Fink, Edwin

Fritz, Hans

Jaumann, Eugen

Schumacher, D. F. B.

Zaneveld, L. J. D.

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Reprinted from PROTIDES O F T H E B I O L O G I C A L F L U I D S -20TH C O L L O Q U I U M Edited by H . P E E T E R S P E R G A M O N PRESS - O X F O R D A N D N E W Y O R K 1973 J . Reprod. Feti, (1973) 32, 525-529 INTERACTION OF HUMAN SPERM ACROSOMAL PROTEINASE WITH HUMAN SEMINAL PLASMA PROTEINASE INHIBITORS L . J . D . Z A N E V E L D , G . F. B. S C H U M A C H E R , H . F R I T Z , E . F I N K AND E . J A U M A N N Section of Reproductive Biology, Department of Obstetrics and Gynecology, The University of Chicago, Pritzker School of Medicine, 5841 South Maryland Avenue, Chicago, I l l i n o i s 60637, U . S . A . , and The Institute f o r Clinical Chemistry and Clinical Biochemistry, University of München, D 8 München E - 2 , Nussbaumstrasse 20, Germany {Received 29th August 1972) Summary. H u m a n sperm acrosomal proteinase is immediately i n -hibited by the Fract ion I (mol. wt. 12,700) and Fract ion I I (mol. wt. 5,400) proteinase inhibitors of human seminal plasma. The Fract ion I I inhibitor inhibits acrosomal proteinase much more effectively than the Fract ion I inhibitor, having an association constant of 5-0 x 10 8 M " 1 and a dissociation constant of 2-0 x 10 " 9 M . The results provide further evidence for the possible rôle of the Fract ion II inhibitor i n the reproductive process. Evidence has been presented that i n some species, seminal plasma proteinase inhibitor is added to spermatozoa and removed again while the spermatozoa reside i n the female genital tract (Zaneveld, Srivastava & Wi l l iams, 1969, 1970). It was further established that the presence of proteinase inhibitors on spermatozoa prevents fertilization (Stambaugh, Brackett & Mastro ianni , 1969; Zaneveld, Robertson & Wil l iams, 1970; Zaneveld, Robertson, Kessler & Wi l l iams, 1971). This antifertility effect is probably obtained by inhibi t ion of the sperm acrosomal proteinase (acrosin), an enzyme that appears essential for the penetration of the spermatozoon through the zona pellucida. Recently, the acrosomal proteinase of ejaculated human spermatozoa was part ial ly purified and characterized (Zaneveld, Dragoje & Schumacher, 1972). The enzyme is released from the spermatozoon i n complex wi th an inhibitor. Two proteinase inhibitors (Fraction I, mol . wt. = 12,700 and Fract ion II , mol . wt. = 5400) have been isolated from human seminal plasma (Fink, Jaumann, Fr i tz , Ingrisch & Werle, 1971). The objective of the present study was to evaluate the kinetics and strength of interaction of the human seminal plasma proteinase inhibitors wi th human sperm acrosomal proteinase. Acrosomal proteinase was obtained by detergent treatment of washed, ejaculated human spermatozoa. The enzyme was partial ly purified by Sephadex G-50 gel filtration using an acidic buffer to separate the enzyme from inhibitor (Zaneveld et a l . , 1972). The proteinase inhibitors were isolated from seminal plasma by CM-Sephadex C-25 adsorption, affinity chromatography and Sepha-525 526 L . J. D . Zaneveld et a l . fro o o o — —o T ime (min) T E X T - F I G . 1. T ime relationship of the inhibit ion of human acrosomal proteinase by human seminal plasma proteinase inhibitors. O, Saline control ; A , Fract ion I ; #, Fraction I I . dex G-75 gel filtration (Fink et al.> 1971). Enzyme activity was evaluated by ad-dit ion of the test solution to 3-0 m l benzoyl arginine ethyl ester ( B A E E , Sigma Chemica l Co. , 0-2 mg/ml) i n 0-1 M-borate buffer, p H 8 0 , containing 0-05 M - C a C l 2 and measuring the change in absorbance at a wavelength of 253 n m for 5 m i n (Schwert & Takenake, 1955). One mil l i -unit (mU) of activity was defined as that amount of enzyme that causes a rate of change of 0-001 /anol B A E E / m i n . Protein estimations were made by absorption readings at a wavelength of 280 nm, assuming that 1 mg of protein/ml has an optical fj.q inhibitor T E X T - F I G . 