Abstract
The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.
Highlights
Sperm capacitation is characterized by a set of physiological changes preparing the male gamete for fertilization [1]
To address the physiological role of HVCN1 channels during in vitro capacitation of pig sperm, some samples were incubated in the presence of 2-guanidino benzimidazole (2-GBI) blocker at 1, 5 or 10 mM added at time 0 (Experiment 1)
In order to understand the functional relationship between progesterone and HVCN1 channels, 2-GBI blocker was added together with progesterone at 240 min of incubation, in a second group of samples (Experiment 2)
Summary
Sperm capacitation is characterized by a set of physiological changes preparing the male gamete for fertilization [1]. These physiological changes include increased beat frequency and curvilinear motility, intracellular pH (pHi) alkalinization, rise in intracellular calcium levels, augmented membrane lipid disorder and tyrosine phosphorylation of specific proteins, which are required for sperm to trigger acrosomal exocytosis [1,2,3,4,5,6,7,8]. Several channels are implicated in the increase of pHi during capacitation and acrosomal reaction in mammalian spermatozoa, including HCO3− membrane transporters, Na+-H+ exchangers (NHEs), monocarboxylate transporters (MCTs) and voltage-gated proton channels (HVCN1) [9,10]. HVCN1 channels drive protons more quickly and efficiently than transporters or exchangers do, and lead them unidirectionally to the extracellular medium [12]
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