95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding

Cell, Vol. 57, 1123-1130, June 30, 1989, Copyright 0 1989 by Cell Press

95 kd Sperm Proteins Bind ZP3 and Serve as Tyrosine Kinase Substrates in Response to Zona Binding Lisette Leyton* and Patricia Saling’t Department of Obstetrics and Gynecology t Department of Cell Biology Duke University Medical Center Durham, North Carolina 27710 l

In the mouse, the zona pellucida (ZP) glycoprotein ZP3 both binds intact sperm and induces acrosomal exocytosis. The subsequent signaling pathway(s) is still uncertain, but G-like proteins have been implicated. By analogy with other signal transduction mechanisms, we examined anti-phosphotyrosine antibody reactivity in mouse sperm. Antibodies reacted with thn?e proteins of 52,75, and 95 kd. Indirect immunofluorescence localized reactivity to the acrosomal region of the sperm head. The 52 kd and 75 kd phosphoproteins are detected only in capacitated sperm, whereas the 95 kd protein is detected in both fresh and capacitated sperm. For the 95 kd protein, the level of immunoreactivity is not related to sperm motility but is enhanced by both capacitation and sperm interaction with solubilized ZP proteins. In addition, binding of radiolabeled whole ZP or purified ZP3 to blots of separated sperm proteins identified two ZP binding proteins of 95 W and 42 kd. 95 kd sperm proteins that bind to ZP3 also react with anti-phosphotyrosine antibodles (in a ZP concentration-dependent manner), supporting the idea that the same 95 W sperm protein serves as a ZP3 receptor and as a tyroslne kinase substrate. These findings and our evidence on acrosome reaction triggering via sperm receptor aggregation suggest that a 95 kd protein in the sperm plasma membrane is aggregated by ZP3, which stimulates tyrosine kinase activity leading to acrosomal exocytosis. Introduction Mammalian sperm undergo an obligate exocytotic event during fertilization. This process, termed the acrosome reaction (AR), involves fusion of the plasma and outer acrosomal membranes with consequent liberation of the acrosomal contents. It has been shown for many mammalian species that the AR is induced by the zona pellucida (ZP), an extracellular matrix surrounding the oocyte (Bleil and Wassarman, 1983; Cherr et al., 1986; O’Rand and Fisher, 1987; Cross et al., 1988; Florman and First, 1988). Extensive investigation using mouse gametes suggests that the AR is induced specifically by one of the ZP glycoproteins, ZP3, which binds to the plasma membrane overlying the acrosome through its O-linked oligosaccharide groups (Florman and Wassarman, 1985). We have provided evidence recently that the polypeptide chain of ZP3 plays an important role in AR triggering by cross-linking the mouse sperm plasma membrane

components recognized by ZP3 (Leyton and Saling, 1989). These membrane components, the sperm’s receptor(s) for ZP3, are not yet well defined (reviewed in Saling, 1989) and little is known about the intracellular signals that are transmitted after ligand-receptor interaction. Recent work, however, indicates that proteins similar to guanine nucleotide binding inhibitory proteins (Gi -like proteins) are present in mouse sperm (Kopf et al., 1986) and have been implicated as participants in the physiological cascade leading to the AR (Endo et al., 1987, 1988). For many receptors, including several that are known to be coupled to G proteins, phosphorylation by a variety of endogenous protein kinases represents an important mode of regulation (Sibley and Lefkowitz, 1985; Huganir et al., 1986). Furthermore, the receptors for several hormones and growth factors (e.g., insulin, EGF, and PDGF) are themselves tyrosine-specific protein kinases that are activated by ligand binding (Hunter and Cooper, 1985; Yarden and Ullrich, 1988). An unusual aspect of these signal transduction systems is receptor autophosphorylation which, in some cases, depends upon receptor aggregation (O’Brien et al., 1987). Considering the similarity between sperm and these other systems with regard to ligand-mediated receptor aggregation as initiator of the signaling process, we were eager to examine whether other common characteristics also exist. In this paper, we have used anti-phosphotyrosine (anti-P.Tyr) antibodies to detect proteins that serve as substrates for tyrosine kinases. We have identified a 95 kd protein of the acrosomal region of the sperm head that is phosphorylated on tyrosine residues; this protein’s level of phosphorylation increased following capacitation and following exposure to solubilized ZP proteins. In addition, ‘2%ZP3 specifically recognizes a 95 kd sperm protein. Finally, 95 kd sperm proteins that bind to solubilized ZP also react with anti-!?Tyr antibodies, consistent with the suggestion that ZP3 receptor activity and tyrosine kinase substrate activity are found in the same protein. Together with other recent findings (Leyton and Saling, 1989), these results suggest that a 95 kd receptor protein in the sperm plasma membrane binds ZP3, and the resulting receptor aggregation stimulates tyrosine kinase activity leading to the biochemical pathway that results in the acrosome reaction. Results Interaction of Labeled ZP Proteins with Sperm We have used a Western blot assay to look for sperm proteins that bind ZP3. For this assay, it was necessary to optimize the conditions for sperm preparation to preserve the plasma membrane overlying the acrosome. This is the region of the cell that first interacts with the ZP, but it is shed during the acrosome reaction, and spontaneous ARs can be readily triggered during sample preparation. Mouse sperm exposed briefly to Ca2+ in the absence of capacitating conditions (TN plus 1.7 mM Ca2+ for 30 min)

