Conserved Mechanism of Bicarbonate-Induced Sensitization of CatSper Channels in Human and Mouse Sperm

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.

Strategies to safely target widely expressed soluble adenylyl cyclase for contraception

“…These changes are essential for sperm to pass through the cervix to leave the inhospitable environment of the vagina and enter the permissive environment of the uterus (Visconti et al, 1995;Visconti et al, 2002;Suarez and Pacey, 2006;Buffone et al, 2014;Balbach et al, 2018). sAC's essential role in male reproduction has been validated genetically (Esposito et al, 2004;Hess et al, 2005;Akbari et al, 2019;Balbach et al, 2021) and pharmacologically (Hess et al, 2005;Mannowetz et al, 2012;Ramos-Espiritu et al, 2016;Balbach et al, 2021;Ferreira et al, 2021) in both mice and men. Two different strains of sAC knockout (KO) mice show male-specific sterility; their sperm are immotile and unable to fertilize an oocyte in vitro (Esposito et al, 2004;Hess et al, 2005;Xie et al, 2006;Chen et al, 2013).…”

“…sAC’s essential role in male reproduction has been validated genetically ( Esposito et al, 2004 ; Hess et al, 2005 ; Akbari et al, 2019 ; Balbach et al, 2021 ) and pharmacologically ( Hess et al, 2005 ; Mannowetz et al, 2012 ; Ramos-Espiritu et al, 2016 ; Balbach et al, 2021 ; Ferreira et al, 2021 ) in both mice and men. Two different strains of sAC knockout (KO) mice show male-specific sterility; their sperm are immotile and unable to fertilize an oocyte in vitro ( Esposito et al, 2004 ; Hess et al, 2005 ; Xie et al, 2006 ; Chen et al, 2013 ).…”

“…sAC’s essential role in fertility, and as a target for contraception, has also been validated pharmacologically in humans and mice. Multiple, structurally independent, sAC-specific inhibitors prevent the capacitation-induced changes in mouse and human sperm, including stimulation of cAMP production, protein kinase A (PKA) activation, alkalinization, increased beat frequency, hyperactivated motility, and ability to undergo a physiologically induced acrosome reaction ( Hess et al, 2005 ; Mannowetz et al, 2012 ; Ramos-Espiritu et al, 2016 ; Balbach et al, 2021 ; Ferreira et al, 2021 ). sAC-specific inhibitors also prevent in vitro fertilization in mice ( Ramos-Espiritu et al, 2016 ; Balbach et al, 2021 ).…”

“…Thus, inhibiting sAC in sperm inside the vagina would trap them in this inhospitable environment and prevent fertilization. This idea is already validated in vitro with post-ejaculated sperm ( Balbach et al, 2021 ; Ferreira et al, 2021 ). Specifically, sAC inhibitors interrupt capacitation, inhibit progressive motility, and block acrosome reaction in human sperm when added post-ejaculation (i.e., after capacitation has begun), which is exactly when a vaginally delivered contraceptive would encounter activated sperm.…”

Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin

“…It is generally believed that dramatic changes occur during sperm capacitation and are manifested as the deconstruction of sperm membrane [ 21 ], increase in intracellular pH (pH i ) [ 22 ], influx of Ca 2+ [ 23 ], membrane hyperpolarization [ 24 ], and tyrosine phosphorylation [ 25 ]. Capacitation is induced by different factors in in-vitro media or female reproduction tract, such as heparin [ 26 ], bicarbonate [ 27 ], bovine serum albumin (BSA) [ 28 ], and calcium ions [ 29 ]. However, the molecular mechanism about how different substrates cause sperm’s capacitation has not been fully unveiled.…”