Summary. Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD), 4′-nitrophenyl 4-guanidinobenzoate (NPGB), and 4′-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards α-N-benzoyl-l-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose—response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 μm for BD, 27 μm for ABD, 35 nm for MUGB, and 13 nm for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose—response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.
S. A. Beyler and L. J. D. Zaneveld
Gail S. Prins and L. J. D. Zaneveld
Summary. The movement of radio-opaque medium in the vas deferens of rabbits during sexual rest, after sexual stimulation, and after ejaculation, was followed. Bilateral injections of 20 μl Ethiodol were given at the vas–epididymal junction in the urethral direction. Serial radiographs revealed proximal transport of the dye (towards the testis) within 24 h and total containment in the cauda epididymidis within 1–4 days. Subsequently, small amounts of dye moved from the epididymis through the vas and out of the urethra during sexual rest until no dye remained (11 days–8 weeks). Animals with a ligated vas deferens showed no decrease in dye density. Sexual stimulation moved the dye from the epididymis into the vas. The dye was then either rapidly transported proximally during subsequent rest or removed distally if ejaculation followed stimulation. Ejaculation removed some vasal and epididymal dye via the urethra; however, dye left in the vas after ejaculation was rapidly (< 30 min) transported to the proximal duct and then into the epididymis by 24 h. It is concluded that vasal contents are transported in both urethral and testicular directions during sexual rest and that, after stimulation and ejaculation, the rate of proximal transport is increased. This may be indicative of a sperm removal mechanism by the vas deferens which involves the maintenance of an optimal sperm number in the cauda epididymidis at all times.
L. J. D. ZANEVELD, P. N. SRIVASTAVA and W. L. WILLIAMS
The existence of a trypsin-like enzyme (TLE) in acrosomes of epididymal spermatozoa was confirmed and was further demonstrated to be present in acrosomes of ejaculated and capacitated spermatozoa. TLE rapidly removes the zona pellucida of the ovum. Extracts of acrosomes of ejaculated spermatozoa contain an inhibitor that is separated from the TLE by purification of the TLE.
The inhibitor of TLE is also present in seminal plasma. This enzyme— enzyme inhibitor relationship appears analogous to the corona-removing enzyme-decapacitation factor relationship and part of capacitation very likely involves removal of the inhibitor from TLE and decapacitation factor from the corona-removing enzyme. TLE is inhibited by soybean trypsin inhibitor and less effectively by mercaptoethanol.
J. M. Reddy, R. A. Stark and L. J. D. Zaneveld
Summary. The presence of a high molecular weight antifertility factor in human seminal plasma was established. The factor can be precipitated by centrifugation at 104 000 g. Its activity is maximal when the protein concentration reaches 150 μg/105 spermatozoa using the mouse in-vitro fertilization assay as the test system. The factor is heat labile but its activity is not affected by dialysis. It prevents the penetration of the spermatozoa through the layers surrounding the egg but has no effect on the fusion of the spermatozoa with the vitelline membrane. The factor is only partly removed from spermatozoa by washing but is completely dispersed when the spermatozoa are incubated in capacitation medium. The pellet that is precipitated from the seminal plasma does not contain any particles or vesicles. However, it is significantly contaminated with low molecular weight material. This material includes the acrosin inhibitor which is present in large enough quantities to hinder fertilization. Washing the pellet twice with H2O removes these low molecular weight compounds, as indicated by the absence of the acrosin inhibitor, but has no effect on the antifertility properties of the pellet. Therefore, before further study or purification of the factor, it is essential that the pellet is washed to remove such low molecular weight material. The washed pellet consists of at least 7 components as judged by disc gel electrophoresis.
Sally Perreault, L. J. D. Zaneveld and B. Jane Rogers
Summary. Sodium aurothiomalate, a low molecular weight inhibitor of hyaluronidase, blocked in-vitro fertilization in hamsters at the level of the zona pellucida: concentrations of 25–250 pg inhibitor/ml prevented fertilization of cumulus-intact and cumulus-denuded eggs. Fertilization of zona-free ova was not affected. In-vivo fertilization was also reduced (from 100% in controls to 37·5%) by 10 mg inhibitor/ml added to an epididymal sperm suspension before artificial insemination into the uterus. These findings suggest that hyaluronidase may play a role in zona penetration or that sodium aurothiomalate blocks sperm penetration of the zona by inhibiting an enzyme(s) other than hyaluronidase.
L. J. D. ZANEVELD, L. WAGNER, H. D. SCHLUMBERGER and G. F. B. SCHUMACHER
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.
R. F. Parrish, J. C. Goodpasture, L. J. D. Zaneveld and K. L. Polakoski
Summary. The conversion of human proacrosin to acrosin was inhibited by polyamines. The order of effectiveness was spermine > spermidine > cadaverine > putrescine > 1,3-diaminopropane. These results are similar to those obtained for the conversion of boar proacrosin to acrosin. Unlike the effects on boar acrosin, however, polyamines did not affect the esterolytic activity of human acrosin but had a slight stimulatory effect on the proteolytic activity of human acrosin.
L. J. D. ZANEVELD, R. T. ROBERTSON, M. KESSLER and W. L. WILLIAMS
Treatment of capacitated rabbit spermatozoa with pancreatic trypsin inhibitor or partially purified seminal plasma trypsin inhibitor and subsequent insemination of such spermatozoa into the oviducts of ovulated rabbits markedly inhibited fertilization. Washing the spermatozoa to remove excess inhibitor did not affect the antifertility action of pancreatic trypsin inhibitor. Seminal plasma trypsin inhibitor was purified by specific binding to a trypsin-maleic anhydride-ethylene copolymer and Sephadex G-25 and G-50 column chromatography. A 500-fold purification was obtained.
P. F. Tauber, L. J. D. Zaneveld, D. Propping and G. F. B. Schumacher
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.
P. F. TAUBER, L. J. D. ZANEVELD, D. PROPPING and G. F. B. SCHUMACHER
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.