HISTOCHEMICAL SUBCELLULAR LOCALIZATION OF THE ACROSOMAL PROTEINASE EFFECTING DISSOLUTION OF THE ZONA PELLUCIDA USING FLUORESCEIN-LABELED INHIBITORS*

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1 FERTILITY AND STERILITY Copyright 1972 by The Williams & Wilkins Co. Vol. 23, No.5, May 1972 Printed in U.S.A. HISTOCHEMICAL SUBCELLULAR LOCALIZATION OF THE ACROSOMAL PROTEINASE EFFECTING DISSOLUTION OF THE ZONA PELLUCIDA USING FLUORESCEIN-LABELED INHIBITORS* R. STAMBAUGH, PH.D., AND J. BUCKLEY, B.S. Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Although the association of hyaluronidase with the acrosome has been known for a number of years,i-5 this enzyme dissolves the matrix of the cumulus oophorus, but does not disperse the zona pellucida. More recently, we demonstrated that a trypsin-like enzyme in rabbit spermatozoa is the enzyme which effects penetration of the zona pellucida by its dissolution action. 6, 7 This enzyme was found to be localized in the acrosomes by subcellular fractionation, and it was extracted as a 59,000 molecular weight complex with hyaluronidase. Subsequently, a similar enzymic complex was found in rhesus monkey (Macaca mulatta) and human spermatozoa, 8, 9 and it was demonstrated that in vitro fertilization of rabbit ova can be inhibited by trypsin inhibitors. 1o The presence of this trypsinlike enzyme in acrosomes has now been confirmed in bull sperm by Multamaki and Niemi,11 in rabbit sperm by Zaneveld, Srivastava, and Williams,12 in rabbit, rat, mouse, guinea pig, hamster, and human sperm by Gaddum and Blandau,13 in fowl sperm by Ho and Meizel,14 and in bull sperm by Garner, Salisbury, and Graves. 15 Since this acrosomal enzyme proved to be similar but not identical to pancreatic trypsin,9 we gave this important enzyme the identifying name of acrozonase,16 * Supported by National Institutes of Health Contract , Ford Foundation Grant B, and National Institutes of Health Grant RROO Received August 10, 1971; revised November 4, while Williams 17 suggested the name acrosin. However, we now find that the simple, but descriptive, name of acrosomal proteinase is the official nomenclature of the International Commission of Biochemical Nomenclature,18 and we will use this name in this and future publications from our laboratory. The high stability of the complexes formed between pancreatic trypsin and the naturally occurring trypsin inhibitors at ph suggested to us that it might be possible to use fluorescein-labeled trypsin inhibitors for histochemistry to confirm our subcellular localization of acrosomal proteinase and this is the subject of this article. METHODS AND MATERIALS Ejaculated spermatozoa were collected from New Zealand White rabbits with an artificial vagina and washed twice with isotonic sodium chloride. Rhesus monkey (Macaca mulatta) spermatozoa were collected by electroejaculation,20 and the clot was allowed to liquify at room temperature for 1 hr. The free sperm were then washed twice with isotonic sodium chloride. Sea urchin (Lytechinus) spermatozoa were collected by teasing the sperm from the dissected testes, and the acrosome reaction was initiated with egg-sea water prior to staining. Fluorescein labeling of the soybean and lima bean trypsin inhibitors was carried out using fluoresceinisothiocyanate on celite by the procedure of Rinderknect. 21 The labeled trypsin inhibitor

2 May 1972 FLUORESCEIN-LABELED INHIBITORS 349 was separated from the unlabeled inhibitor using an 18.0 x 2.8 cm. column of Sephadex G-25 and 0.05 M Tris buffer, ph 8.2. The inhibitors retained all of their inhibitory properties for trypsin and acrosomal protease using this experimental procedure. Acrosomal proteinase activity was measured as previously described on the synthetic substrates benzoyl-l-arginine ethyl ester and N -(\' ben z oy 1- D L- arginine p-ni troanilide. 9 Bovine and human serum albumins were labeled with fluorescein by the same procedure to serve as controls in these experiments. Staining of the washed spermatozoa was carried out on air-dried smears using microscope slides. The sperm were stained first for 15 min. with either 0.01% Congo Red or 0.01% Evans Blue in 0.9% NaCl, rinsed and air-dried. The slides were then stained for proteinase activity with a 2% solution of the fluoresceinlabeled trypsin inhibitor in 0.05 M Tris buffer, ph 8.2. Rhesus monkey or rabbit spermatozoa required 1 hr. of staining, but the sea urchin sperm required only 30 min. RESULTS The staining experiments with the fluorescein-labeled trypsin inhibitors confirmed the subcellular localization of the trypsin-like enzyme in the acrosomes of rabbit and rhesus monkey sperm (Figs. la and b), and the green fluorescence of fluorescein was never observed in the control experiments using albumins (Fig. Ie). Also, no differences in staining were observed between rabbit epididymal, ejaculated, or capacitated sperm obtained after 6 hr. in the estrous uterus, indicating that acrosomal proteinase occurs in an active form in all three types of spermatozoa. Although we have not attempted to extract the acrosomal proteinase from sea urchin sperm, we found that the acrosomes of these spermatozoa also stained rapidly with the fluorescein-labeled trypsin inhibitors (Fig. Id), indicating that a similar acrosomal peptidase probably occurs in this marine organism. DISCUSSION Although the earlier work of Yamane 22, 23 and Buruiana 24 established that corona cell dispersion and zona pellucida dissolution activities are present in extracts of whole sperm, no definite relationship of these properties with the fertilization process was established. Most, and probably all cells contain some proteolytic activity, so the demonstration of zona dissolution by extracts of whole sperm does not establish a physiologic function for the enzyme. The identity of the enzyme(s) must be ascertained, and their subcullular localization and participation in a physiologic event established. Srivastava, Adams, and Hartree 25, 26 also observed that extracts of ram sperm acrosomes remove the corona radiata and sometimes the zona pellucida from rabbit ova, but no subcellular fractionation or histochemical demonstration of the subcellular localization of these activities was made. More recently, the trypsin-like enzyme, acrosomal proteinase, which effects penetration of the zona pellucida by its dissolution action was isolated and characterized. 6-9 This enzyme occurs in a 59,000 molecular weight complex with hyaluronidase in the acrosomes of rabbit, rhesus monkey, and human spermatozoa. 6-9 Subsequently its participation in fertilization was demonstrated by the use of trypsin inhibitors to inhibit fertilization. 10 The histochemical experiments described here, using fluorescein-labeled trypsin inhibitors, have provided visible confirmation of the subcellular localization of this proteinase in the acrosomes of rabbit and rhesus monkey spermatozoa.

