 activity that is exacerbated when serum folate is low