Cell, Vol. 20, 873-882.

July 1980,

Copyright

0 1980

by MIT

Mammalian Sperm-Egg Interaction: Identification of a Glycoprotein in Mouse Egg Zonae Pellucidae Possessing Receptor Activity for Sperm
Jeffrey D. Bleil and Paul M. Wassarman Department of Biological Chemistry Laboratory of Human Reproduction and Reproductive Biology Harvard Medical School Boston, Massachusetts 02115 perivitelline space (Endo, 1961) and these modify the vitelline envelope (Monroy, 1965; Vacquier, Tegner and Epel, 1973; Foerder and Shapiro, 1977) such that sperm no longer bind to nor penetrate this structure (Schmell et al., 1977; Glabe and Vacquier, 1978). Although less well documented, analogous events apparently occur following fertilization of mammalian eggs (Gwatkin, 1976,1977). Subsequent to activation of the egg by sperm or by other methods, sperm no longer fuse with the mammalian eggs plasma membrane (Zinor and Wolf, 1979). In addition, the zona pellucida, an acellular coat surrounding the plasma membrane, is modified as a result of,fertilization such that sperm can attach loosely, but no longer bind tightly to nor penetrate this structure (Braden, Austin and David, 1954; Pike, 1969; Barros and Yanagimachi, 1972; lnoue and Wolf, 1975). This second block to polyspermy in mammals has been termed the zona reaction and is thought to involve the masking, modification or destruction of a sperm receptor in the zona pellucida (Braden et al., 1954; Gwatkin, 1976, 1977). Several lines of evidence suggest that the zona pellucida contains a sperm receptor. For example, capacitated sperm bind to zonae pellucidae of mammalian eggs and the binding is relatively species-specific (Hanada and Chang, 1972; Hartmann, Gwatkin and Hutchinson, 1972); it is inhibited by pretreating eggs with proteases (Hartmann and Gwatkin, 1971) lectins which bind to zonae pellucidae (Barros and Yanagimachi, 1972; Oikawa, Yanagimachi and Nicolson, 1973) or antisera directed against whole ovaries or eggs (Shivers et al., 1972; Shivers, 1974; Jilek and Pavlok, 1975; Oikawa and Yanagimachi, 1975; Tsunoda and Chang, 1976; Sacco, 1977). Moreover, it has been demonstrated that sperm fail to penetrate hamster eggs in vitro in the presence of solubilized zonae pellucidae (Gwatkin and Williams, 1976). Recently, we reported that zonae pellucidae of mouse oocytes and eggs are composed of three different glycoproteins, designated ZPl , ZP2 and ZP3, having apparent molecular weights of 200,000, 120,000 and 83,000, respectively (Bleil and Wassarman, 1978, 1980a, 1980b). All three of these glycoproteins are synthesized and secreted by mouse oocytes during their growth phase (Bleil and Wassarman, 1980b). To determine whether any of the zona pellucida glycoproteins possesses sperm receptor activity, we have purified each glycoprotein and evaluated its effect on the binding of sperm to eggs in vitro utilizing a competition assay. The results of the experiments presented here demonstrate that ZP3 purified from zonae pellucidae of mouse oocytes or unfertilized eggs is the only one of the three glycoprotein components that intereferes with the binding of sperm to eggs in vitro. On the other hand, ZP3 purified from zonae pellucidae of 2-cell embryos has no effect on the binding of sperm to eggs in vitro. The behavior of

Summary Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZPl, ZP2 and ZP3, having apparent molecular weights of 2DD,DW, 120,DDD and 83,DDD, respectively (Bleil and Wassarman, 1978, lg8Da, 198Db). In this investigation, ZPl , ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2tell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2cell embryos, reduced binding of sperm to as little as loo/ of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-tell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-tell embryos to affect sperm binding. In no case did purified ZPl and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm. Introduction The results of studies on fertilization of echinoderm, amphibian and mammalian eggs suggest that a block to polyspermy is effected at two different sites on the egg (Gwatkin, 1976, 1977; Epel, 1977). For example, in sea urchins, fusion of sperm and egg triggers plasma membrane depolarization (Jaffe, 1976) that correlates with the autocatalytic release of calcium from the eggs cortex (Vacquier, 1975; Steinhart, Zucker and Schatten, 1977) and prevents further sperm fusion. In addition, fertilization results in the release of cortical granule contents into the eggs

