Sperm capacitation involves a series of biochemical and physiological changes essential for fertilization. A critical regulator of capacitation, the soluble adenylyl cyclase (sAC; ADCY10)-dependent production of the second messenger cyclic AMP (cAMP), drives key downstream events such as protein kinase A (PKA) substrate phosphorylation. sAC activity is directly stimulated by bicarbonate (HCO3-) and calcium (Ca2+). CatSper, a sperm-specific Ca2+ channel, is considered the primary pathway for Ca2+ influx during capacitation; however, emerging evidence suggests additional pathways exist. This study reveals that bovine serum albumin (BSA) influences the dynamics of intracellular Ca2+ concentration ([Ca2+]i) in CatSper1 knockout (KO) sperm and plays a novel role in sAC activation. Using single-cell live imaging and flow cytometry, we observed a rapid [Ca2+]i rise in the head of CatSper1 KO sperm under capacitating conditions, indicating an alternative Ca2+ entry mechanism. BSA alone, in the absence of HCO3-, triggered a significant [Ca2+]i rise. Removal of extracellular Ca2+ abolished this [Ca2+]i rise, confirming the necessity of Ca2+ influx. This BSA-induced [Ca2+]i rise was upstream of sAC activation, since it was not affected by sAC inhibitors and led to increased cAMP production and PKA substrate phosphorylation. Our findings provide new insights into the regulatory mechanisms of sAC, highlighting the existence of a CatSper-independent Ca2+ entry pathway activated by BSA during sperm capacitation. This rapid [Ca2+]i rise is initiated in the sperm head and propagates throughout the cell, and is sufficient to activate sAC and stimulate cAMP synthesis independently of HCO3-. KEY POINTS: Sperm capacitation, essential for fertilization, is regulated by sAC, which produces cAMP in response to HCO3- and Ca2+, driving key events like protein kinase A substrate phosphorylation. We demonstrate the existence of a CatSper-independent Ca2+ entry pathway that initiates in the sperm head and propagates throughout the cell, occurring rapidly after sperm encounters albumin, a critical component of the capacitation medium used in in vitro fertilization procedures in mammals. This albumin-induced Ca2+ influx is sufficient to activate sAC and stimulate cAMP synthesis independently of HCO3-. We further reveal a novel role for albumin, beyond its well-established function as a cholesterol acceptor, in triggering this rapid Ca2+ influx and downstream signalling events essential for sperm capacitation. By demonstrating a CatSper-independent regulatory pathway, we expand the current paradigm of Ca2+ signalling in sperm physiology.
