Identification of human sperm proteins involved in the interaction with homologous zona pellucida.

OBJECTIVE

To identify human sperm proteins involved in homologous zona pellucida (ZP) interaction.

DESIGN

Prospective study.

SETTINGS: Basic research laboratory.

PATIENT(S): Semen samples from normozoospermic donors, tissue sections from surgical pieces, and ZP from nonfertilized oocytes.

INTERVENTION(S): Antibodies for sperm proteins (HSE; high salt extract) were developed (anti-HSE) and partially characterized. Participation of sperm proteins on ZP-interaction was tested with the hemizona assay (HZA). Antigens were immunolocalized in sperm and tissues.

MAIN OUTCOME MEASURE(S): Sperm and tissue immunostaining; Western blotting; and number of sperm bound to the ZP.

RESULT(S): Anti-HSE antibodies recognized several polypeptides in HSE (9 to 200 kd). Specific antibodies for 49 and 66 kd proteins (p49, p66) were obtained. Both (anti-p49 and anti-p66) stained the head of ejaculated and capacitated sperm. In the HZA, sperm preincubation with a mixture of anti-p49 and anti-p66 (100 micro g/mL) resulted in a decrease in the number of spermatozoa bound to the ZP. Presence of p66 (10 micro g/mL) inhibited sperm-ZP interaction. In contrast, p49 did not alter sperm binding to the ZP. Immunohistochemical analysis showed that p66 is present in the epididymis. No staining was observed in testicular sections.

CONCLUSION(S): We found that p66 is an epididymal protein that participates in human sperm interaction with homologous ZP.