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We prepared four monoclonal antibodies raised against mouse spermatozoa. The ability of the hybridoma culture supernatants to inhibit in vitro mouse fertilization was determined. One of these monoclonal antibodies, termed A-1, strongly inhibits sperm penetration into zona pellucida of egg in a concentration-dependent fashion. On the other hand, even in the presence of the same concentration of A-1 antibody, sperm binding to comulus-free eggs was slightly inhibited and sperm-egg fusion was not inhibited at all. Immuno-fluorescence studies with A-1 showed that it recognizes an antigen(s) localized in the acrosome of mouse spermatozoa. In the case of unfixed sperm, the acrosome-intact sperm (i.e., sperm bound to egg zona pellucida) and unbound sperm were slightly immunostained with A-1 antibody (1.6% and 9.0%, respectively). All the full acrosome reacted sperm (i.e., sperm penetrating into the perivitelline space) were not immunoreacted. On the other hand, when sperm were fixed with methanol, approximately 80% of unbound sperm were immunoreacted with this antibody. Furthermore, almost all of zona-binding sperm were immunostained, whereas acrosome reacted, penetrating sperm were not. These data suggested that the antigen recognized by A-1 was located inside the plasma membrane of spermatozoa, and appeared on the surface of the spermatozoa during the period of penetration into egg zona pellucida. After sperm penetration into the perivitelline space this antigen must be released from the sperm.

Effect of a Monoclonal Anti-sperm Antibody (A-1) on in Vitro Fertilization in the Mouse.