BSA-mediated CatSper-independent rapid calcium uptake initiates sAC activation during mouse sperm capacitation

Abstract

Mammalian sperm require capacitation to fertilize oocytes, whichcan be induced in vitro using capacitating medium (CAP) containingenergy sources, albumin, Ca2+, and HCO3-. Previous studieshave shown that only a subpopulation of mouse sperm exhibits arapid increase in intracellular Ca2+ concentration ([Ca2+]i) duringCAP incubation, becoming evident within 1 minute. This study investigatedearly [Ca2+]i changes by analyzing live sperm loadedwith the Ca2+ dye Fluo‐4 AM to determine if this early [Ca2+]i isrequired for the initial activation of soluble adenylyl cyclase (sAC).When extracellular Ca2+ was removed, either by using a mediumwith no added Ca2+ or by adding EGTA, this early Ca2+ influx wasabolished, indicating that Ca2+ is incorporated from the extracellularenvironment through an ion channel or transporter. The rapid risewas independent of the CatSper channel, as CatSper1 KO spermincubated in CAP showed a significant increase in [Ca2+]i similarto wild-type sperm. To identify which CAP component is responsiblefor this initial Ca2+ uptake, CatSper1 KO sperm were exposedto either HCO3- or bovine serum albumin (BSA), revealing that theCa2+ increase occurred only in response to BSA. As an indirect assessmentof sAC activation, we measured substrates phosphorylatedby PKA (pPKAs) in CatSper1 KO sperm after BSA addition andfound that BSA induced a significant increase in pPKAs. Additionally,pharmacological inhibition of sAC with 11861 did not affect the early[Ca2+]i increase, indicating that sAC activation occurs downstreamof this Ca2+ uptake. These findings suggest that a subpopulation ofmouse sperm rapidly increases [Ca2+]i during capacitation due to aCatSper-independent influx of extracellular Ca2+ induced by BSA.This rise in [Ca2+]i is critical for the initial activation of sAC, offeringnew insights into the molecular mechanisms behind sAC regulationand the early events that trigger capacitation signaling pathways.