Atienza-Samols, S. B.

Fink, Edwin

Fritz, Hans

Ludwig, Hans

Schleuning, W. D.

English

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

Human Fertilization International Workshop Essen, July 1976 Edited by Hans Ludwig and Peter F. Tauber 36 With Contributions by S. B. Atienza-Samols V. Frick D. Krebs I. Schier M . Badawi E. Friedrich G. Kuppe H . Schiessler K. J. Beck H . Fritz F. Lehmann W.-B. Schill H . M . Beier Joan Fu H . Ludwig A . E. Schindler R. P. Bernard R. Göser T. Mann W.-D. Schleuning J. Bodenstein A . Grösser L. Mettler H . Schmidt-Eimendorff B. G. Boving E. S. E. Hafez A . Mezkel S. T. Shaw Renée L. Boving A . A . Haspels K. S. Moghissi M . I. Sherman M . Breckwoldt F. Hefnawi M . Molls C. Streffer D. van Beuningen M . L'Hermite D. L. Moyer P. F. Tauber K. H . Broer W. Herting K. Ohlsson M . Thiery A . Caufriez N . Hofmann C. Olmedo M . N . Thomas F. C. Chretien W. R. Hohman W. van Os R. S. Thompson P. Delvoye V. Insler H . van der Pas O. Wallner H.-W. Denker E. W. Joel C. J. Pauerstein R. G. Wheeler R. Dorn R. Kaiser G. Preissler R. M . Wynn C. A . Eddy E. Keller D. Propping M . M . Yacout G. Ehrig E. Kessel A . Psychoyos E. C. Yurewicz M . Elstein H . van Kets C. Robyn L. J. D . Zaneveld E. Fink H . Koester L. Rüping W. Zubke 152 Figures, 64 Tables Reprint Reprinting only with permission of the publisher Georg Thieme Publishers Stuttgart 1978 142 Kaiser, R., P. Citoler, K . H . Broer: Relative increase in Y-chromatin bearing spermatozoa after in vitro penetrations into human cervical mucus. IRCS Med. Sci. 2:1100, 1974 Kremer, J.: A simple sperm penetration test. Int. J. Fertil. 10:209, 1965 Pawlowitzki, I.H., Pearson, P.L.: Aneuploidy in human spermatozoa. Humangenetik 16:119, 1972 Rohde, W., T. Porstmann, S. Prehn, C. Dörner: Migration of Y-bearing human spermatozoa in cervical mucus. J. Reprod. Fert. 42:587, 1973 Shettles, L.B. : Factors influencing sex ratios. Int. J. Gynecol. Obstet. 8:643, 1970 Sumner, A.T., J.A. Robinson, H.J. Evans: Distinguishing between X , Y and Y Y bearing human spermatozoa by fluorescence and DNA content. Nature (New Biol.) 229:231, 1971 Spermatozoa Acrosin and Seminal Plasma Acrosin Inhibitors E. Fink, W.-D. Schleuning, and H. Fri tz, Munich, F R G Properties of Acrosin Inhibitors Acid-stable inhibitors of acrosin, the trypsinlike enzyme localized in the acrosome of the spermatozoon's head, are found in spermatozoa, in seminal plasma and in male sexual organs (Fritz et al. 1975). These inhibitors are polypeptides with molecular weights in the range of 6,000 to 7,000 and consist of approximately 60 amino acids. They can form an equi-molar complex with acrosin by binding to the active site of the enzyme, thus blocking its hy-drolytic activity towards natural and synthetic substrates. A l l known acrosin inhibitors are al-so inhibitors for trypsin (Table 1). Table 2 summarizes for some species the trypsin inhibiting activities found in the sex organs and in seminal plasma. The glands which contribute most to the inhibitory content of seminal plasma are the seminal vesicles. Table 1 Inhibition properties of inhibitors of the male genital tract and porcine pancreatic secretory inhibitor Source Acrosin Inhibition of Trypsin Plasmin Chymotrypsin Human seminal plasma Boar seminal plasma Guinea pig seminal vesicles Porcine pancreas Table 2 Trypsin inhibiting activities in male sex organs and seminal plasma Species ml U per g tissue or ml plasm Testes Epididymis Seminal Seminal vesicles plasma Man 7 0 - 1 0 0 5 0 - 8 0 5 0 - 1 0 0 1 5 0 - 3 3 0 Pig 9 0 - 1 2 0 7 0 - 1 1 0 500 -1 ,000 8 0 0 - 1 , 2 0 0 Cattle 4 0 - 7 0 5 0 - 8 0 9 0 0 - 1 , 5 0 0 2 ,400 -3 ,100 Guinea