Cell 874

purified ZP3 in the competition assay compares very favorably with that of solubilized zonae pellucidae from unfertilized eggs and 2-cell embryos, suggesting that it is the zona pellucida receptor recognized by sperm. Results Experimental Design We have used an in vitro binding assay (competition assay) to determine whether one or more of the three glycoproteins which comprise the mouse eggs zona pellucida has the properties expected for a sperm receptor. A comparable assay has been used by other investigators to study the sperm receptor activity of sea urchin eggs (Schmell et al., 1977). In our experiments, capacitated mouse sperm (Wolf and Inoue, 1976) were first cultured in vitro, either in the presence of solubilized zonae pellucidae isolated individually from mouse oocytes, eggs or 2-cell embryos, or in the presence of purified zona pellucida proteins. Ovulated unfertilized eggs were then added to the cultures and the number of sperm bound to the eggs was quantitated by counting the number of sperm tightly associated with eggs after gentle pipetting of the eggs to remove loosely associated or attached sperm (Hartmann et al., 1972). In this manner, we were able to assess whether solubilized zonae pellucidae or individual zona pellucida proteins interfered with the binding of sperm to eggs. In each experiment, 2-cell mouse embryos were also added to the cultures and served as internal controls since sperm should not bind to zonae pellucidae of fertilized eggs or embryos (Barros and Yanagimachi, 1972; lnoue and Wolf,. 1975); 2-cell embryos were used here since they can be distinguished easily from unfertilized eggs. Effect of Solubilized Zonae Pellucidae on the Binding of Sperm to Eggs Zonae pellucidae were isolated individually with mouth-operated micropipettes from mouse eggs and embryos as previously described (Bleil and Wassarman, 1980a, 1980b) and solubilized in 5 mM NaH2P04 brought to pH 2.5 with HCI, such that the final concentration was l-40 zonae pellucidae per pl. In a typical experiment, aliquots of this solution were added to drops of culture medium (Ml99-M), an aliquot of medium containing capacitated mouse sperm was added to each drop such that the final concentration was about 1 OS sperm per ml, and the suspensions were incubated for 1 hr at 37C under oil in a humidified atmosphere of 5% COn in air. At the end of this period, ovulated unfertilized eggs and 2-tell embryos in M 199-M were added to the sperm suspensions and incubated for 40 min under the conditions just described. Solubilized zonae pellucidae from unfertilized eggs and embryos appeared to have no significant

effect on sperm motility during the incubation period. Sperm cultured in vitro in the absence of solubilized zonae pellucidae were bound to unfertilized eggs, but not to embryos (Figures 1 A and 1 B). In fact, under comparable culture conditions at lower sperm concentrations (about lo4 sperm per ml), the eggs were fertilized in vitro and progressed to the 2cell embryo stage within 36 hr of culture. As anticipated from the inability of sperm to bind to fertilized eggs and embryos, sperm exposed to solubilized zonae pellucidae from 2-cell embryos bound to ovulated eggs to the same extent as untreated sperm, regardless of the concentration of embryo zonae pellucidae added (Figures 1C and 2). Exposure of 2-cell embryos to hyaluronidase had no effect on these results. On the other hand, sperm exposed to solubilized zonae pellucidae from unfertilized eggs exhibited greatly diminished binding to eggs as compared to untreated sperm (Figure 1D). Identical results were obtained using solubilized zonae pellucidae isolated from eggs matured in vitro that were not treated with hyaluronidase. Titration of sperm with egg zonae pellucidae revealed that binding of sperm could be reduced to as little as 10% of the control values (Figure 2). Solubilized zonae pellucidae from unfertilized eggs were shown to affect sperm rather than eggs, since mouse eggs which failed to bind sperm exposed to solubilized zonae pellucidae could be removed from these cultures and were shown to bind untreated sperm to the same extent as control eggs. These results suggest that zonae pellucidae from unfertilized mouse eggs, but not from embryos, contain a component that behaves in the manner expected for a sperm receptor. It should be noted that a comparison of the results of several different experiments revealed that, at a constant concentration of 1 egg zona pellucida per ~1, binding of sperm to eggs was inhibited from 10 to 50%. This variability is attributable to several factors. For example, in many cases, values for the number of sperm bound to eggs in the absence of added solubilized zonae pellucidae were underestimates of the actual situation. This was due to difficulties inherent in attempting to discriminate between two or more closely associated sperm tails and a single tail when large numbers of sperm (that is, 50-l 20) were bound to eggs in the plane of focus examined (for example see Figures 1 A and 1 B). When low numbers of sperm were bound to eggs, less overlapping of sperm tails occurred and, consequently, estimates of these numbers were made more accurately (see Figure 1 D). In addition, since no attempt was made to separate motile and immotile sperm in our experiments, the values for maximal sperm binding varied by as much as a factor of three (see legend to Figure 1). Despite these technical difficulties, the dramatic inhibitory effect of solubilized zonae pellucidae from unfertilized eggs on the binding of sperm to eggs was observed consistently. Solubilized zonae pellucidae from 2-tell

