EFFECT OF ENZYMES AND ACIDITY ON THE ZONA PELLUCIDA OF THE MOUSE EGG BEFORE AND AFTER FERTILIZATION

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1 EFFECT OF ENZYMES AND ACIDITY ON THE ZONA PELLUCIDA OF THE MOUSE EGG BEFORE AND AFTER FERTILIZATION R. B. L. GWATKIN King Ranch Laboratory of Reproductive Physiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 4, Pennsylvania, U.S.A. (Received 5th July 1963) Summary. A survey was made of the effectiveness of acid media and of twelve different enzyme preparations in dissolving the zona pellucida of the mouse egg. It was found necessary to lower the ph of the medium to nearly 4 in order to dissolve the zona pellucida. Purified papain, crude ficin and crude Streptomyces griseus protease (`pronase') were found to be effective enzymes for zona pellucida removal. A crude preparation of \g=b\-glucuronidase also dissolved the zona pellucida, but its activity was shown not to be due to its \g=b\-glucuronidasecontent. A difference in response between fertilized and unfertilized eggs was it dissolved the zona noted with the crude \g=b\-glucuronidasepreparation : pellucida of unfertilized eggs more rapidly than that of fertilized eggs. Purified elastase did not dissolve the zona pellucida of either group, but caused the zona pellucida of some unfertilized eggs to swell. The following enzyme preparations had no apparent effect on the mouse zona pellucida: purified trypsin, \g=a\-chymotrypsin and carboxypeptidase, crude preparations of trypsin, pancreatic lipase, lecithinase D, \g=b\-1,4-glucosidaseand lysozyme. INTRODUCTION Austin & Lovelock (1958) reported that the zona pellucida of rabbit and rat eggs was permeable to compounds of molecular weight 1200, or less, but not to heparin, which has a molecular weight of 16,000. These results suggest that large molecules do not pass through the zona pellucida. As we wished to study the interaction of viruses with mouse eggs, some means of removing the zona pellucida was required. A method for doing this by rapid pipetting has been described by Tarkowski (1961), but the eggs have to be 'shelled' one at a time. The only data on chemical removal of the zona pellucida appeared in an abstract by Smithberg (1953), who found that trypsin, chymotrypsin and ficin, but not papain were effective. Accordingly, the studies described in this paper were undertaken to determine the best method of dissolving the zona pellucida from fertilized as well as unfertilized mouse eggs. Work had begun when Mintz (1962) described the use oí Streptomyces griseus protease (pronase) for dissolving 99

2 100 R. B. L. Gwatkin the zona pellucida of mouse eggs, and as soon as this enzyme became commer cially available, it was included in our study. METHODS Three-month-old Swiss mice were superovulated by the injection of 5 i.u. pregnant mare serum gonadotrophin ('Gestyl', Organon), followed 48 hr later by 3 i.u. human chorionic gonadotrophin ('Pregnyl', Organon). When fertilized eggs were required, a mature fertile male was placed with each female at the time of the second injection and the animals were checked for the presence of vaginal plugs 16 hr later. This method, of course, gives only presumptive evidence of fertilization. Six to 8 hr after the expected time of ovulation (18 to 20 hr after the second injection) the mouse was killed and the Fallopian tubes were removed and placed under a dissecting microscope. A small cut in the wall of the tube was made with a cataract knife to permit extrusion of the eggs, embedded in the cumulus oöphorus. The eggs and the cumulus were stored in Medium BGJé (Biggers, Gwatkin & Heyner, 1961). When enough eggs had been collected, they were transferred to Medium BGJé containing 300 U.S.P. units of hyaluronidase (bovine testicular, Nutritional Biochemical Corp.) per ml. This concentration of hyaluronidase was used because Rowlands (1944) showed that it removed the cumulus cells of their surrounding the rat egg. After 5 min at 37 C the eggs, now stripped cumulus, were transferred through two washings of saline solution (NaCl 8-00 g, KC g, glucose 1-00 g, phenol red 0-01 g) to remove the hyaluronid ase. A number of eggs, usually five to ten, were finally deposited in 0-5 ml of the appropriate acid medium, or enzyme solution, in a watch glass. In the enzyme experiments, controls at ph and were always included. After to min at 37 C the condition of the zona pellucida was examined under a dissecting microscope. Acid media These consisted ofsaline solution with 5% Mcllvaine's citric acid-phosphate buffer. The final concentration of the phosphate was 0-1 M. Enzyme solutions Enzymes were dissolved at 0-05 or 0-5 % (w/v) in saline solution Z, buffered with the same concentration of Mcllvaine's citric acid-phosphate buffer as was used for preparing the acid media. By using buffers prepared at ph and it was possible to test the enzymes at, or near to, other ph optima, as given by Dixon & Webb (1958), Spector (1956) and Boyer, Lardy & Myrbàck (19). The enzymes used, and their sources, were as follows: carboxypeptidase (3x cryst.), cellulase (crude), -chymotrypsin (3x cryst.), ß-glucuronidase (crude, 70 to 100 Fishman units per ml), papain (2x cryst.) and lysozyme (2x cryst.) from Worthington Biochemical Corporation; hyaluronidase (crude, bovine testicular), elastase (2x cryst.), lecithinase D (crude) and trypsin (salt free, 2x cryst.) from Nutritional Biochemicals Corporation; ficin (fig latex), lipase (crude, porcine pancreas) and pronase (crude) from California Corporation for