2 . Inhibit ion of human acrosomal proteinase by varying amounts of human seminal plasma proteinase inhibitors. A , Fract ion I ; • , Fraction I I . Human sperm proteinase and proteinase inhibitors 527 density of 1-0 using a 1-cm light path. O n this basis, the activity of the acro-somal proteinase preparation used was 2100 mU/mg protein. Inhibitor tests were performed by two different methods: (1) the acrosomal proteinase pre-paration (0-6 m l , 65 fig) i n the substrate buffer was incubated wi th inhibitor (3 /ig, 0-3 ml) i n 0 1 5 M N a C l at room temperature. Al iquots (0-15 ml) were tested for activity at various times (Text-fig. 1); (2) the acrosomal proteinase preparation (0-15 ml , 30 fig i n the case of the Fract ion I inhibitor and 55 fig i n the case of the Fract ion I I inhibitor) was mixed wi th various amounts of inhibitor (0-15 ml) . After incubation at room temperature for 5 min , 0-2 m l of the solution was tested for activity (Text-fig. 2). As control, the acrosomal proteinase preparations were incubated wi th 0-15 M N a C l under the conditions described for each test. The Fract ion I I inhibitor inhibits human acrosomal proteinase rapidly (Text-fig. 1) and i n very small amounts (Text-fig. 2). F r om the linearity of the inhibi t ion curve (Text-fig. 2), it can be concluded that 1 mol enzyme reacts with 1 mol inhibitor (Laskowski & Sealock, 1971). Extrapolat ion of the l inear part of the curve shows that 2-2 fig inhibitor should completely inhibit 78 m U acrosomal proteinase. In practice, only 8 8 % of the enzyme is inhibited. F rom this, the association constant of the reaction can be calculated as 5 0 x IO 8 M " 1 and the dissociation constant as 2-0 x 1 0 " 9 M (Green & Work, 1953). The Fract ion I inhibitor is also an ' immediate' inhibitor but inhibits human acrosomal proteinase much less effectively than the Fract ion I I inhibitor (Text-fig. 1). This correlates wi th the relative effect of these inhibitors on bu l l (Suominen & N iemi , 1972) and boar (Fritz, Forg-Brey, F ink, Schiessler, Jaumann & Arnho ld , 1972) acrosomal proteinase. Increasing amounts of Fract ion I inhibitor init ia l ly do not diminish enzyme activity significantly unt i l a certain point when a rapid decrease i n enzyme activity occurs (Text-fig. 2). Higher amounts of enzyme than that used i n Text-fig. 2 necessitated proportionally larger amounts of Fract ion I inhibitor to reach this point of rapid decrease. No explanation can be given at the present time, although the possibility exists that the Fract ion I inhibitor interacts first wi th another enzyme that does not hydrolyse B A E E before the inhibitor reacts w i th the acrosomal proteinase. It has indeed been shown (Fink et a l . , 1971; Suominen & N iemi , 1972) that the Fract ion I inhibitor is an excellent inhibitor of chymotrypsin as well as trypsin, whereas the Fract ion I I inhibitor inhibits only trypsin. It is also possible, however, that the rather short incubation time of enzyme and inhibitor resulted i n poor inhibit ion at low concentrations of inhibitor. The association constant of the Fract ion I inhibitor cannot be cal-culated from the curve in Text-fig. 2, but appears to be much smaller than that of the Fract ion I I inhibitor. The results show that the Fract ion I I inhibitor effectively inhibits the human acrosomal proteinase. Since, i n addition, the properties of the inhibitor prepared from human sperm acrosomes are identical to those of the Fract ion I I inhibitor (Zaneveld et a l . , 1972), it appears l ikely that the Fract ion I I inhibitor is the one involved i n the reproductive process. The high association constant of the Fract ion I I inhibitor, which i n reality may be even somewhat higher than that calculated for 5 m in of incubation since complete inhib i t ion 528 L . J . D . £aneveld e t a l -was not obtained unt i l a few minutes later (Text-fig. 1), explains the rapid complex formation between the acrosomal proteinase and the inhibitor under physiological conditions. Spontaneous dissociation of this complex i n the female genital tract during capacitation is, however, somewhat difficult to imagine. Either a factor must be present i n the female genital tract secretions to induce the dissociation, or the acrosomal proteinase and inhibitor are not actually i n a complex on the spermatozoon, i.e. the enzyme may be located within the acrosome and the inhibitor on the surface of the sperm head. In the second case, a complex would be formed as the enzyme is released from the spermato-zoon due to the high affinity of the enzyme for the inhibitor. Residence of the spermatozoon in the female genital tract causes changes i n the surface com-ponents of the sperm head (Ericsson, 1969; Ol iphant & Brackett, 1972), possibly resulting i n the removal of inhibitor from the spermatozoon, thus al lowing the enzyme to exert its activity. Some evidence for the localization of the inhibitor on the surface of the acrosome can be surmised from the results of Hirschhauser & Baudner (1972), which showed that seminal plasma