~7~ammalian

Eggs

Sperm

Receptor

Figure 1. Binding of Sperm to Eggs in the Presence of Solubtlized Zonae Pellucidae Isolated from Unfertilized Eggs and 2-Ceil Embryos Zonae pellucidae isolated from unfertilized eggs and 2cell embryos were dissolved in 5 mM NaH*PO, (pH 2.5) at a concentration of 10 ronae pellucidae per pl as described in Experimental Procedures. 5 f.rI of solubtlired zonae pellucidae and 5 cl of a sperm suspension (10 sperm per ml) were added to 40 ~1 drops of Ml 99-M, the sperm were cultured for 1 hr. and ovulated eggs and 2cell embryos were added to the drops and cultured for an additional period as described in Experimental Procedures. Binding of sperm to eggs was compared after exposing sperm to 5 mM NaH*PO, (pH 2.5) alone (A. 8). solubilized zonae pellucidae from P-cell embryos (0, or solubilized zonae pellucidae from unfertilized eggs (D). Shown are photomicrographs obtained using a 10X Planochromat Phase 1 objective (Zeiss) with conventional illumination through a Phase 1 condenser (A-D). Magnification 133X. (B-D) represent the results of a single experiment. The differences seen in (A) and (6) illustrate the variation in 100% sperm binding levels between individual experiments.

embryos always sperm binding.

failed to have a significant

effect on

Effect of Individual Zona Pellucida Proteins on the Binding of Sperm to Eggs SDS-polyacrylamide gel electrophoresis was used to purify ZPl , ZP2 and ZP3 from zonae pellucidae isolated from unfertilized eggs. Approximately 600 zonae pellucidae were isolated from ovulated mouse eggs, 10% of these were radiolabeled with *?-iodosulfanilic acid, and the entire sample was prepared for and subjected to SDS-polyacrylamide gel electrophoresis as previously described (Bleil and Wassarman, 1980a, 1980b). The positions of the proteins on the gel were localized by autoradiography and the appropriate sections of the gel were excised. The proteins were then electroeluted from the gel slices and subjected to extensive dialysis, first against urea and then distilled water, prior to lyophilization. Each of the isolated zona pellucida proteins was subjected to SDS-polyacrylamide gel electrophoresis to assess its

purity; densitometer tracings of autoradiographs of these gels are shown in Figure 3. ZPl , ZP2 and ZP3, partially purified by SDS-PAGE, were each tested for their ability to prevent the binding of sperm to eggs by using the competition assay described above. Whereas ZPl and ZP2 did not have a significant effect on the extent of sperm binding, ZP3 was nearly as effective as solubilized zonae pellucidae from unfertilized eggs in reducing the number of sperm bound to eggs (Figures 4 and 5). Bovine serum albumin and two glycoproteins, bovine submaxillary mucin and transferrin. were also subjected to the same preparative procedures as the zona pellucida proteins and then tested for their ability to reduce sperm binding. In no case, even at concentrations as high as 100 pg/ml (that is, 10 times the highest concentration of purified zona pellucida protein tested), did these proteins have any effect on the binding of sperm to eggs. These results suggest that ZP3 is the component of zonae pellucidae from unfertilized eggs that possesses sperm receptor activity.