pig 1 0 0 - 2 2 0 3 0 0 - 4 0 0 3 ,500-5 ,000 Mouse 9 0 - 1 3 0 1 0 0 - 2 0 0 2 ,200-2 ,700 Hamster 6 0 - 9 0 8 0 - 1 2 0 3 0 0 - 6 0 0 Rat 1 0 0 - 2 0 0 9 0 - 1 3 0 1,400-1,600 1 m i l l inhibits the activity of approximately 1 jjg trypsin Novo 143 Biochemically best characterized are the inhibitors from boar seminal plasma, guinea pig seminal vesicles and human seminal plasma. The amino acid sequence of the boar seminal plasma inhibitor (Fig. 1 ; Tschesche et al. 1976), is homologous to the sequences of pan-creatic secretory inhibitors; the same holds true for one of the inhibitors of guinea pig seminal vesicles and very probably also for the acrosin inhibitor of human seminal plasma. A Thr-Arg-Lys-Gln-Pro-Asn- Cys Asn-Val-Tyr-Arg-Ser-Hls-Leu-Phe-Phe- Cys-Thr-B Phe-Ala-Pro-Ser-Lys-Val-Asx-Ser- Cys-Arg-Pro-Asx-Ser-Asx-Arg-Tyr-Val-Glx-His-Leu-Tyr-Met- Cys-Thr-C Thr-Ser-Pro-Gln-Arg-Glu-Ala-Thr- Cys Thr-Ser-Glu-Val-Ser-Gly Cys-Pro-A >4r$r-Gln-Met-Asp-Pro-lle- Cys-Gly-Thr-Asn-Gly-Lys-Ser-Tyr-Ala-Asn-Pro- Cys-lle-Phe-Cys-Ser-Glu-Lys-B Lys-Glx-Leu-Asx-Pro-Val, C y s , G l y T h r Asx ,G ly ,H is ,Thr ,Ty r , C Lys-\\e -Tyr-Asn-Pro-Val- Cys-Gly-Thr-Asp-Gly- l le -Thr-Tyr-Ser-Asn-Glu- Cys-Val-Leu-Cys-Ser-Glu-Asn-A Gly-Leu-Arg-Asn-Gln-Lys-Phe-Asp-Phe-Gly-HisTrp-Gly-His-Cys-Arg-GluTyr^hr-Ser-Ala-Arg-Ser B Phe-Thr-Phe-Ser -His-Tyr -Gly-Arg,Cys C Lys-Lys-Arg-GIn-Thr-Pro-VaI-Leu-1le -Gin-Lys-Ser -GIy-Pro-Cys A = boar seminal plasma acrosin inhibitor B = guinea pig seminal vesicle acrosin trypsin plasmin inhibitor C = porcine pancreatic secretory trypsin inhibitor Fig. 1 Alignment of the homologous amino acid sequences of the boar seminal plasma acrosin inhibitor and the partial sequence of the guinea pig seminal vesicle acrosin trypsin plasmin inhibitor with the sequence of porcine pancreatic secretory trypsin inhibitor Physiological Function of Acrosin Inhibitors The physiological role of the acrosin inhibitors is not yet fully understood. But it is a rea-sonable assumption that the main function of the inhibitors is to protect spermatozoa as well as cell membranes and secretory proteins of the male and female genital tract against acrosin which might be liberated from disintegrating spermatozoa during maturation and after ejaculation. The physiological function of the trypsin-chymotrypsin inhibitor present in human seminal plasma which has no effect on acrosin is discussed elsewhere in this volume (see Schiessler et al. this volume). Isolation and Properties of Acrosin Acrosin was demonstrated to be present in the spermatozoa of all species investigated. There was only one report claiming that mouse spermatozoa did not contain acrosin (Erickson and Martin 1974). However, recently Brown and Hartree (1976) published results showing that acrosin is present also in mouse spermatozoa. These results were confirmed in our laboratory by an experimentally different approach (Fink and Wendt, in press). Acrosin can be extracted from spermatozoa by neutral detergent-containing buffers or by acidic solutions. We usually use the acidic extraction procedure (Fink et al. 1972) and isolate the acrosin from the extract by gel filtration followed by affinity chromatogrophy on p-aminobenzamidine aminoalkyl cellulose (Schleuning et al. 1975). Boar acrosin isolated by this method from ejaculated spermatozoa showed only one sub-stantial protein band in acrylamide gel electrophoresis at pH 4.8 and one sharp digestion zone when gelatin was incorporated in the gel (Fig. 2). In SDS electrophoresis the molecular weight of the main protein band was calculated as 38,000. Two additional minor fractions had molecular weights of 37,000 and 34,000. These two