3 Zona pellucida of the mouse egg 101 Biochemical Research, and finally, crude trypsin from British Drug Houses. Papain and ficin were activated with M-cysteine and lipase with 0-1 mg CaCtyml. RESULTS Crude hyaluronidase (300 U.S.P. units/ml) removed the cumulus oöphorus from the eggs in about 3 min at 37 C. This enzyme had no apparent effect on the zona pellucida even after 2\ hr exposure. Table 1 effect of acidity on the zona pellucida of fertilized and unfertilized mouse eggs PH Proportion of eggs which lost zona pellucida during exposure 6/6 5/6 30 min 6/6 6/6 min Acidity The effect of acidity upon the mouse zona pellucida is presented Prolonged contact with buffer at ph 4-32 did not visibly pellucida, but at ph in Table 1. affect the zona 4T5 it dissolved within 30 min. No consistent difference was detected between the response of fertilized and unfertilized eggs. Enzymes Table 2 presents the effect of enzymes which hydrolyse peptide bonds. Crystalline papain, crude ficin and crude pronase removed the zona pellucida in min or less. Crystalline elastase did not remove the zona pellucida from eggs within a 30-min period of exposure. However, the zona pellucida of some of the unfertilized ova became distended within the 30-min period. In one experiment the swelling of some zonae pellucidae progressed until they were lost entirely. No reaction was detected in the fertilized eggs. Trypsin, whether crystalline or crude, crystalline cc-chymotrypsin and crystalline carboxypeptidase had no apparent effect on the zona pellucida. Table 3 presents the effect of enzymes which hydrolyse ester or glycosidic bonds. Neither crude pancreatic lipase nor crude lecithinase D visibly affected the zona pellucida. Among the group of enzymes hydrolysing glycosidic bonds, crude lysozyme and crude ß-l,4-glucosidase were ineffective. When eggs exposed

