Washed, ejaculated bull spermatozoa were separated by sonication and sucrose density gradient centrifugation into three fractions, one containing a protein mixture (Fraction P), another only sperm tails (Fraction T), and the third only sperm heads (Fraction H). Detergent extracts containing mostly acrosomal and plasma membrane material were prepared from whole bull spermatozoa (Extract S) and the isolated sperm heads (Extract E). Extracts S and E and Fraction P contained acrosin (acrosomal proteinase) that appeared to be associated with an inhibitor in Extract S and Fraction P. Fraction P and Extract S, but not Extract E, possessed hyaluronidase. Extract P was absent if detergent-treated spermatozoa were sonicated and centrifuged using a sucrose density gradient.
Heteroantisera against Fractions P, T and H, and Extract S were prepared by immunizing rabbits. Immunodiffusion experiments revealed the formation of precipitin bands between bull seminal plasma and Fraction H or Extract S antisera but not with antisera of Fractions P and T. These bands did not occur after absorption of the antisera with bull seminal plasma. Cross-reactions occurred among all sperm preparations. The precipitating antibodies appeared to be species specific. The Extract S antisera caused the highest degree of sperm agglutination, followed in decreasing order by the antisera against Fractions H, T and P. Extract S antisera also produced the most rapid sperm immobilization. No difference among the immobilization rates of the other antisera was observed. Removal of the seminal plasma specific antibodies by absorption with seminal plasma resulted in a significant decrease in sperm agglutination but had no effect on sperm immobilization. Thus, sperm agglutinating antigens are present in both spermatozoa and seminal plasma, whereas the sperm immobilizing antigens are only associated with spermatozoa. Complement was shown to be a necessary part of the sperm immobilization process.
Extract S antisera inhibited the activity of testicular and acrosomal hyaluronidase. Immunoglobulins prepared from Extract S antisera by (NH4)2SO4 precipitation inhibited the activity of acrosin but only if a high molecular weight substrate was used. Such immunoglobulins did not inhibit bovine pancreatic trypsin.
The detergent extraction of spermatozoa seems to be the most suitable method to prepare surface and acrosomal antigens that induce sperm-agglutinating, sperm-immobilizing and enzyme-inhibiting antibodies after injection into animals of a heterologous species.
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