bands may represent "degraded" molecules with one or more peptide bonds hydrolyzed either by acrosin itself or by other proteinases. Human acrosin was isolated by the same procedure as boar acrosin. By electrophoretic analysis (Schleuning et al. 1976) of the preparations four forms of acrosin were distinguish-144 Fig. 2 Polyacrylamide gel electrophoresis of boar acrosin preparations Gel 1 : pure acrosin, Coomassie blue stained; Gel 2: acidic sperm extract; Gel 3: pure acrosin. The unstained bands in Gel 2 and 3 represent zones of gelatinolytic activity able. The ratio of the four forms in the acrosin preparations differed from batch to batch. By SDS electrophoresis of the four forms protein bands with the apparent molecular weights of 64,000, 38,000, 25,000, 15,000 and 12,000 were observed. The bands with 15,000 and 12,000 daltons can be interpreted as two peptides originating from an acrosin form with a cleaved peptide bond. The low molecular weight acrosin forms can be produced from the high molecular weight form by incubation at pH 8. During this incubation the enzymic activity increases twofold. We cannot yet discriminate whether the high molecular weight form is already enzymatic-ally active or, at least partly, represents the inactive precursor of acrosin, called proacrosin. Molecular forms of different molecular weights for acrosins of various species have been observed by several authors, the occurrence of an active acrosin form with similar mole-cular weight as proacrosin was recently shown for rabbit testis acrosin by Mukerji and Meizel (1975). Inactive proacrosin was demonstrated by these authors first in testes and later on in epididymal spermatozoa (Meizel and Mukerji 1975). Proacrosin has also been found in ejaculated spermatozoa by other groups (Zaneveld et al. 1975) and in our laboratory (Fritz et al. 1975a). Presumably, the activation of proacrosin takes place via limited pro-teolysis, a mechanism which is well established for several proteases involved in the blood clotting system. During such an activation process intermediate enzyme forms are produced with molecular weights between those of the proenzyme and the fully activated enzyme. The enzymic properties of boar and human acrosin and most probably of the acrosins from other species are very similar to those of trypsin (Table 3); to our knowledge, only quantita-tive but thus far no qualitative differences have been observed. There are striking similarities also in the amino acid compositions of some forms of acrosin and trypsin (Schleuning et al. 1976, Stambaugh and Smith 1975), but in contrast to trypsin acrosin is a glycoprotein. Localization of Acrosin The subcellular localization of acrosin is not yet definitively known. Several groups could demonstrate a relatively strong binding of acrosin to acrosomal membranes, probably the inner acrosomal membrane and/or the equatorial segment (for further references, see Fritz 145 Table 3 Properties of acrosin and trypsin Acrosin Trypsin Specif ic activity U/mg B A P A ^ 18 .8 e 1.2 B A E E 214 36 Equal casein digestion rate is caused by 4.5 mg 1 mg Bimolecular rate constant of inhibi t ion, l /mole x min D F P C 630 300 T L C K 23 34 Dissociation constants Kj (mole/1) of complexes wi th: 7x1 O j _7 leupeptin and antipain 2 x 1 0 ^ seminal acrosin inhibitors < 1 x 1 0 y < 1 x 1 0 y kinin liberation f rom kininogen + + a N-a-benzoyl-DL-arginine p-nitroanilide b N-a-benzoyl-L-arginine ethyl ester c Diisopropylf luorophosphate d N-a-tosyl-L-lysyl-chloromethane e Protein was estimated by the biuret method et al. 1975a and Zaneveld et al. 1975). This assumption is especially supported by the following observations. 