Large cyclic AMP-dependent increases in tyrosine-phosphorylated proteins are not absolutely necessary for the induction of full-type hyperactivation and penetration into eggs in ejaculated boar spermatozoa in vitro.

Abstract

Although it is well-known that sperm tyrosine-phosphorylated proteins increase in a cyclic AMP (cAMP)-dependent manner during capacitation, it remains unclear whether or not this reaction is involved directly in sperm fertilization-related events. This study aimed to examine the effects of pharmacological suppression of cAMP-dependent increases in tyrosine-phosphorylated proteins on the induction of full-type hyperactivation and penetration into eggs in boar spermatozoa. Ejaculated spermatozoa were treated with a cAMP analog (cBiMPS) and subsequently used for Western blotting, flagellar beating assays, and egg penetration assays. Marked increases in tyrosine-phosphorylated proteins and full-type hyperactivation were observed in spermatozoa treated with cBiMPS in the presence of CaCl₂. These spermatozoa effectively penetrated eggs. A spleen tyrosine kinase (SYK) inhibitor OXSI2 suppressed large cBiMPS-dependent increases in sperm tyrosine-phosphorylated proteins, in contrast to the ineffectiveness of SU6656 (a SRC family kinase inhibitor) and dasatinib (a c-ABL inhibitor). However, this suppression was not associated with changes in the state of full-type hyperactivation or the outcomes of egg penetration assays conducted in insemination medium without OXSI2. Conversely, adding OXSI2 to both the cBiMPS treatment medium and the sperm insemination medium suppressed increases in sperm tyrosine-phosphorylated proteins during insemination and significantly decreased the average number of spermatozoa that penetrated eggs. These results suggest that high SYK activation and large cAMP-dependent increases in tyrosine-phosphorylated proteins are not absolutely necessary for inducing full-type hyperactivation and penetration into eggs in boar spermatozoa in vitro, but may influence sperm functions associated with the outcomes of egg penetration assays.