Cell 076

A. TOTAL

zp2

Figure 2. Ouantitation of Binding of Sperm to Eggs in the Presence of Various Concentrations of Solubilized Zonae Pellucidae Isolated from Unfertilized Eggs and 2-Cell Embryos These experiments were carried out as described in the legend to Figure 1, except that stock solutions of solubilized zonae pellucidae were prepared at a concentration of 40 zonae pellucidae per ~1. The average number of sperm bound per egg after 40 min of culture was determined by counting sperm tails associated with eggs in fixed preparations (see Experimental Procedures). Values for binding of sperm to eggs in the absence of solubilized zonae pellucidae varied from 50 to 120 sperm per egg in four different experiments, with an average of 96 sperm per egg representing the 100% binding level in the experiment shown (D. It should be noted that these numbers represent the number of bound sperm in the plane of focus, a value much lower than the number of sperm bound to the entire egg (see Experimental Procedures). The effect of solubilized zonae pellucidae from 2cell embryos (0) and unfertilized eggs (0) on % sperm binding is plotted as a function of zona pellucida concentration. In each case, a minimum of eight eggs were examined and included in the calculations.

Figure

3. SDS-PAGE

Analysis

of Purified

Zona

Pellucida

Proteins

ZP1, ZP2 and ZP3 were radiolabeled and purified as described in Experimental Procedures. An aliquot (approximately 1000 cpm) of the starting material (A) and of each of the purified zona pellucida proteins (B-D) was subjected to SDS-PAGE, and densitometer tracings of such a gel are shown. The yield of purified proteins varied from 65 to 80%.

On the basis of SDS-PAGE under nonreducing conditions, ZPl, ZP2 and ZP3 are present in zonae pellucidae isolated from mouse oocytes, unfertilized eggs and 2-cell embryos (Figure 6). Since sperm do not bind to fertilized eggs or embryos, we purified each of the zona pellucida proteins from zonae pellucidae isolated from 2-cell embryos, using the procedures described above, and tested their ability to prevent sperm binding to unfertilized eggs. If ZP3 indeed possessed the unfertilized eggs sperm receptor activity, we anticipated that it would have lost the ability to prevent sperm binding in the competition assay as a consequence of fertilization. This proved to be the case, since none of the three proteins

purified from zonae pellucidae isolated from 2cell embryos had any effect on the number of sperm bound to eggs in vitro (Figure 7). In the same experiment, solubilized zonae pellucidae from unfertilized eggs and ZP3 purified from egg zonae pellucidae had a marked inhibitory effect on the binding of sperm to eggs (Figure 7). It would therefore appear that ZP3 is altered as a result of fertilization such that it no longer exhibits sperm receptor activity. Binding By using hamster bind not surface of Sperm to Growing Mouse Oocytes fragments of zonae pellucidae isolated from eggs, it has been demonstrated that sperm only to the outer surface but also to the inner of zonae pellucidae (Gwatkin and Williams,

;$ammalian

Eggs

Sperm

Receptor

I 1

I 2

I 3

I 4

I 5

I 6

I 7

ZONAE

PEUUCIDAE/pL

Figure 5. Quantitation of Binding of Sperm to Eggs in the Presence of Various Concentrations of Zona Pellucida Proteins Purified from Zonae Pellucidae Isolated from Unfertilized Eggs These experiments were carried out as described in the legends to Figures 1 and 2 using purified ZPl (0). ZP2 (0) and ZP3 (A) from zonae pellucidae isolated from unfertilized eggs. There were an average of 50 sperm bound per egg at the 100% binding level 0 and, in each case, a minimum of eight eggs were examined and included in the calculations.