Cell 1124

ABC

DEF

zoo97-

Lmci

6843-

29dfFigure 1. Autoradiographic teins before (A) and after

Analysis Following SDS-PAGE (6) Purification via Electroelution

of ZP Pro-

After structurally intact ZP were labeled with ‘s51-Bolton-Hunter reagent, the proteins were solubilized in SB, electrophoresed under nonreducing conditions, and the bands were resolved after 20 min of exposure of the gel to a Kodak AR-5 X-ray film at 4% (A). The three ZP proteins were identified on the basis of electrophoretic mobility, cut out of the gel, electroeluted from the polyacrylamide, and re-electrophoresed separately: ZPI, 200 kd; ZP2, 120 kd; and ZP3, 83 kd (B). The sharp horizontal lines in (A) indicate where the gel was cut for ZPl, ZP2, and ZP3 recovery; the faint band in (B) that appears just below the 45 kd marker corresponds to the dye front of the gel. Molecular weight markers (Pharmacia LKB) are indicated to the right of (B).

demonstrate minimal levels of spontaneous ARs. Such preparations also display maximal ZP binding activity regardless of capacitation status (Saling and Storey, 1979; Lee and Storey, 1985). Sperm prepared using these con-

A

B

C

Figure 3. Autoradiographic Phosphotyrosine Antibody of Capacitation

Analysis with Mouse

of the Reactivity of AntiSperm Proteins as a Function

Sperm were recovered from the cauda epididymis in TN (lanes A and D) or in CM (lanes B and E) and dissolved immediately in SB, or were incubated in CM for 80 min (lanes C and F) and then dissolved in SB. After electrophoresis on SDS-PAGE and transfer to nitrocellulose, separated sperm proteins were incubated with anti-phosphotyrosine antibody (unblocked; lanes A-C) or anti-phosphotyrosine antibody previously blocked with 40 mM o-phospho-DL-tyrosine for 1 hr (blocked, lanes D-F). The presence of phosphorylated proteins was detected by incubation of both blots with ‘a51-goat anti-rabbit IgG. Molecular weight markers (~10~~; prestained standards from BRL) are indicated to the left of lane A; df indicates the dye front.

ditions were dissolved in SDS and the proteins were separated by SDS-PAGE. Western blots of these gels were probed directly with 12%whole ZP or 1251-ZP3. The probes are shown in Figure 1, which presents the autoradiographic analysis following SDS-PAGE of mouse ZP proteins before (Figure 1A) and after (Figure 16) purification via electroelution. When blots containing separated sperm proteins were probed directly with 1251-whale ZP or 12VZP3 (Figure 2), two sperm proteins, at 95 kd and 42 kd, were prominent; faint reactivity was observed with low M, components running near the dye front of the gel. 1251ZP2 did not bind to either the 95 kd or the 42 kd protein, nor to any other sperm protein, when the cells were prepared in this manner.

