Plain Language Summary
This study showed that thymosin alpha-1 and anti-FA-1 monoclonal antibody affect sperm fertilizing capacity by modulating protein phosphorylation during capacitation. Thymosin alpha-1 enhanced phosphorylation of multiple sperm proteins, particularly increasing tyrosine phosphorylation of 95 and 51 kDa proteins, while anti-FA-1 antibody inhibited these same phosphorylation events. The findings reveal a signal transduction pathway involving sequential protein phosphorylation during sperm capacitation, where thymosin alpha-1 acts as a positive modulator of this process.
Abstract
The present investigation was conducted to investigate the modulation of phosphorylation pattern of human sperm membrane proteins during capacitation by thymosin alpha-1 (T alpha 1) (which enhanced sperm penetration index) and anti-FA-1 monoclonal antibody (anti-FA-1 mAb) (which completely blocked sperm penetration) using 32P metabolic labeling, in vitro kinase assay and Western immunoblot analysis. In 32P metabolic labeling experiments, T alpha 1 (0.25 and 0.5 microgram/100 microliters) enhanced phosphorylation of 7 proteins in four molecular regions namely one protein (190 kDa) in 200-kDa, two proteins (112 and 104 kDa) in 97-kDa, two proteins (48 and 42 kDa) in 43-kDa and two proteins (31 and 25 kDa) in 29-kDa molecular regions, respectively. Anti-FA-1 mAb (10 micrograms/100 microliters) resulted in a general decrease in the 32P labeling of these sperm proteins. In in vitro kinase assay using non-capacitated sperm extracts, T alpha 1 (0.5 microgram/100 microliters) enhanced autophosphorylation of 14 proteins in various molecular regions (122, 105, 95, 89, 73, 62, 48, 46, 40, 33, 30, 28, 25 and 22 kDa, respectively). The same concentration of T alpha 1 did not affect autophosphorylation of proteins in capacitated sperm extract. Anti-FA-1 mAb (10 micrograms/100 microliters) inhibited autophosphorylation of a subset of 8 proteins (122, 104, 95, 89, 73, 62, 48 and 46 kDa, respectively) in non-capacitated sperm membrane extracts, and 12 proteins (112, 104, 95, 89, 73, 62, 48, 46, 33, 30, 28 and 25 kDa, respectively) in capacitated sperm membrane extracts. In the Western immunoblot analysis, T alpha 1 resulted in a concentration-dependent increase in tyrosine phosphorylation of two proteins (95 and 51 kDa) during capacitation of human sperm, whereas anti-FA-1 mAb inhibited tyrosine phosphorylation of both proteins. These results indicate that T alpha 1 and anti-FA-1 mAb affect the fertilizing capacity of human sperm by modulating phosphorylation of proteins especially tyrosine phosphorylation of 95- and 51-kDa proteins during capacitation. These findings also suggest that there may be a signal transduction pathway(s) involved in phosphorylation of membrane proteins during capacitation and that an exogenous stimulus affecting a single membrane protein component can modulate phosphorylation of all the relevant proteins involved in capacitation/acrosome reaction of human sperm.