Figure 4. Binding of Sperm to Eggs in the Presence Proteins Purified from Zonae Pellucidae Isolated

of Zona Pellucida from Unfertilized

Eggs Zona pellucida proteins were purified as described in Experimental Procedures. Each of the lyophilized proteins was resuspended in Ml 99-M and aliquots. at a relative concentration of two zonae pellucidae per pl (calculated from the yields of each protein). were tested for their ability to reduce sperm binding to eggs as described in the legend to Figure 1. Binding of sperm to eggs was compared afler exposure of sperm to purified ZPl (A). ZP2 (B) or ZP3 0. Shown are photomicrographs obtained using a 10X Planochromat objective (Zeiss) with conventional illumination (left, 65X) or illumination through a Phase 3 condenser (right, 104X). Arrows denote 2cell embryos.

1976). This suggests that the sperm receptor is distributed throughout the zona pellucida. Consistent with such a distribution of receptor, we found that all three of the zona pellucida glycoproteins were synthesized and secreted continuously during oocyte growth; only fully grown mouse oocytes were inactive

in the synthesis and secretion of these glycoproteins (Bleil and Wassarman, 1980b). These observations suggest that oocytes at various stages of growth, with incomplete zonae pellucidae, should be able to bind sperm and that solubilized zonae pellucidae from growing oocytes should prevent sperm binding to unfertilized eggs in the competition assay described above. Oocytes at various stages of growth were isolated from the ovaries of juvenile mice as previously described (Bleil and Wassarman, 1980b) and cultured with capacitated sperm as described above. We found that sperm could bind to growing oocytes ranging from 40 to 72 pm in diameter, and that, when sperm were exposed to solubilized zonae pellucidae isolated from oocytes 55 pm in diameter, they would no longer bind to unfertilized eggs to the same extent as untreated sperm (Figure 8). The results of these experiments are consistent with the observations outlined above and indicate that sperm receptor activity is present in zonae pellucidae of growing mouse oocytes. It should be noted that, at equivalent concentrations, zonae pellucidae from growing oocytes were less effective than those from fully grown oocytes at preventing the binding of sperm to eggs. This difference is probably attributable to the smaller size of zonae pellucidae surrounding growing as compared to fully grown oocytes. For example, zonae pellucidae from fully grown oocytes are about 8 pm thick, whereas those from growing oocytes 55 pm in diam-

Cell 878

ZP1 Zp2 ZP3

ZP2

Figure 6. SDS-PAGE Analysis of 251-lodosulfanilic Acid-Labeled Zonae Pellucidae Isolated from Mouse Docytes. Unfertilized Eggs and Z-Cell Embryos Zonae pellucidae were isolated from fully grown oocytes, ovulated eggs and 2cell embryos, labeled with 251-iodosulfanilic acid, and processed for and subjected to SDSPAGE and autoradiography as described in Experimental Procedures. Shown are autoradiographs and corresponding densitometer tracings of proteins radiolabeled in zonae pellucidae isolated from oocytes (A). ovulated eggs (8) and 2-tell embryos(C). Each lane of the gel contained approximately 8000 cpm.

eter are only about sefowicz. 1978). Discussion

4 pm thick (Wassarman

and Jo-

The results of experiments presented here suggest that ZP3, a glycoprotein having an apparent molecular weight of 83,000, an isoelectric point of 3.7, and representing about 15% of the total protein of zonae pellucidae (Bleil and Wassarman, 1978, 1980a), possesses the receptor activity responsible for sperm binding to unfertilized mouse eggs. Since the purification of zona pellucida proteins involved the use of denaturants, it is possible, of course, that any sperm receptor activity associated with ZPl and/or ZP2 in intact zonae pellucidae was lost. However, the likelihood of this possibility is diminished in view of our finding that the concentration dependence of purified ZP3 in reducing the binding of sperm to eggs in vitro closely resembled that of solubilized zonae pellucidae from unfertilized eggs. In fact, there are numerous reports of the isolation of proteins involved in cell-cell recognition that retained biological activity even after exposure to denaturing conditions. These include an avian hepatic receptor which binds to glycoproteins possessing a terminal galactose that was solubilized in the presence of Triton X-l 00 (Lunney and Ashwell, 1976); contact sites A from D. discoideum that were extracted from membranes with 1% sodium desoxycholate (Huesgen and Gerisch, 1975); the sexual agglutinin from flagellae of C. reinhardii that was extracted with octyl glucoside (Adair, Hwang and Goodenough, 1979; Mesland et al., 1980); and the sperm binding factor from H. pulcherrimus that was isolated by extraction of eggs with 1 M urea (Tsuzuki et al., 1977). Finally, bacteriophage receptors that are, for the most part, specific oligosaccharide components of the bacterial cell wall have been isolated under

