Antibodies against mouse ovaries and their effect on fertilization in vitro and in vivo in the mouse.

zona pellucida, the theca interna and in atretic follicles (Porter, 1965; Porter, Highfill & Winovich, 1970a, b; Ownby & Shivers, 1972; Sacco & Shivers, 1973a, b, c, d). Antisera (by precipitate formation) modified the surface of the zona pellucida of hamster and rabbit ova to such an extent that it even became inert to the lytic effects of trypsin (Ownby & Shivers, 1972; Sacco & Shivers, 1973a). In hamsters, these antibodies blocked penetration of the zona pellucida by spermatozoa in vitro (Shivers, Dudkiewicz, Franklin & Fussell, 1972). Shahani, Padbidri & Rao (1969, 1972) demonstrated inhibition of conception in vivo in mice by antisera against mouse ovaries, but did not explain the mechanism of their effect in detail. The aim of the present study was to investigate the specificity and mechanism of the effect of heteroimmune antibodies against mouse ovaries in fertilization in vitro and in vivo. Mouse ovarian extract for immunization of rabbits was prepared by homogenization of washed ovaries with phosphate-buffered saline (pH 7-2) at 4°C in the ratio of 1 part ovaries to 3 parts buffer (w/w). The homogenate was centrifuged for 15 min at 670 g and the supernatant was used for immuniza¬ tion. The sediments of the tissue homogenates were used for absorption. Intramuscular injection of 0-5 ml supernatant with 0-5 ml complete Freund's adjuvant was given every week. Blood was collected from the marginal ear vein on the 10th day after the sixth injection. The resultant serum was in¬ activated for 30 min at 56°C and was absorbed by normal mouse serum in a 1:1 ratio and by the sediment of mouse liver, spleen and kidney tissue homo¬ genate, in the same ratio. This was termed specific immune serum (SIS). This serum absorbed twice by mouse ovarian tissue (ovary-absorbed immune serum—OAIS) served as the control. Since the reaction of the antisera in Ouchterlony's test (Ouchterlony, 1949) and immunoelectrophoresis was relatively weak, they were tested by indirect immunofluorescence only, using pig anti-rabbit IgG labelled with fluorescein isothiocyanate (SwAR/FITC) supplied by SEVAC (Prague).