554336df-

Figure 2. Autoradiographic Mouse Sperm

Analysis

of

ZP3

Binding

Proteins

in

Sperm incubated in TN plus Ca*+ (1.7 mM) for 30 min were dissolved in SB and the proteins were separated by SDS-PAGE. After transfer of the proteins to nitrocellulose, the sperm proteins were probed with (A) ‘*sl-whole ZP, (6) ‘*?-ZP2, or (C) t*sl-ZP3. Both t*sl-whole ZP and r2sl-ZP3 bind to sperm proteins at 95 kd and 42 kd (arrowheads), whereas t*sl-ZP2 does not bind to these proteins. All ZP probes bind faintly to unresolved material running at or close to the dye front. Molecular weight markers (~10~~; Pharmacia LKB) are indicated to the left of lane. A; df indicates the dye front.

Sperm Proteins Phosphorylated on Tyrosine Residues Proteins phosphorylated on tyrosine residues are present in mouse sperm, and the level of phosphorylation appears to increase markedly following capacitation (Figure 3). Fresh, uncapacitated sperm, either in TN or CM, demonstrated a low level of phosphorylation on tyrosine residues in a 95 kd protein (Figure 3, lanes a and b) compared with sperm that were capacitated for 80 min in CM (lane c). Comparison of the counts found in the 95 kd protein bands from these samples revealed that lanes a and b in Figure 3 differ insignificantly (3% difference), whereas in lane c, a 1.8-fold increase was observed. If the same immunoblot is exposed to film for a longer interval, the capacitated sperm sample can be seen to consist of three tyrosinephosphorylated proteins, at 95 kd, 75 kd, and 52 kd. An example of this may be seen in Figure 4, lane b. Non-

The Acrosome 1125

Reaction

and Signal Transduction

ABC

ABCD

DEF

E

FG

H

20097684329-

29df -

df -

Figure 4. Western Blot Analysis of the Reactivity of Anti-Phosphotyrosine Antibody with Mouse Sperm Proteins as a Function of ZPBinding Ability Versus Motility Mouse sperm were incubated in TN plus 1.7 mM Ca*+ at 3PC (lanes A and D), CM at 37% (lanes B and E), or TN plus 25 uM La3+ at 4% (lanes C and F). After 60 min, sperm proteins were dissolved in SB, electrophoresed, transferred to nitrocellulose. and incubated with antiphosphotyrosine antibody (unblocked, lanes A-C; blocked, lanes D-F). Subsequent incubation with rz51-goat anti-rabbit IgG was used to detect anti-phosphotyrosine antibody reactivity. Molecular weight markers (x10-? prestained standards from BRL) are indicated to the left of lane A; df indicates the dye front.

capacitated cells were never observed to display the two lower M, phosphoproteins, regardless of the length of autoradiographic exposure. The reactive bands appeared specific for l?Tyr since the same sperm samples, probed with anti-myr antibody that had been incubated previously with 40 mM o-phosphoDL-tyrosine, displayed no immunoreactivity (Figures 3, 4, 5, and 7, lanes d-f). Additional evidence that the 52,75, and 95 kd proteins contain PTyr residues was obtained when we observed that these three proteins continue to be recognized by anti-PTyr antibodies following alkaline hydrolysis of capacitated sperm samples blotted onto lmmobilon nylon sheets following SDS-PAGE, as described by Kamps and Sefton (1989) (data not shown). Soon after sperm are liberated from the cauda epididymis, two major changes occur: one is the acquired ability to bind to ZP and the second is motility. To distinguish whether the observed increased phosphorylation on tyrosine (Figure 3, lanes a and b versus lane c) was due to the acquisition of motility or ZP binding activity, we have manipulated the extracellular ionic conditions. Both motility and ZP binding require extracellular calcium. However, in the case of sperm motility, transport of Ca2+ into the cell is also required and the addition of La3+ inhibits motility rapidly (Heffner et al., 1980). In contrast, for ZP binding, Ca2+ transport is unnecessary and La3+ is not inhibitory but will substitute for Ca2+ (Saling, 1982). The level of phosphorylation on tyrosine, therefore, was compared between sperm recovered after a 60 min incubation in TN buffer containing either Ca2+ (1.7 mM) at 37°C or La3+ (25 uM) at 4%; neither of these conditions capacitate sperm. Reactivity of anti-PFyr antibody with these cells was compared with that of fully capacitated sperm, incubated in