1. Denuded, i.e. acrosome-free ram spermatozoa contain nearly as much acrosin activity as can be extracted from intact spermatozoa (Brown and Hartree 1974) 2. In spermatozoa of frozen-thawed semen samples usually a significant increase in extract-able acrosin activity is observed. By this treatment the plasma membrane and the outer acrosomal membrane are extensively disrupted and removed (Schill and Wolff 1974) 3. Immunochemical studies (Morton 1975, Schill and Wolff 1974) give strong evidence that acrosin is bound to the inner acrosomal membrane. Physiological Role of Acrosin (Fig. 3) It is generally accepted that acrosin is involved in the penetration of sperm through the zona pellucida of the egg (Zaneveld et al. 1975, Fritz et al. 1975a, Stambaugh and Smith 1975). In the presence of acrosin inhibitors the zona layer is not dissolved by acrosin and penetration of spermatozoa through the zona is prevented. Enhancement of spermatozoa Induction or support of the Penetration of the zona pel-moti l i ty via bradykinin acrosome reaction lucida by the spermatozoon liberation Fig. 3 Possible effects of acrosin Very recently Meizel and Lui (1976) presented evidence for a possible involvement of acrosin in the acrosome reaction. The acrosome reaction is a process during which fusion of the outer acrosomal membrane and the adjacent plasma membrane occurs followed by vesiculation and consequently loss of the acrosomal content. This sequence of events is essential to mammalian spermatozoa penetration of the zona pellucida and takes place in spermatozoa that are within or close to the cumulus oophorous cell layer and have under-146 gone capacitation. Meizel and Lui (1976) demonstrated that the acrosome reaction in ca-pacitated hamster spermatozoa is drastically inhibited by low molecular weight trypsin inhibitors (a-N-tosyl-L-lysine chloromethylketone and p-nitrophenyl-p-guanidinobenzoate). The authors conclude that a trypsinlike enzyme of the spermatozoa, possibly acrosin, is in-volved in the acrosome reaction. Another function of acrosin may be the enhancement of spermatozoa motility by the release of kinins since acrosin is a potent bradykinin liberating protease. This is discussed in more detail in this volume (see Schill et al.). Summarizing the present knowledge one can draw the following speculative conclusions on the role of acrosin (Fig. 3) and acrosin inhibitors in fertilization: Acrosin is synthesized as proacrosin and bound to the inner acrosomal membrane of the spermatozoa. To prevent premature autoactivation and liberation of acrosin, there are in-hibitors present in the acrosome and in the adjacent fluids. After ejaculation, further acrosin inhibitors are picked up from the seminal plasma and are adsorbed to the outer surface membranes of the acrosome. These adsorbed inhibitors protect intact spermatozoa against acrosin released from disintegrated spermatozoa. During migration of the spermatozoa through the female genital tract or during capacitation, the inhibitor (s) is removed from the spermatozoa surface and proacrosin is converted to acrosin. The acrosome reaction, in which acrosin plays a role, takes place within or close to the cumulus oophorous of the egg and then the spermatozoon penetrates the zona pellucida by the assistance of acrosin. References Brown, C R . , E.F. Hartree: Distribution of a trypsin-like proteinase in ram spermatozoa. J. Reprod. Pert. 36:195, 1974 Brown, C.R., E.F. Hartree: Identification of acrosin in mouse spermatozoa. J. Reprod. Fert. 46:249, 1976 Erickson, R.P., S.R. Martin: Demonstration of the lack of acrosin in mouse spermatozoa by a radioassay. Contraception 9:539, 1974 Fink, E., H. Schiessler, M. Arnhold, H. Fritz: Isolation of a trypsin-like enzyme (acrosin) from boar spermatozoa. Hoppe-Seyler's Z. Physiol. Chem. 353:1633, 1972 Fink, E., V. Wendt: Demonstration of acrosin in mouse spermatozoa, J. Reprod. Fert.: in press, 1978 Fritz, H., W.