denaturing conditions without altering their receptor activity (Linberg, 1973). For example, M. lysodeikticus cell walls treated with 2% SDS did not lose their capacity to bind the tail tips of bacteriophage Nl (Lovett and Shockman, 1970) and, similarly, a lipoprotein fraction extracted from E. coli B with 2 M urea inactivated bacteriophages T2 and T6, and lost receptor activity only after heat or protease treatment (Michael, 1968). In view of these analogous observations it is not surprising that ZP3 retained receptor activity even after exposure to SDS and urea. It is possible, of course, that an oligomer of the 83,000 molecular weight polypeptide chain of ZP3 actually serves as the receptor in intact zonae pellucidae; this possibility is being examined in our laboratory. On the basis of SDS-PAGE analysis, ZP3 is present in zonae pellucidae isolated from 2-cell embryos. However, unlike ZP3 purified from egg zonae pellucidae, ZP3 purified from embryo zonae pellucidae does not exhibit sperm receptor activity. These observations are consistent with both the inability of sperm to bind to fertilized mouse eggs and embryos and the inability of solubilized zonae pellucidae isolated from 2-cell embryos to reduce the binding of sperm to eggs in vitro. It seems probable that ZP3 undergoes a structural change at the time of fertilization that is sufficient to abolish its receptor activity, but insufficient to alter significantly its mobility on SDS-polyacrylamide gels. Since certain lectins bind to zonae pellucidae and prevent fertilization in vitro and Yanagimachi, (Oikawa et al., 1973; Oikawa 1975), it is possible that this structural change involves the carbohydrate moiety of ZP3 rather than the polypeptide portion itself. Such a situation would be compatible with the suggestion that the binding of sperm to eggs in the sea urchin is mediated via a lectin-glycoprotein receptor interaction (Vacquier and Moy, 1977; Schmell et al., 1977; Glabe and

$7lgMammalian

Eggs

Sperm

Receptor

I A Ir 0 0 *----A-------*-

I l0

I 0 A

120 100 80 60 :
8.

Figure 7. Quantitation of Binding of Sperm to Eggs in the Presence of Various Concentrations of Zona Pellucida Proteins Purified from Zonae Pellucidae Isolated from 2-Cell Embryos These experiments were carried out as described in the legends to Figures 1 and 2 using purified ZPl (O), ZP2 (0) and ZP3 (A.) from zonae pellucidae isolated from 2-tell embryos, purified ZP3 (A) from zonae pellucidae isolated from unfertilized eggs and solubilized zonae pellucidae 0 isolated from unfertilized eggs. There were an average of 56 sperm bound per egg at the 100% binding level 0 and, in each case, a minimum of eight eggs were examined and included in the calculations.

Figure 8. Binding of Sperm to Growing Oocytes Gocytes in the Presence of Solubilized Zonae from Growing Oocytes

and to Fully Grown Pellucidae Isolated

Vacquier, 1978; Glabe, 1979). The results presented here indicate that zonae pellucidae of growing oocytes, although not completely formed, possess sperm receptor activity. Such a situation is consistent with the results of previous studies from our laboratory which demonstrated that all three zona pellucida proteins, including ZP3, are synthesized and secreted by mouse oocytes during their growth phase (Bleil and Wassarman, 1980b). In addition, we have found that ZPl , ZP2 and ZP3 are all present in zonae pellucidae of fertilized mouse eggs and embryos, suggesting that only limited proteolysis or crosslinking of zone pellucida proteins occurs as a result of fertilization. This is of interest in view of the reports that artificially activated mammalian eggs release proteases (Gwatkin et al., 1973) as well as peroxidases that can crosslink tyrosine residues (Schmell and Gulyas, 1979). After binding of mouse sperm to the zona pellucida of unfertilized eggs, the plasma membrane and outer acrosomal membrane of the sperm head fuse (ac-