Figure 5. Effect of Solubilized ZP Glycoproteins tion of Sperm Proteins on Tyrosine Residues

on the Phosphoryla-

Sperm recovered in TN (lanes A and E) or in CM (lanes Band F) were solubilized immediately in SB; sperm recovered in CM were capacitated and then incubated without (lanes C and G) or with solubilized ZP (1 ZP/nI; lanes D and H) for 30 min and dissolved in SB. The samples were then electrophoresed and transferred to nitrocellulose. A-D, protein reactivity with unblocked anti-phosphotyrosine antibody; E-H, nitrocellulose incubation with blocked anti-phosphotyrosine antibody. The antibody reactivity was followed with 1251-goat anti-rabbit IgG. The arrow indicates the 95 kd sperm protein that is tyrosine-phosphory lated in response to capacitation and ZP binding. Molecular weight markers (~10~~; prestained standards from BRL) are indicated to the left of lane A; df indicates the dye front.

CM for 60 min. The results presented in Figure 4 show that capacitated sperm (lane b) expressed three proteins (95, 75, and 52 kd) recognized by anti-F!Tyr antibody, whereas the 95 kd protein alone was detected in both the Ca2+and the La3+-incubated sperm (lanes a and c, respectively). The relative level of phosphorylation in the La+3incubated sperm, estimated by counting the 95 kd protein band cut from the blot, did not differ from that of the other preparations, despite their lack of motility. Solubilized whole ZP is known to induce ARs in capacitated sperm, and ZP3 has been identified as the active agent (Bleil and Wassarman, 1983). We have recently provided evidence that ZP3 triggers this event by aggregating sperm receptors in a mechanism presumed to be analogous to that for receptor-effector coupling in many hormone- and growth factor-responsive cells (Leyton and Saling, 1989). To examine whether ligand (ZP3) binding in this system promotes phosphorylation on tyrosine residues, capacitated sperm were incubated for 30 min in the absence or presence of solubilized ZP In the presence of ZP, three phosphorylated proteins of 95, 75, and 52 kd were found as in capacitated sperm without exposure to ZP, but only the anti-PTyr antibody’s reactivity with the 95 kd protein is enhanced (Figure 5). The amount of f?Tyr reactivity appeared to double in response to ZP binding, estimated from a 1.9-fold increase in counts when the 95 kd bands of lanes c and d (in Figure 5) were compared. For the image presented in Figure 5, the film was exposed briefly to appreciate maximally the differences in F?Tyr levels observed among the samples. Longer exposure of the same blot demonstrated the three phosphoproteins characteristic of capacitated preparations (of

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Figure 6. Indirect lmmunofluorescence Analysis of the Distribution of Anti-Phosphotyrosine Antibody on Mouse Sperm Capacitated sperm were fixed in formaldehyde, and incubated with anti-phosphotyrosine antibody (unblocked, A-D; blocked, E-F). Paired phase contrast and epifluorescence micrographs indicate that only unblocked anti-phosphotyrosine antibody localized to the acrosomal region of the sperm head. Faint staining of the midpiece can be observed in all epifluorescence micrographs (6, D, and F).

sizes 95, 75, and 52 kd) in lanes c and d of Figure 5 (data not shown). lmmunofluorescent Localization of Phosphotyrosine Residues Indirect immunofluorescence was used to localize the anti-PTyr antibody on fixed sperm. The antibody reacted specifically with the sperm head in the acrosomal region (Figures 6b and 6d). A punctate appearance of fluorescence was often observed. Weak midpiece fluorescence caused by the second antibody, but not sperm head fluorescence, can be seen in ceils incubated with antil?Tyr antibody that had been blocked previously with o-phosphotyrosine (Figure 6f). In preparations fixed at T = 0, very few cells (<50/o) displayed acrosomal fluorescence. Capacitation appeared to increase the proportion of labeled cells to approximately 15% and, after exposure to solubilized ZP, approximately 35% of the population displayed acrosomal fluorescence. Relationship between the 95 M ZP3 Binding Protein and the 95 kd Tyrosine Kinase Substrate A dot blot assay was used to examine the relationship of the 95 kd sperm proteins identified earlier. By analogy with findings for receptors of various growth factors, we were interested in investigating the question: Does the same 95 kd sperm protein serve as a ZP3 receptor and as a tyrosine kinase substrate in response to ZP binding? The results of our initial test of this possibility, which used a dot blot to assay whether 95 kd proteins from capacitated sperm that bind ZP are also recognized by anti-myr antibodies, are consistent with the idea that both activities are found in the same 95 kd sperm protein. Increasing concentrations of solubilized ZP proteins (O-100 ZP/well) were blotted onto a nitrocellulose sheet. After washing, a constant amount of sperm protein was applied to the dot blots. For this purpose, capacitated sperm proteins were separated on an SDS gel, and material from the 95 kd region was electroeluted and then dialyzed to remove the SDS (see Experimental Procedures).

