| Abstract: |
The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94 000 +/- 3000 and 46 000 +/- 3000 M(r) on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94 000 +/- 3000, 46 000 +/- 3000, 25 000 +/- 7000 and 12 000 +/- 2000 M(r), respectively, were labeled with P-32 when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher P-32 incorporation into 94 000 +/- 3000 M(r) proteins and 17% less incorporation into 12 000 +/- 2000 M(r) proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94 000 +/- 3000 M(r) proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead. PTA showed a significant (P < 0.001) inhibition of human sperm binding and penetration of zona-free hamster eggs. These results indicate that the human sperm membrane proteins are phosphorylated at tyrosine residues during capacitation, and the degree of autophosphorylation of 94 000 +/- 3000 M(r) proteins increases after exposure to zona proteins, implying a vital role for tyrosine phosphorylation in the development of the fertilizing capacity of human sperm cells. |