Growing oocytes were collected and cultured with ovulated eggs, 2cell embryos and sperm for 10 min as described in the legend to Figure 1 and in Experimental Procedures. The binding of sperm to oocytes 40-72 pm in diameter is shown in (A) and (B). The binding of sperm to unfertilized eggs in the presence of solubilized zonae pellucidae isolated from either 2cell embryos or growing oocytes (55 pm in diameter) is compared in (0 and (D), respectively. The concentration of zonae pellucidae in the experiments shown in (C) and (D) was four zonae pellucidae per Al. Photomicrographs were obtained using a 10X Planochromat objective (Zeiss) illuminated through a Phase 3 condenser. Magnification 100X.

rosome reaction) which, in turn, exposes the inner acrosomal membrane (McRorie and Williams, 1974; Gwatkin, 1976, 1977; Hartree, 1977; Saline and Storey, 1979; Saling, Sowinski and Storey, 1979). Acrosin, the protease apparently responsible for digesting a path through the zona pellucida in front of the advancing sperm head, is associated with the inner acrosomal membrane of sperm (McRorie and Williams, 1974; Brown, Andani and Hartree, 1975; Brown and Hartree, 1978). Sperm which have undergone the acrosome reaction prior to exposure to unfertilized eggs fail to bind to the zona pellucida, suggesting that it is the plasma membrane that specifically interacts with the zona pellucidas receptor (Saling and Storey, 1979). In this connection, a species-specific sperm protein thought to be involved in the binding of sea

Cell 880

urchin sperm to eggs has been identified and characterized (Vacquier and Moy, 1977; Bellet, Vacquier and Vacquier, 1977; Schmell et al., 1977; Frazier and Glaser, 19791, and evidence for a mammalian sperm plasma membrane receptor for the zona pellucida has recently been presented (Peterson et al., 1979). Since we have found that purified ZP3 from egg zonae pellucidae exhibits sperm receptor activity in vitro, it is possible that ZP3 may induce the acrosome reaction and, as a result, prevent the binding of sperm to unfertilized eggs. We are currently investigating this possibility.
Experimental Procedures

extensive dialysis against 7 M urea followed by extensive dialysis against distilled water, and ftnally lyophilization. The lyophiiized material was stored at -20C and resuspended in Ml 99-M for use in binding studies. The yield of zona pellucida proteins was determined by analyzing gel slices and resuspended proteins in a Beckman gamma counter. in Vitro Binding Assay (Competition Assay) 5 FI of 5 mM NaH*PO, (pH 2.5) containing various concentrations of solubilized zonae pellucidae were added to 40 ~1 drops of Ml 99-M Following a 10 min incubation, 5 pl of a sperm suspension in Ml 99M (IO sperm per ml) were added to the drop and the experimental samples were cultured for 1 hr. At the end of this period, four 2ceil embryos and about ten unfertilized eggs were added to each drop in about 0.5 PI of Ml 99-M and the samples were cultured for an additional 40 min. Finally, eggs and embryos were removed from the drops and washed together in Ml 99-M using mouth-operated, large bore micropipettes in order to remove loosely attached sperm; O-5 sperm remained attached to the 2-tell embryos following this treatment. Cells were fixed in 1% glutaraldehyde made up in phosphatebuffered saline (pH 7.5) containing 0.1% polyvinylpyrroiidone-40, transferred to glass slides and examined by light microscopy. Final concentrations of the gametes were 10 sperm per ml and O-4 zonae pellucidae per PI in the experiments presented. All cell culture was carried out at 37C under paraffin oil in a humidified atmosphere of 5% CO? in air. Measurements of sperm binding were made by counting the number of sperm bound to eggs in one plane of focus with fixed preparations. Such measurements ranged from 50 to 120 sperm at the 100% binding level. which corresponds to about 2000 sperm bound to the entire zona pellucida surface. Acknowledgments This research was supported by grants from the National Institute of Child Health and Human Development and the NSF. J.D.B. is a predoctoral trainee supported by a National Research Service Award in Cell and Developmental Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received References Adair. W. S.. Hwang, C. and Goodenough, the sexual agglutinin from Cblamydomonas branes. J. Cell Biol. 82. 75a. Barros. C. mechanisms 288. U. W. (1979). Isolation of gametic flagellar memMarch 28. 1980