Table 1. Reactivity ZP Binding Sperm

Experimental

specific

cpm

Antibody

A

BCDEFG

0 -a --

0 +

50 ++

0 +

0

0

0

23

with 95 kd

conditions

ZPlwell 95 kd sperm protein anti-P.Tyr Ab Radioactivity

of Anti-Phosphotyrosine Proteins

50 + +

100 + +

50 + +b

452

0

bound 164

Nitrocellulose was spotted with solubilized ZP or buffer, as indicated, and blocked with 1% BSA. After washing, a 10 ul aliquot of sperm protein or of buffer was applied, as indicated. The proteins were electroeluted from the 95 kd region of an SDS gel used to separate proteins of capacitated mouse sperm and then dialyzed to remove the SDS. After washing, and blocking with 1% BSA, wells were incubated with a murine monoclonal anti-phosphotyrosine antibody (PY12, 2 uglml; Glenneyet al., 1966), as indicated. After washing, the dot blot apparatus was disassembled and the complete nitrocellulose sheet was incubated with rabbit anti-mouse lg, followed by ls%goat anti-rabbit IgG. Bound radioactivity was determined by counting each individual spot directly in a gamma counter. Specific counts are defined as cpm greater than those bound in column G, where phosphotyrosine-blocked antiphosphotyrosine antibody was used; the actual number of counts bound in this case was 65. a - indicates addition of the relevant buffer only; + indicates the addition of the reagent specified. b Hapten-blocked anti-phosphotyrosine antibody was used.

The presence of F!Tyr in the ZP bound material was probed by reactivity with anti-PTyr antibodies. Compared with the absence of ZP (Table 1, column D), anti-PTyr antibody reactivity increased 6-fold in the presence of 50 ZP (column E) and 20-fold in the presence of 100 ZP (column F). No specific counts were associated with a variety of controls, including the absence of anti-PTyr antibody (Table 1, column B) or of sperm protein (column C) or when the antimyr antibody is preincubated with PTyr (column G). These results indicate that sperm proteins with sizes in the 95 kd region that interact with ZP in a concentration-dependent manner also react with anti-l?Tyr antibodies. The findings

The Acrosome 1127

Reaction

and Signal

Transduction

abc

def

200-

4s

--

I

Figure 7. Western Blot Analysis of the Effect of M42 MAb on AntiPhosphotyrosine Antibody’s Reactivity with Mouse Sperm Sperm shown in lanes a and d were recovered in TN and dissolved immediately in SB. Other samples, shown in lanes b, c, e, and f, were incubated in CM. After 30 min of incubation, M42 IgG was added at a final concentration of 200 uglml to samples in lanes c and f. All CMincubated samples were dissolved in SB after 60 min. After electrophoresis and transfer to nitrocellulose. blots were incubated with antiphosphotyrosine antibody (unblocked, lanes a-c; blocked, lanes d-f). Subsequent incubation with 1251-goat anti-rabbit IgG was used to detect anti-phosphotyrosine antibody reactivity. Molecular weight markers (~10~~; prestained standards from BRL) are indicated to the left of lane A; df indicates the dye front.

