Isoantibodies to whole semen or epididymal spermatozoa of rabbits inhibit fertilization in vitro (Russo & Metz, 1974a). This inhibition is attributed to a primary blocking of one or more sperm antigens essential for the sperm-egg interaction, and not to a secondary action (e.g. agglutination, complement-dependent immobilization), because univalent (Fab) antibody fragments, which do not agglutinate or immobilize spermatozoa, are also effective. Hyaluronidase, the sperm enzyme required for penetration of the cumulus surrounding the egg, could be one such sperm antigen. This possibility is supported by the fact that cumulus dispersion was inhibited in the experiments of Russo & Metz (1974a) and that hyaluronidase-inhibiting antibodies are present in isoantisera to rabbit semen (Metz, Seiguer & Castro, 1972). It is necessary to employ antibody preparations specific for rabbit sperm hyaluronidase to examine critically the inhibition of fertilization by antihyaluronidase antibodies. We report here such inhibition of rabbit fertilization in vitro by Fab isoantibodies to purified rabbit sperm hyaluronidase. MethodsHyaluronidase was purified from rabbit epididymides (Pel-Freez, Rogers, Arkansas). These were homogenized in batches of approximately 200-300 in 500 ml triton-X buffer (0-1 m-P04, 0-15 M-NaCl, 01 % Triton-X, pH 5-3) using a Waring-type blender. After further homogenization in a glass homogenizer the material was frozen and thawed (10 cycles) and centrifuged (10,000 g, 30 min, 4°C). The hyaluronidase in the supernatant was purified by a modification of the procedure of Borders & Raftery (1968); an initial precipitation with 50% saturated (NH4)2S04 was followed by DEAE chromatography. Hyaluronidase activity was determined by the turbidimetric (Tolksdorf, McCready, McCullough & Schwenk, 1949) or the N-acetylglucosamine (Aronson & Davidson, 1967) assay procedure. The purity of the hyaluronidase obtained was tested for some preparations by acryla¬ mide gel electrophoresis and immunodiffusion against isoantisera to whole semen of rabbits. The least pure hyaluronidase preparations (sp. act. 3600 turbidity reducing units/mg protein) used as immunizing antigens and so tested produced up to 6 protein-staining bands in acrylamide gel elec¬ trophoresis but only one precipitin band, which was identical to the band containing hyaluronidase activity in the acrylamide gel. It was therefore concluded that the hyaluronidase preparations were isoantigenically pure. Isoantisera were prepared by injecting virgin female rabbits of the New Zealand White breed once a week for 3-4 weeks with this isoantigenically pure hyaluronidase (0-2-0-46 mg in 0-5 ml phosphate-buffered saline emulsified in 0-5 ml complete Freund's adjuvant per injection). The isoantisera utilized from these rabbits strongly inhibited rabbit sperm hyaluronidase activity, and did not produce more than one precipitin band during immunodiffusion against concentrated sperm extract, or the immunizing antigen which was sometimes tested. These results are consistent with ...
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