Collection and Cuiture of Mouse Gametes Mouse sperm were collected by mincing cauda epididymides from male CD-l mice (Charles River Breeding Laboratories) in Eartes modified medium 199 (pH 7.5; Grand Island Biological Supply) supplemented with bovine serum albumin (2 mg/ml; Sigma) and pyruvate (30 &ml: Sigma) (M199-M). Sperm were capacitated (Wolf and moue, 1978) for 1 hr in Ml 99-M at 37C in a humidified atmosphere of 5% COn in air and were then diluted appropriately for each experiment. Ovulated eggs were obtained from oviducts of female CD-l mice which had been superovulated by injection of pregnant mares serum (PMS. 5 IU; Sigma) and human chrorionic gonadotropin (HCG. 5 IU; Sigma) 48 hr later (Rafferty, 1970). Cumulus-enclosed unfertilized eggs were freed of surrounding cumulus by treatment with hyaluronidase (0.2 mg/ml in Ml99M: Sigma, Type V) for 3 min, followed by extensive washing with Ml99M. 2cell embryos were obtained as previously described (Schultz, Letourneau and Wassarman. 1979) 38 hr after mating. Fully grown oocytes (Schultz and Wassarman. 1971; Schultz et al.. 1979) and growing oocytes (Bleil and Wassarman. 1980b) were obtained and cultured in Ml 99-M as previously described. isolation of Zonae Peiiucidae Zonae pellucidae were isolated from oocytes, eggs and 2-tell embryos cultured in Ml94M using mouth-operated micropipettes as previously described (Bieil and Wassarman. 1980a). For experiments requiring solubilized zonae pellucidae, zonae pellucidae were isolated, washed thoroughly, dissolved in 5 mM NaH2P04 (pH 2.5) at a concentration of 40 zonae pellucidae per pl and stored at -20-C. Radiolabeling and Eiectrophoretic Analysis of Zonae Peiiucidae Isolated zonae pellucidae were washed thoroughly with phosphatebuffered saline (pH 7.5) containing 0.1% polyvinylpyrrolidone-40 (Sigma) and radiolabeled with the diazonium salt of Z51-iodosulfanilic acid (>lOOO Ci/mmole; New England Nuclear) in the manner previously described (Bleil and Wassarman. 198Oa, 1980b). Zonae peilucidae were radiolabeled to an average specific activity of 1800 cpm per zona pellucida (approximately 3.3 x 1 O5 cpm/r.rg protein). SDS-polyacrylamide gel eiectrophoresis of zona pellucida proteins was carried out according to the method of Laemmli (1970) using 7 or 9% separating gels as previously described (Bieil and Wasaarman. 1980a. 1980b). To purify individual zona pellucida proteins, 50 251-iodosulfanilic acid-labeled and 550 unlabeled zonae pellucidae were combined, dissolved in solubilization buffer and subjected to SDS-PAGE as previously described (Bleil and Wassarman. 1980a. 1980b). Positions of the proteins ZPl, ZP2 and ZP3 on the gel were identified by exposing Kodak XR-5 film in the presence of a Kodak X-Omatic fine intensifying screen to the gel for 18 hr at 4C. Each protein band was excised from the gel and the gel slices were subjected to electroelution in SDS-electrophoresis buffer at 1 watt constant power for 3 hr. The electroeluted material was then subjected to electrodialysis at 1 watt for 4 hr against electrophoresis buffer not containing SDS,

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