The effects of protein kinase A catalytic subunit on sperm motility regulation in Pacific abalone Haliotis discus hannai

Protein kinase A plays a central role in the regulation of sperm motility from echinoderms to mammals, but the information about its regulatory role in molluscs is very limited. In this study, a protein kinase A catalytic subunit (designated as HdPKA-C) was identified from Pacific abalone Haliotis discus hannai. The open reading frame of HdPKA-C was of 1,077 bp, encoding a peptide of 358 amino acids with a typical protein kinase domain. HdPKA-C shared 82%-87% sequence similarities with other PKA-Cs, and it was clustered first with gastropod PKA-Cs in the phylogenetic tree. The mRNA of HdPKA-C was constitutively expressed in examined tissues, with the highest level detected in hepatopancreas. The phosphorylated form of HdPKA-C (p-HdPKA-C) was localized at the acrosome, connecting piece and flagellum of spermatozoa with variable intensity. Its phosphorylated substrates were also detected in these regions with much lower intensity at the connecting piece. The inhibition of HdPKA-C activity with H-89 led to a significant reduction in the percentage of motile sperm and sperm velocities. p-HdPKA-C was detected by Western blot in strip-spawned sperm, naturally spawned sperm and H-89-treated sperm with almost the same intensity. The intensity of p-HdPKA-C substrates in naturally spawned sperm was higher than that in strip-spawned sperm, and it was roughly the same as that in H-89-treated sperm except for two bands at 50 and 60 kDa. These results collectively indicated that HdPKA-C played an important role in the regulation of abalone sperm motility by altering its substrates phosphorylation.