Regulation of intracellular Ca2+ in bovine spermatozoa: relevance of NCX and SERCA

MAIA RODRÍGUEZ; ENRIQUE L PORTIANSKY; ALEJANDRO ORLOWSKI; PAOLA FERRERO

Congreso; XXXIV Meeting Chilean Society of Reproduction and Development; 2023

Background. Spermatozoa undergo biochemical and physiological changes known ascapacitation, allowing them the ability to fertilize the oocyte. Modulation of the intracellularcalcium (Ca 2+i ) is essential for capacitation. This work studied the participation of theNa+/Ca 2+ exchanger (NCX) and the Ca 2+ ATPase pump (SERCA) in managing Ca 2+i incryopreserved bovine spermatozoa. Methods. Cryopreserved bovine sperm (obtainedthrough CIAVT) were thawed and resuspended in TALP medium. Subcellular localization ofthese proteins was evaluated in three different samples by indirect immunofluorescence,using anti NCX and anti SERCA primary antibodies and a cyanine conjugated secondaryantibody. Furthermore, we utilized inhibitors of NCX (KB R7943) and SERCA (thapsigargin:Tg) to test their contr ibution to the regulation of Ca 2+i levels. We used the FLUO 3AM asCa 2+i dye and a Varioskan multimode reader to detect the fluorescent signal. Fluorescenceintensity was measured for 10 minutes in four groups: untreated or treated with Tg, KB R,and Tg+KB R. We calculated the fluorescence difference between the intensity recordedafter 5 minutes and the initial intensity. The data were analyzed by one way ANOVA. Results.The NCX was mainly detected in the post acrosomal region, while the SERCA was observedin the acrosome. Treatment of spermatozoa with Tg and Tg + KB R produced a significantincrease in fluorescence intensity relative to control. However, we not detected changes incells treated with KB R alone. The combination of inhibitors slightly increa sed Ca 2+icompared to treatment with Tg alone. Conclusions. The results showed that SERCA, locatedin the acrosome, contributes significantly to the increase in cytosolic Ca 2+i . In contrast, theNCX, situated in the post acrosomal region, plays a less rele vant role, or its function mightbe compensated by other components not identified in this study promoting Ca 2+ release outof cells while NCX is blocked.