4 102 R. B. L. Gwatkin to crude ß-glucuronidase were examined after 30 min, only the unfertilized eggs had lost their zonae pellucidae. When the eggs were re-examined after min exposure to the enzyme the zonae pellucidae of the fertilized eggs also were dissolved. Table 2 effect on mouse zona pellucida of enzymes which hydrolyse peptide BONDS Enzyme Trypsin* Lot 1 Lot 1 Lot 2 Lot 2 (0-5%) Crude trypsin a-chymotrypsin* Carboxypeptidase* Papain* Ficin S. griseus protease pronase Elastase* Optimum ph 8 to 9 7 to 8 ph at which tested Approximate period of exposure (min) Proportion of eggsfrom which zona pellucida was removed 0/8 0/10 13/13 0/8 0/10 10/10 7/7 7/7 2/5 Crystalline enzymes. Unless otherwise stated the enzyme concentration was 005%. Each horizontal line of data represents one experiment. Controls at ph and were included in each experiment and were invariably negative. Purified ß-glucuronidase An extremely pure preparation of ß-glucuronidase, containing 0,000 units of ß-glucuronidase activity (Fishman, Springer & Brunetti, 1948) per mg of protein, was obtained from Dr G. A. Lewy of the Rowett Research Institute, Bucksburn, Aberdeen. This purified enzyme had no visible effect on the zona pellucida of either fertilized or unfertilized mouse eggs even in concentrations 1000 times higher than that at which the crude preparation was active. It was concluded that the efficacy of the crude enzyme preparation was probably due to a contaminant, perhaps a proteolytic enzyme. DISCUSSION The effect of acidity on the zona pellucida of the mouse has not been reported previously. The acid conditions (ph 4-1 ) needed to dissolve the zona pellucida of the mouse egg are intermediate between those required for dissolving the

5 Zona pellucida of the mouse egg 103 zona pellucida of the rat egg, ph 4-5 to, and the zona pellucida of the rabbit egg, ph 3-0 (Austin, 1961). Smithberg (1953) reported that trypsin, chymotrypsin and ficin dissolved the zona pellucida of the mouse egg, but that papain, erepsin and a mould protease were ineffective. In the only other study of the effect of enzymes on the mouse zona pellucida, Mintz (1962) described trypsin, chymotrypsin, ficin and pronase as effective; lysozyme, elastase, collagenase and hyaluronidase as Table 3 effect on mouse zona pellucida of enzymes which hydrolyse ester and glycosidic bonds Enzyme (0-0b%) Hydrolysing ester bonds Pancreatic lipase C. ««/c/ifilecithinase (lecithinase D) Optimum ph 7 to 7-5 ph at which tested Approxi mate period of ex posure (min) Proportion of eggsfrom which zona pellucida was removed 0/3 0/8 0/3 0/7 Hydrolysing glycosidic bonds a-1,4-glucosidase (cellulase) Lysozyme ß-Glucuronidase and 30 and 30 and 0/7 0/4 0/4 0/7 and 5/7 and and 0/4 2/6 and 6/6 Enzyme concentration was 0-05%. Each horizontal line ofdata represents one experiment. Controls at ph and were included in each experiment and were invariably negative. ineffective. Neither author gives the concentration of enzyme used, the ph, time of exposure, or other conditions, so comparison with our results must be made in general terms. In spite of a favourable ph and the testing of several batches of enzyme, we found that trypsin did not visibly affect the zona pellucida and we also obtained negative results with chymotrypsin. The reason for the discrepancy between these results and those of the authors quoted above is not clear. Furthermore we, unlike Smithberg, found that purified papain was effective in removing the zona pellucida. On the other hand, our finding that ficin and pronase dissolve the zona pellucida of the mouse egg is in agreement with those of Smithberg and Mintz, respectively. So far it is only the proteolyte enzymes which have been found to dissolve the zona pellucida ofthe mouse egg. However, in addition to protein the mouse zona pellucida probably contains other components. Histochemically, Braden (1952) has shown the presence of polysaccharide in the zona pellucida ofrat and rabbit eggs. Evidence has been reported for the presence of ascorbic acid in the rat