suggest that the 95 kd protein recognized by ZP3 and the 95 kd protein that serves as a substrate for tyrosine kinase may be the same protein. Effect of M42 MAb on Tyrosine Phosphorylation The extent of phosphorylation on tyrosine residues as a consequence of sperm exposure to M42 MAb was also examined. This antibody recognizes a 200/220 kd protein located in the acrosomal region of the sperm head (Saling and Lakoski, 1985) and specifically inhibits ZP3-induced ARs in mouse sperm (Leyton et al., 1989). M42 MAb (200 ug IgGlml) was added at the midpoint of the capacitation incubation. Sperm capacitated in the absence or presence of M42 MAb demonstrated the same three tyrosinephosphorylated proteins, but the intensity of the signal in the M42 MAb-treated sperm is decreased markedly in all three proteins when compared with parallel samples that were not exposed to M42 MAb (Figure 7). Direct counting of the three reactive protein bands in lanes band c (Figure 7) revealed a e-fold decrease in the PTyr signal intensity as a consequence of M42 MAb treatment. Discussion Definition of the sperm receptor(s) for ZP3 has remained elusive for several years. Various candidates for this function have been suggested (e.g., galactosyltransferase, fucosyltransferase, fucose/fucose sulfate, and a trypsin inhibitor-sensitive site), but none of these activities have been isolated from the sperm plasma membrane and characterized. Recent work using boar sperm indicates that proacrosin is both a fucose binding and ZP binding protein (Jones and Brown, 1987; Jones et al., 1988; TopferPetersen and Henschen, 1987) and is probably employed

during secondary binding between the acrosome reacting sperm and ZP2 (Bleil et al., 1988; Saling, 1989). In this paper, we report the results of using directly labeled ZP3 in Western blots to identify ZP3 binding proteins of sperm. Using either purified ZP3 or whole ZR we have identified two major ZP binding proteins, at 95 kd and 42 kd. The relationship of the 42 kd to the 95 kd polypeptide remains to be clarified. It may be pertinent that an anti-sperm monoclonal antibody (MAb) has identified a 95 kd human sperm protein thought to participate in sperm-ZP binding (Moore et al., 1987). No other reports have utilized individual ZP proteins to probe electrophoretically separated sperm proteins. However, O’Rand et al. (1985) conducted similar experiments using whole ZP as a probe and reported that mouse ZP recognize small (14-18 kd) sperm components. The weakly reactive material running close to the dye front in our blots (Figure 2) may represent the low M, material reported by these other investigators. Substantial differences in the preparation of both the ZP probe and the sperm cells could also account for the different results obtained. Preparation of bioactive radiolabeled ZP proteins can be difficult; on several occasions, we have inadvertantly destroyed ZP reactivity during the labeling procedure. When this occurred, no reactivity on blots was observed, even with prolonged exposure of the X-ray film. Sperm preparation, also, is known to affect reactivity with a ZP probe; the identification of proacrosin as a ZP binding protein was aided by using acid extracts of sperm to prevent autoactivation of the zymogen (Jones et al., 1988). In the experiments reported here, we employed great care to preserve the labile plasma membrane overlying the acrosomal region of the mouse sperm head since this is the presumed iocation of primary receptors for the ZP However, when we prepared an acid extract of mouse sperm, 1251-ZP2 bound to a distinct set of sperm proteins with M, unrelated to those sperm proteins that 12%ZP3 bound (Leyton and Saling, unpublished data). Considering ZP3’s binding activity (Florman and Wassarman, 1985) its consequent AR triggering by the aggregation of receptors for ZP3 in the sperm plasma membrane (Leyton and Saling, 1989) and the finding that the cell surface receptors for various extracellular ligands are tyrosine kinases (Yarden and Ullrich, 1988) we wondered whether triggering of the AR might also involve elevated tyrosine kinase activity. In the studies presented here, several PTyr-containing proteins have been identified, with the most prominent migrating at 95 kd. Although we have not yet demonstrated directly that this protein is the same as the 95 kd protein that binds ZP3, two independent lines of evidence argue that this may be the case. It is striking that the anti-PTyr antibody localized to the acrosomal region, the area of the sperm shown to bind ZP3 (Bleil and Wassarman, 1986). Furthermore, we have shown that sperm proteins with M, in the 95 kd region bind to solubilized ZP immobilized on nitrocellulose and that these sperm proteins react in a ZP-dependent manner with antiPTyr antibodies (Table 1). It is possible that two or more different 95 kd proteins associate following electroelution and that these are responsible for the distinct activities.

