MONOCLONAL-ANTIBODIES AGAINST UNFERTILIZED ZONA-FREE MOUSE OOCYTES - CHARACTERIZATION AND EFFECTS ON FERTILIZATION

A panel of anti-oocyte antibodies was raised against unfertilized zona -free mouse oocytes by intrasplenic immunization and checked for their effects on in vitro fertilization. Four antibodies decreased the fert ilization rate from about 90% in controls to 8% (B5-2 F7), 12% (A2-2 A 7), 13% (4-G1), and 25% (A2-2 F2), when the sperm cell concentration w as 1 x 10(5) to 1 x 10(6). Antigen localization: All the antibodies la belled components in the cell membrane of zona-free oocytes as demonst rated by indirect immunofluorescence and/or by complement-mediated ooc yte lysis. In various patterns, the ooplasm and zona pellucida were al so labelled with different intensities. Western blotting: A2-2 A7 and A2-2 F2 recognized a protein with a molecular weight of approximately 65 kDa, while antibody B5-2 F7 bound a 97 kDa protein. Complement acti vation and complement-mediated oocyte lysis: Systemically injected ant ibodies, C3 and C4 were detected on zona-free oocytes recovered from t he mouse oviduct indicating the activation of C3 and C4 by antigen-ant ibody complexes. The recovered oocytes were not damaged, suggesting a presence of complement-regulating factors. In vitro, however, a large number of zona-free oocytes preincubated with antibodies were lysed or protruded ooplasma vesicles in complement-active serum. Stage, tissue , and species specificity: None of the antibodies, except A2-2 A7, sho wed a positive immunolabelling to the pronuclear stage. Antibodies 4-G 1 and A2-2 F2 cross-reacted with the ovarian oocytes. No antibodies bo und to any of the tissues tested, indicating that the corresponding an tigen epitopes are not commonly expressed. A2-2 A7, A2-2 F2, and B5-2 F7 cross-reacted with hamster and human unfertilized oocytes, suggesti ng the presence of developmentally conserved molecules and the possibi lity to apply these antibodies in hamster and human in vitro fertiliza tion. It is concluded that the approach used could be a useful strateg y in searching for anti-fertilization antibodies for human contracepti on. (C) 1996 Wiley-Liss, Inc.