Effect of cryopreservation on phosphorylation state of proteins involved in sperm motility initiation in sea bream

We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing–thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS–PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen–thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.