6 104 R. B. L. Gwatkin zona pellucida (Deane, 1952) and of hyaluronic acid and lipoprotein in the zona pellucida of the cat (Koneckny, 1959). It was hoped to detect a difference in response between the zona pellucida of fertilized and unfertilized eggs, thereby indicating some physico-chemical basis for the 'zona reaction' (Braden, Austin & David, 1952). This expectation was to some extent realized. The crude ß-glucuronidase preparation acted more rapidly on the zona pellucida ofunfertilized eggs than on that offertilized eggs. However, this effect is not due to ß-glucuronidase itself. A second difference was the swelling reaction induced only in the zona pellucida of unfertilized eggs by elastase. It is not known why only some unfertilized eggs acted in this way. In addition to these relatively clear-cut differences, we observed instances when unfertilized eggs kept at ph 4 lost their zonae pellucidae before fertilized eggs. However, this effect was not consistent. Smithberg (1953) noted that his proteolytic enzymes removed the zona pellucida from unfertilized eggs more rapidly than from fertilized ones. Our results did not show this. It seems probable that the changes induced by sperm entry must be rather subtle and require just the right enzyme concentration and timing for their demonstration. Although we did not determine accurately the relative speeds with which the enzymes acted on the zona pellucida, it was clear that pronase acted more rapidly than any other enzyme. A 0-05 % solution of the crude enzyme removed the zona pellucida in 3 to 5 min. This is not surprising since pronase has the least specificity and greatest activity of any known protease (Nomoto, Narahaski & Murakami, 19). Because of its rapid action, which apparently does not harm the egg (Mintz, 1962; Gwatkin, 1963) pronase is now being used to investigate the possible role of the zona pellucida as a barrier to virus infection (Gwatkin, 1963). However, since we do not know the effect of pronase on virus receptors, the availability of alternative enzymes for the removal of the zona pellucida may prove a valuable asset. ACKNOWLEDGMENTS I should like to thank Dr G. A. Lewy of the Rowett Research Institute, Aberdeen, for the purified ß-glucuronidase and Dr W. J. Tindall of Organon Laboratories Ltd, London, for a supply of the gonadotrophins. Mrs Pamela Yates and Mr Dorsey Williams gave skilled technical assistance. This research was supported by grants from the National Science Foundation (GB 617) and the Population Council. REFERENCES Austin, C. R. (1961) The mammalian egg, p Blackwell Scientific Publications, Oxford. Austin, C. R. & Lovelock, J. E. (1958) Permeability of rabbit, rat and hamster egg membranes. Exp. Cell Res., 2. Biggers, J. D., Gwatkin, R. B. L. & Heyner, S. (1961) Growth of embryonic avian and mammalian tibiae on a relatively simple chemically defined medium. Exp. Cell Res. 25, 41. Boyer, P. D., Lardy, H. & Myrbäck,. (19) The enzymes, 2nd edn, vol. 5. Academic Press, New York. Braden, A. W. H. (1952) Properties of the membranes of rat and rabbit eggs. Aust. J. sci. Res. B, 5, 4. Braden, A. W. H., Austin, C. R. & David, H. A. (1954) The reaction of the zona pellucida to sperm penetration. Aust. J. biol. Sci. 7, 391.

7 Zona pellucida of the mouse egg 105 Deane, H. W. (1952) Histochemical observations on the ovary and oviduct of the albino rat during estrous cycle. Amer. J. Anat. 91, 363. DrxoN, M. & Webb, E. C. (1958) Enzymes. Academic Press, New York. Fishman, W. H., Springer,. & Brunetti, R. (1948) Application of an improved glucuronidase assay method to the study of human blood ß-glucuronidase. J. biol. Chem. 173, 449. Gwatkin, R.. L. (1963) Effect of viruses on early mammalian development, I. Action of Mengo encephalitis virus on mouse ova cultivated in vitro. Proc. nal. Acad. Sci., Wash., 576. Konegny, M. (1959) Etude histochemique de la zone pellucide des ovules de chatte. C.R. Soc. Biol., Paris, 3, 893. Mintz, B. (1962) Experimental study of the developing mammalian egg: removal of the zona pellucida. Science, 138, 594. Nomoto, M., Narahasi, Y. & Murakami, M. (19) A proteolytic enzyme of Streptomyces griseus. VI. Hydrolysis by Streptomyces griseus protease. J. Biochem. 48, 593. VII. Substrate specificity of Streptomyces griseus protease. J. Biochem. 48, 906. Rowlands, I. W. (1944) Capacity of hyaluronidase to increase the fertilizing power of sperm. Nature, Lond. 4, 332. Smithberg, M. (1953) The effect of different proteolytic enzymes on the zona pellucida of mouse ova. Anat. Ree. 117, 554. Spector, W. S. (1956) Handbook of biological data, p. 28. Saunders, Philadelphia. Tarkowski, A. K. (1961) Mouse chimaeras developed from fused eggs. Nature, Lond. 190, 857.