trypsin inhibitor antibodies bind to the sperm head. Both hypotheses explain the fact that natural trypsin inhibitors do not prevent fertilization i f added to ejaculated spermatozoa before deposition i n the vagina (Schumacher, Swartwout & Zuspan, 1971), but do interfere wi th fertility on addition to capacitated spermatozoa before insemination into the oviduct (Zaneveld et a i , 1971). I n contrast, synthetic proteinase inhibitors such as tosyl-lysine chloromethyl ketone ( T L C K , mol. wt. = 363) that most l ikely pass through the outer acrosomal membrane and form an irreversible complex wi th the acrosomal proteinase, prevent fertilization on incubation with ejaculated as well as capacitated spermatozoa (Zaneveld, Robertson & Wi l l iams, 1970). The research upon which this publication is based was performed pursuant to Contract No. N I H 70-2317 and N I H Grant No . 1-RO1-HD-06315 with the Nat ional Institutes of Heal th, Department of Hea l th , Educat ion and Welfare (G.F.B.S. and L .J .D.Z . ) and by funds of the Sonderforschungsbereich 51 of the Deutsche Forschungsgemeinschaft (H.F. , E .F . and E.J . ) . R E F E R E N C E S ERICSSON, R . J . ( 1 9 6 9 ) Methods for measurement of sperm capacitation i n different species. Adv. Biosci. 4 , 5 1 . F I N K , E . , J A U M A N N , E . , F R I T Z , H . , I N G R I S C H , H . & W E R L E , E . ( 1 9 7 1 ) Protease-Inhibitoren i m men-schlichen Spermaplasma. Isolierung durch Affinitätschromatographie und Hemmverhalten. Hoppe-Seyler's Z- physiol. Chem. 3 5 2 , 1 5 9 1 . F R I T Z , H . , F O R G - B R E Y , B. , F I N K , E . , SCHIESSLER , H . , J A U M A N N , E . & A R N H O L D , M . ( 1 9 7 2 ) Charak-terisierung einer Trypsin-ahnlichen Proteinase (Akrosin) aus Eberspermien durch ihre H e m m -barkeit mit verschiedenen Protein-Proteinase-Inhibitoren. I. Seminale Trypsin-Inhibitoren und Trypsin-Kal l ikrein-Inhibitor aus Rinderorganen. Hoppe-Seyler's Z- physiol. Chem. 3 5 3 , 1 0 0 7 . G R E E N , N . M . & W O R K , E . ( 1 9 5 3 ) Pancreatic trypsin inhibitor I I . React ion wi th trypsin. Biochem. J . 5 4 , 3 4 7 . H I R S C H H A U S E R , C . & B A U D N E R , S. ( 1 9 7 2 ) Immunologic localization of the human seminal plasma protease inhibitor i n human spermatozoa. F e r t . Steril. 2 3 , 3 9 3 . L A S K O W S K I , M . J . & S E A L O C K , R . W . ( 1 9 7 1 ) Protein p r o t e i n a s e i n h i b i t o r s - m o l e c u l a r aspects. I n : The E n -zymes, V o l . I l l , p. 3 7 5 . E d . P. D . Boyer. Academic Press, New York . Human sperm proteinase and proteinase inhibitors 529 O L I P H A N T , G . & B R A C K E T T , B. G . (1972) Immunological approach for assaying rabbit sperm capacita-t ion. B i o l . Reprod. 7, 102. S C H U M A C H E R , G . F . B. , S W A R T W O U T , J . R . & Z U S P A N , F . P. (1971) Fertility experiments in mice and rabbits with the trypsin-kallikrein inhibitorfrom bovine lung. In : Proc. Int. Res. Gonf. on Proteinase Inhibitors, M u n i c h , Germany, 1970, p. 247. Eds. H . Fr i tz and H . Tschesche. W . DeGruyter, Ber l in and New York . S C H W E R T , G . W . & T A K E N A K E , Y . (1955) A spectropho tome trie determination of trypsin and chymo-trypsin. Biochim. biophys. Acta, 16, 570. S T A M B A U G H , R. , B R A C K E T T , B . G . & M A S T R O I A N N I , L . (1969) Inhibit ion of in vitro fertilization of rabbit ova by trypsin inhibitors. B i o l . Reprod. 1, 223. S U O M I N E N , J . J . & N I E M I , M . (1972) H u m a n seminal trypsin inhibitors. J . Reprod. Fert. 2 9 . 163. Z A N E V E L D , L . J . D . , D R A G O J E , B . M . & S C H U M A C H E R , G . F. B. (1972) Acrosomal proteinase and proteinase inhibitor of human spermatozoa. Science, N . T . 177, 702. Z A N E V E L D , L . J . D. , R O B E R T S O N , R . T . , K E S S L E R , M . & W I L L I A M S , W . L . (1971) Inhibit ion of fertiliza-t ion in vivo by pancreatic and seminal plasma trypsin inhibitors. J . Reprod. F e r i . 2 5 , 387. Z A N E V E L D , L . J . D . , R O B E R T S O N , R . T . & W I L L I A M S , W . L . (1970) Synthetic enzyme inhibitors as antifertility agents. F E B S L e t t . 11, 345. Z A N E V E L D , L . J . D . , S R T V A S T A V A , P. N . & W I L L I A M S , W . L . (1969) Relationship of a trypsin-like enzyme i n rabbit spermatozoa to capacitation. J . Reprod. Fert. 2 0 , 337. Z A N E V E L D , L . J . D. , SRTVASTAVA , P. N . & W I L L I A M S , W . L . (1970) Inhibit ion by seminal plasma of acro-somal enzymes i n intact sperm. Proc. Soc. exp. B i o l . M e d . 113, 1172. 