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However, given the precedent of autophosphorylating tyrosine kinases serving as receptors for various growth factors, it seems more likely that ZP3 receptor activity and tyrosine kinase substrate activity are contained in the same 95 kd sperm protein. The pattern of phosphorylation on tyrosine residues appears to reflect the physiological state of the sperm population. Three distinct polypeptides were identified using the anti-PTyr antibodies, at 52, 75, and 95 kd. The 95 kd protein demonstrated PTyr residues under all of the experimental conditions examined, although its level of phosphorylation appears to increase as a function of both capacitation and ZP binding, but not as a function of motility. In contrast, the 75 kd and 52 kd proteins are detected only in capacitated sperm and may represent capacitation-specific markers. On immunoblots, a 2-fold increase in the level of antiF!Tyr reactivity was detected in the 95 kd protein of capacitated sperm following exposure to ZP proteins when compared with capacitated sperm in their absence. Paralleling our findings, it is interesting to note that, in sea urchin sperm, three phosphoproteins of 100, 75, and 52 kd increase their level of phosphorylation markedly when intact sperm are incubated with egg peptides (Bentleyet al., 1987). The phosphoamino acids involved in this system have not yet been reported. lmmunofluorescence observations on fixed mouse sperm revealed that the acrosomal region of the sperm head contains P.Tyr residues and that increased numbers of cells bear anti-P.Tyr label after ZP exposure. The level of increase in P.Tyr detected in the blot following stimulation with ZP proteins is low compared with the increase that occurs in other systems such as the EGF receptor in response to ligand binding (Yarden and Ullrich, 1988). However, this may reflect the inevitably heterogeneous population of sperm, only a fraction of which can participate in fertilization events, together with a relatively high background of P.Tyr due to the presence of spontaneous ARs occurring within the population. An increase in plasma membrane fluidity occurs during capacitation, which is the sperm’s final maturation phase after release from the male (see Yanagimachi, 1988, for a recent review of this topic), In our current model for sperm-ZP interaction, we suggest that multimeric aggregates formed in the sperm’s plasma membrane constitute the functional units responsible for AR triggering. Formation of such functional aggregates could also occur spontaneously as a result of increased fluidity during capacitation. We view ZP3 as a specific accelerator of aggregate formation by cross-linking at least some of its constituents. As a result of the findings presented here, and by analogy with other systems shown to rely on receptor aggregation for activation, we also suggest that tyrosine kinase activity may be stimulated by receptor aggregation, independent of ligand (ZP3) binding. If this is so, tyrosine kinase activity would be expected to increase with capacitation and to increase further following exposure to ZP3; the latter increase would occur as a consequence of increased aggregate formation rather than being solely the result of ligand binding.

In previous work, we developed a MAb that blocks the mouse sperm’s AR induced by ZP3 (Saling and Lakoski, 1985; Saling, 1986). This MAb, M42, blocks ARs only if it is present prior to the initiation of the AR cascade; once the signals for this event are initiated, the MAb is no longer inhibitory (Leyton et al., 1989). According to our working hypothesis, the current results with M42 MAb can be interpreted to indicate that, under capacitating conditions, spontaneous aggregation of receptors occurs, and this aggregation can be blocked by M42 MAb. If the M42 protein is required for formation of the functional unit that transduces extracellular signals, its inability to participate in aggregate formation by steric hindrance due to MAb binding would prevent ARs. Once aggregates are formed, however, it would be predicted that the MAb could no longer exert an inhibitory effect. The experimental evidence presented here is consistent with this prediction. This novel view of sperm-ZP interaction reconciles much of the information that has emerged in recent years on this topic (see Saling, 1989, for review). It also generates many new questions, including the identity and valency of the aggregate’s constituents and the possible kinase activity of the 95 kd ZP3 receptor. Work is presently directed toward these issues. Experimental Collection