3 FIG. 1. Fluorescence photomicrograph of: (A) rabbit sperm stained with Congo Red and fluoresceinlabeled soybean trypsin inhibitor x 330; (B) rhesus monkey sperm stained with Congo Red and fluoresceinlabeled lima bean trypsin inhibitor x 265; (C) rabbit sperm stained with Congo Red and fluorescein-labeled albumin x 330; and (D) sea urchin sperm stained with Evans Blue and fluorescein-labeled soybean trypsin inhibitor x

4 May 1972 FLUORESCEIN-LABELED INHIBITORS 351 In addition they have provided evidence that a trypsin-like enzyme probably occurs in the acrosomes of sea urchin sperm also, even after the acrosome reaction has taken place. Admittedly, no protein can serve as a completely adequate c.,ntrol for this type of experiment. On the other hand, these inhibitors are highly specific, and the lack of a completely adequate control should not preclude gleaning additional information by their use for the histochemical subcellular localization of this peptidase. Since the outer acrosomal membrane is partially removed by vesiculation during the acrosomal reaction,27-30 the results would seem to indicate that acrosomal proteinase is associated with the inner acrosomal membrane, or perhaps even part of the structure of this membrane. The fact that epididymal and ejaculated rabbit sperm and ejaculated monkey sperm stained just as rapidly as capacitated sperm suggests that this enzyme is not involved directly in the capacitation process, since only the active enzyme binds these inhibitors. Similar conclusions were arrived at by Gaddum and Blandau 13 using timelapse photography to demonstrate the lytic activity of epididymal, ejaculated, and capacitated sperm on gelatin films. This conclusion is also supported by the ability to consistently extract highly active acrosomal proteinase preparations from ejaculated rabbit, rhesus monkey, and human sperm, and from rabbit epididymal sperm.7. 9 Therefore, it seems likely that capacitation involves the preparation of spermatozoa for the vesiculation process of the acrosome reaction, , 32 but the acrosomal enzymes, themselves, are not directly involved in capacitation. In the lower marine organisms a thin apical filament is formed during the acrosomal reaction,33-37 but no fluorescent filaments were observed in our sea urchin sperm after the acrosome reaction had been initiated with egg-sea water. Either the fluorescence of these filaments was below the detectable level, or the filaments are devoid of protease activity. To our knowledge this is the first reported use of fluorescein-labeled enzyme inhibitors for histochemistry, and perhaps this technic may be applicable to other enzymes whenever suitable specific inhibitors are available. SUMMARY Subcellular localization of the trypsinlike acrosomal proteinase by sperm fractionation was confirmed by the use of fluorescein-labeled trypsin inhibitors. Fluorescein-labeled lima bean and soybean inhibitors were specifically bound by the acrosomes of epididymal, ejaculated, or capacitated rabbit sperm, and also by ejaculated rhesus monkey sperm, indicating that the enzyme exists in an active form both before and after capacitation. Acrosomes of sea urchin sperm, pretreated with egg-sea water, also stained rapidly with the inhibitors, indicating that a similar trypsin-like enzyme is present in this marine organism. REFERENCES 1. TYLER, A. Properties of fertilizin and related substances of eggs and sperm of marine animals. Amer Natur 83:195, WADA, S. K., COLLIER, J. R., AND DAN, J. C. Studies on the acrosome. V. An egg-membrane lysin from the acrosomes of Mytilus endulis spermatozoa. Exp Cell Res 10:168, LEUCHTENBERGER, C., AND SCHRADER, F. The chemical nature of the acrosome in the male germ cells. Proc Nat Acad Sci USA 36:677, AUSTIN, C. R., AND BISHOP, M. W. H. Some features of the acrosome and perforatorium in mammalian spermatozoa. Proc Roy Soc Med 148: 234, AUSTIN, C. R., AND BISHOP, M. W. H. Role of the rodent acrosome and perforatorium in fertilization. Proc Roy Soc Med 149:241, STAMBAUGH, R., AND BUCKLEY, J. Zona pellucida dissolution enzymes of the rabbit sperm head. Science 161:585, STAMBAUGH, R., AND BUCKLEY, J. Identification

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