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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)2SO4precipitation 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.

Twenty-two synthetic proteinase inhibitors were tested for their inhibitory properties towards human acrosin. p-Nitrophenyl-p1-guanidino benzoate (NPGB) was the most effective (K1value of 1·5 × 10−8M), producing a non-competitive type of inhibition in contrast to all other inhibitors which showed a competitive type of inhibition. The Michaelis constant for human acrosin on BAEE at pH 8·1 was calculated to be 4·25 × 10−5M.

Lysozyme, α-amylase, neutral proteinase and plasminogen activator were most concentrated in the initial portion of the ejaculate that consists mostly of Cowper's gland and prostate gland fluids as well as spermatozoa. The concentration of the high molecular weight proteinase inhibitors, α1-antitrypsin and α1x-antichymotrypsin, was essentially unaltered throughout the ejaculate fractions, although their absolute amounts showed an increase towards the final fraction. By contrast, the total inhibitory activity towards pancreatic trypsin was highest both in concentration and amount in the last fraction, thus indicating that the seminal vesicles are its primary source. Plasminogen, prothrombin, Factor XIII, and the proteinase inhibitors antithrombin III, α2-macroglobulin, inter-α-trypsin inhibitor and C1s-inactivator could not be detected immunochemically in whole ejaculates, and indicates the dissimilarity between the coagulation/liquefaction processes of semen and blood.

The concentrations of spermatozoa, fructose, IgG, IgA, albumin, lactoferrin, transferrin, secretory piece of IgA, β1C/β1A-globulin (C′3-component of complement), ceruloplasmin and fibrinogen were evaluated in human split ejaculates and/or in whole human seminal plasma. The concentrations of spermatozoa, IgG, IgA, albumin and transferrin decreased from the first portion of the split ejaculate to the last, indicating that these proteins originate mostly from secretions other than the seminal vesicles. By contrast, the highest amounts of fructose and lactoferrin were present in the final portion of the split ejaculates, showing their seminal vesicle origin. No secretory piece, IgM, β1C/β1A-globulin, ceruloplasmin or fibrinogen could be detected in human semen. An unidentified antigen was found that has a relatively high molecular weight and shows β1-mobility on immunoelectrophoresis.

Human sperm acrosomal proteinase is immediately inhibited by the Fraction I (mol. wt. 12,700) and Fraction II (mol. wt. 5,400) proteinase inhibitors of human seminal plasma. The Fraction II inhibitor inhibits acrosomal proteinase much more effectively than the Fraction I inhibitor, having an association constant of 5·0 × 108m−1and a dissociation constant of 2 ·0 × 10 − 9m. The results provide further evidence for the possible rôle of the Fraction II inhibitor in the reproductive process.