Desalted and lyophilized seminal plasma increases protein tyrosine-phosphorylation of frozen-thawed bull spermatozoa incubated with a cell-permeable cyclic AMP (cAMP) analog (cBiMPS)

摘要

The present study investigates the effect of desalted seminal plasma (SP) added to semen extender on hyperactivated motility and protein tyrosine-phosphorylation (PTP) of bull spermatozoa. The SP was harvested by centrifugation and desalted using Sephadex G-25 columns in order to be added to semen extender at 0 (control), 2.5, 12.5 and 25 mg/ml. Frozen-thawed spermatozoa were incubated with a cell permeable cyclic AMP (cAMP) analog (cBiMPS) and examined subjectively for hyperactivated motility and for PTP by Western blotting. Although, the added SP sustains sperm motility at all incubation times especially in the presence of cBilWPS but without significant difference from the control samples. Moreover, total sperm motility of 12.5 and 25 mg/ml in the presence of cBiMPS at 60, 120 and 180 min were similar (P >= 0.05). Surprisingly, cBiMPS-incubated spermatozoa in the presence of desalted SP were capable of exhibiting hyperactivated motility. Addition of SP increased and prolonged intracellular cAMP-induced PTP and in total 21 phosphorylated proteins with molecular weight ranging from 10 to >230 kDa were detected. The most prominent tyrosine-phosphorylated proteins (TPPs) were of 32, 38, 74 and 80 kDa which were more predominant in fertile bulls than subfertile bull. Furthermore, TPPs of 45 and 48 kDa were cBiMPS-dependent in fertile bulls whereas, in subfertile bull the latter was barely detectable and the former was cBiMPS-independent at only 0 min. This increase in PTP not only emphasizing the beneficial roles of desalted SP but excluding any detrimental effect of it on sperm cell functions during storage as well.