Bovine serum albumin‐induced calcium influx triggers soluble adenylyl cyclase activation and cyclic AMP signalling pathways in mouse sperm capacitation

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.