-D. Schleuning, H. Schiessler, W.-B. Schill, V. Wendt, G. Winkler: Boar, bull and human sperm acrosin - isolation, properties, and biological aspects. In: Proteases and biological control, ed. by E. Reich, D.B. Rifkin, E. Shaw. Cold Spring Harbor Laboratory, Cold Spring Harbor 1975a Fritz, H., H. Schiessler, W.-B. Schill, H. Tschesche, N. Heimburger, O. Wallner: Low molecular weight proteinase (acrosin) inhibitors from human and boar seminal plasma and sperma-tozoa and human cervical mucus - isolation, properties and biological aspects. In: Proteases and biological control, ed. by E. Reich, D.B. Rifkin, E. Shaw. Cold Spring Harbor Laboratory, Cold Spring Harbor 1975b Meizel, S., Ch.W. Lui: Evidence for the role of a trypsin-like enzyme in the hamster sperm acrosome reaction. J. Exp. Zool. 195:137, 1976 Meizel, S., S.K. Mukerji: Proacrosin from rabbit epididymal spermatozoa: partial purification and initial biochemical characterization. Biol. Reprod. 13:83, 1975 Morton, D.B. Acrosomal enzymes: immuno-chemical localization of acrosin and hyaluranidase in ram spermatozoa. J. Reprod. Fert. 45:375, 1975 Mukerji, S.K., S. Meizel: The molecular trans-formation of rabbit testis proacrosin into acrosin. Arch. Biochem. Biophys. 168:720, 1975 Schiessler, H., O. Wallner, K. Ohlsson: Inhibitors of granulocyte proteases (antileukoprotease) in human genital tract secretions. This volume Schill, W.-B., H.H. Wolff: Ultrastructure of human sperm acrosome and determination of acrosin activity under conditions of semen preservation. Int. J. Fert. 19:217, 1974 Schill, W.-B., A. Grösser, G. Preissler, O. Wallner: In vitro stimulation of cervical mucus spermatozoa penetration by components of the kallikrein-kinin system. This volume Schleuning, W.-D., R. Hell, H. Schiessler, H. Fritz: Boar acrosin. I. Modified isolation procedure, active site titration and evidence for the presence of sialic acid. Hoppe-Seyler's Z. Physiol. Chem. 356:1915, 1975 Schleuning, W.-D., R. Hell, H. Fritz: Multiple forms of human acrosin: isolation and pro-perties. Hoppe-Seyler's Z. Physiol. Chem. 357:855, 1976 Stambaugh, R., M. Smith: Studies on the enzymatic and molecular nature of acrosomal proteinase. In: Proteases and biological control, ed. by E. Reich, D.B. Rifkin, E. Shaw. Cold Spring Harbor Laboratory, Cold Spring Harbor 1975 Tschesche, H. , S. Kupfer, R. Klauser, E. Fink, H. Fritz: Structure, biochemistry and com-parative aspects of mammalian seminal plasma acrosin inhibitors. In: Protides of the biological fluids, ed. by H. Peeters. Pergamon Press, Oxford 1976 

Acrosomal enzymes: immunochemical localization of acrosin and hyaluranidase in ram spermatozoa.

Boar, bull and human sperm acrosin - isolation, properties, and biological aspects. In: Proteases and biological control,

Demonstration of acrosin in mouse spermatozoa,

Hartree: Identification of acrosin in mouse spermatozoa.

Multiple forms of human acrosin: isolation and properties. Hoppe-Seyler's Z.

Structure, biochemistry and comparative aspects of mammalian seminal plasma acrosin inhibitors. In: Protides of the biological fluids,

The molecular transformation of rabbit testis proacrosin into

Spermatozoa Acrosin and Seminal Plasma Acosin Inhibitors

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Spermatozoa Acrosin and Seminal Plasma Acosin Inhibitors

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