A spectropho tome trie determination of trypsin and chymotrypsin.

Acrosomal proteinase and proteinase inhibitor of human spermatozoa.

Fertility experiments in mice and rabbits with the trypsin-kallikrein inhibitorfrom bovine lung.

Human seminal trypsin inhibitors.

Immunologic localization of the human seminal plasma protease inhibitor in human spermatozoa.

Inhibition by seminal plasma of acrosomal enzymes in intact sperm.

Inhibition of fertilization in vivo by pancreatic and seminal plasma trypsin inhibitors.

Inhibition of in vitro fertilization of rabbit ova by trypsin inhibitors.

Methods for measurement of sperm capacitation in different species.

Pancreatic trypsin inhibitor II. Reaction with trypsin.

Protease-Inhibitoren im menschlichen Spermaplasma. Isolierung durch Affinitätschromatographie und Hemmverhalten.

Protein proteinase inhibitors-molecular aspects. In: The Enzymes,

Relationship of a trypsin-like enzyme in rabbit spermatozoa to capacitation.

Synthetic enzyme inhibitors as antifertility agents.

Interaction of Human Sperm Acrosomal Proteinase with Human seminal Plasma Proteinase Inhibitors

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Interaction of Human Sperm Acrosomal Proteinase with Human seminal Plasma Proteinase Inhibitors

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