THYMOSIN ALPHA-1 AND FA-1 MONOCLONAL-ANTIBODY AFFECT FERTILIZING-CAPACITY OF HUMAN SPERM BY MODULATING PROTEIN-PHOSPHORYLATION PATTERN

The present investigation was conducted to investigate the modulation of phosphorylation pattern of human sperm membrane proteins during cap acitation by thymosin alpha-1 (T alpha 1) (which enhanced sperm penetr ation index) and anti-FA-1 monoclonal antibody (anti-FA-1 mAb) (which completely blocked sperm penetration) using P-32 metabolic labeling, i n vitro kinase assay and Western immunoblot analysis. In P-32 metaboli c labeling experiments, T alpha 1 (0.25 and 0.5 mu g/100 mu l) enhance d phosphorylation of 7 proteins in four molecular regions namely one p rotein (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 kD a) in 29-kDa molecular regions, respectively. Anti-FA-1 mAb (10 mu g/1 00 mu l) resulted in a general decrease in the P-32 labeling of these sperm proteins. In in vitro kinase assay using non-capacitated sperm e xtracts, T alpha 1 (0.5 mu g/100 mu l) enhanced autophosphorylation of 14 proteins in various molecular regions (122, 105, 95, 89, 73, 62, 4 8, 46, 40, 33, 30, 28, 25 and 22 kDa, respectively). The same concentr ation of T alpha 1 did not affect autophosphorylation of proteins in c apacitated sperm extract. Anti-FA-1 mAb (10 mu g/100 mu l) inhibited a utophosphorylation of a subset of 8 proteins (122, 104, 95, 89, 73, 62 , 48 and 46 kDa, respectively) in non-capacitated sperm membrane extra cts, 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-dep endent increase in tyrosine phosphorylation of two proteins (95 and 51 kDa) during capacitation of human sperm, whereas anti-FA-1 mAb inhibi ted tyrosine phosphorylation of both proteins. These results indicate that T alpha 1 and anti-FA-1 mAb affect the fertilizing capacity of hu man sperm by modulating phosphorylation of proteins especially tyrosin e phosphorylation of 95- and 51-kDa proteins during capacitation. Thes e findings also suggest that there may be a signal transduction pathwa y(s) involved in phosphorylation of membrane proteins during capacitat ion and that an exogenous stimulus affecting a single membrane protein component can modulate phosphorylation of all the relevant proteins i nvolved in capacitation/acrosome reaction of human sperm.