Abstract
To fertilize an egg, mammalian sperm must undergo capacitation in the female genital tract. A key contributor to capacitation is the calcium (Ca2+) channel CatSper, which is activated by membrane depolarization and intracellular alkalinization. In mouse epididymal sperm, membrane depolarization by exposure to high KCl triggers Ca2+ entry through CatSper only in alkaline conditions (pH 8.6) or after in vitro incubation with bicarbonate (HCO3–) and bovine serum albumin (capacitating conditions). However, in ejaculated human sperm, membrane depolarization triggers Ca2+ entry through CatSper in non-capacitating conditions and at lower pH (< pH 7.4) than is required in mouse sperm. Here, we aimed to determine the mechanism(s) by which CatSper is activated in mouse and human sperm. We exposed ejaculated mouse and human sperm to high KCl to depolarize the membrane and found that intracellular Ca2+ concentration increased at pH 7.4 in sperm from both species. Conversely, intracellular Ca2+ concentration did not increase under these conditions in mouse epididymal or human epididymal sperm. Furthermore, pre-incubation with HCO3– triggered an intracellular Ca2+ concentration increase in response to KCl in human epididymal sperm. Treatment with protein kinase A (PKA) inhibitors during exposure to HCO3– inhibited Ca2+ concentration increases in mouse epididymal sperm and in both mouse and human ejaculated sperm. Finally, we show that soluble adenylyl cyclase and increased intracellular pH are required for the intracellular Ca2+ concentration increase in both human and mouse sperm. In summary, our results suggest that a conserved mechanism of activation of CatSper channels is present in both human and mouse sperm. In this mechanism, HCO3– in semen activates the soluble adenylyl cyclase/protein kinase A pathway, which leads to increased intracellular pH and sensitizes CatSper channels to respond to membrane depolarization to allow Ca2+ influx. This indirect mechanism of CatSper sensitization might be an early event capacitation that occurs as soon as the sperm contact the semen.
Highlights
After ejaculation, human and mouse sperm cannot fertilize an egg until they undergo a maturation process in the female genital tract known as capacitation (Austin, 1951, 1952; Chang, 1951)
As expected, bypassing the increase in pH that occurs during capacitation by incubating the sperm at alkaline pH allowed 77% of the non-capacitated sperm to respond to KCl
As with mouse epididymal sperm, bypassing the increase in pH that occurs during capacitation with alkaline pH allowed a high percentage (81%) of sperm to respond to KCl
Summary
Human and mouse sperm cannot fertilize an egg until they undergo a maturation process in the female genital tract known as capacitation (Austin, 1951, 1952; Chang, 1951). Capacitation involves several molecular events such as hyperpolarization of the plasma membrane (Zeng et al, 1995; Santi et al, 2010; de la Vega-Beltran et al, 2012; Chavez et al, 2013; López-González et al, 2014) and increases in intracellular pH, cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, tyrosine phosphorylation (Visconti et al, 1995; Battistone et al, 2013; Puga Molina et al, 2018), and intracellular calcium concentration ([Ca2+]i) (Baldi et al, 1991; Suarez et al, 1993; Breitbart, 2002a,b; Luque et al, 2018) Some of these events take place as soon as as the sperm are ejaculated, whereas others occur over a longer period of time in the female tract or in a medium that support in vitro capacitation (Visconti, 2009)
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