Abstract
Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.
Highlights
Alkalinization of inner pH is considered a central event for eliciting the sequence of changes related to sperm capacitation, since it is necessary for further activation of CatSper channels and Ca2+ entrance [1,2,3,4]
While HVCN1 channels are not present in the plasma membrane of mouse sperm [8,9], they have been identified in the flagellum of human, pig, and cattle sperm, where they exert a key role in the regulation of hypermotility [2,6,9]
HVCN1 channels are functionally related to their CatSper counterparts [10], whereas, in pigs, the activity of HVCN1 channels does not seem to be related to Ca2+ influx [2], suggesting that other H+ transporters could participate in the regulation of pHi during sperm capacitation in this species
Summary
Alkalinization of inner pH (pHi ) is considered a central event for eliciting the sequence of changes related to sperm capacitation, since it is necessary for further activation of CatSper channels and Ca2+ entrance [1,2,3,4]. Different ion channels and transporters are implicated in pHi regulation of sperm cells, being the Na+ /H+ exchangers (NHEs), HCO3 −. While HVCN1 channels are not present in the plasma membrane of mouse sperm [8,9], they have been identified in the flagellum of human, pig, and cattle sperm, where they exert a key role in the regulation of hypermotility [2,6,9]. HVCN1 channels are functionally related to their CatSper counterparts [10], whereas, in pigs, the activity of HVCN1 channels does not seem to be related to Ca2+ influx [2], suggesting that other H+ transporters could participate in the regulation of pHi during sperm capacitation in this species.
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