Procedures of Gametes

Mouse sperm were obtained by rupturing the excised caudae epididymides of mature CD-I mice (Charles River Breeding, Co., Wilmington, MA) in 20 mM Tris/ldO mM NaCl buffer (pH 7.4; TN buffer), washed once by centrifugation at 100 x g for 10 min, and resuspended in CM (Krebs Ringer-bicarbonate medium supplemented with pyruvate, lactate, glucose, and bovine serum albumin [BSA]; Saling and Storey, 1979). Capacitation was achieved by incubating sperm (approximately IO6 sperm/ml in CM) for 60 min at 37oC in 5% COP in air. Mouse ZP were isolated from ovarian follicles of PI-day-old CD-l mice following the procedure described previously (Leyton et al., 1969). i!P were solubilized in TN buffer containing 1.7 mM CaC12 and 0.4% polyvinyl-pyrrolidone by incubation for 1 hr at 60%. Structurally intact ZP were radiolabeled with 1*51-Bolton-Hunter reagent (New England Nuclear) at O°C (Bleil and Wassarman, 1963); individual ZP proteins were then purified by electroelution and subsequently dialyzed to remove the SDS, as described by Florman et al. (1964). Immunological Reagents Anti-Phosphotyrosine Antibodies Two different preparations of polyclonal anti-P.Tyr antibodies and three different preparations of monoclonal anti-P.Tyr antibodies were used, with identical results. Dr. Patricia Maness (Department of Biochemistry, UNC, Chapel Hill) donated a polyclonal anti-P.Tyr antibody, produced in rabbits as described by Wang (1965); the IgG fraction was affinity purified against P.Tyr. It was used in immunoblots at a final concentration of 0.5 pglml and in immunofluorescence at 1 W/ml. Dr. Shelton Earp (Lineberger Cancer Center, UNC, Chapel Hill) provided another polyclonal anti-P.Tyr antibody that was produced in rabbits as described (Kamps and Sefton, 1966; McCune and Earp, submitted). This affinity purified preparation was used at a final concentration of 2 pg/ml for immunoblots. Both of the polyclonal preparations were detected by subsequent incubation with msl-goat anti-rabbit IgG (see below). Dr. John Glenney (Markey Cancer Center, University of Kentucky, Lexington) generously supplied three different murine monoclonal anti-P.Tyr antibodies, PY12, PY20, and PY69, produced as described (Glenney et al., 1966). Each was used for blots at a concentration of 2 pglml. For detection of the murine monoclonal anti-P.Tyr antibodies, a two-step method was used. Following incubation with mouse MAb, blots were incubated with rabbit anti-mouse lg (serum fraction, 1:lOO

The Acrosome 1129

Reaction

and Signal

Transduction

dilution, Cappel Laboratories, Malvern, PA). After washing, blots were incubated subsequently with lz51-goat anti-rabbit IgG (see below). ‘z52SI.Goat Ant/-Rabbit IgG Affinity purified goat anti-rabbit IgG, iodinated using lodogen, was donated by Dr. Keith Burridge (Department of Cell Biology and Anatomy, UNC, Chapel Hill). The preparation was used at lo6 cpmlml with blots. M42 Monocfonal Antibody M42 MAb was isolated from spent tissue culture medium by affinity chromatography on goat anti-mouse IgG coupled to agarose (Lakoski et al., 1988), dialyzed against PBS, and used at a final concentration of 200 Nglml. SDS-Polyacrylamide Gel Electmphoresis and lmmunoblot of Sperm Pmteins Sperm were incubated as described below and then prepared for SDS-PAGE (Laemmli, 1970). Samples of sperm recovered promptly after dispersal of the cells from the epididymis in TN buffer were considered as time zero (T = 0) samples in TN. The remaining sperm were then centrifuged gently (100 x g, for 10 min) and resuspended in CM. Another sample was withdrawn, representing T = 0 in CM. The remainder of the sample was capacitated and then incubated in the absence or the presence of solubilized ZP (1 ZPIpI) or M42 MAb (ZOO pglml). Other treatments included incubation for 30-80 min in TN buffer containing either 1.7 mM CaCI, or 25 KM LaCI, at 4°C or 37oC, as indicated. Treated sperm were centrifuged (12 000 x g, for 5 min) and resuspended in an equal volume of 2x sample buffer (SB; lx SB = 62.5 mM Tris at pH 6.8, 2% SDS, 10% glycerol) at a concentration of 3 x lo6 sperm/20 PI of SB. The suspension was vortexed, boiled for 5 min, and loaded (20 PI/lane) onto either 7.5% or 10% polyacrylamidebisacrylamide gels. Prestained molecular weight standards (see Figures 3, 4. 6, and 7) were purchased from BRL Life Technologies, Inc. (Gaithersburg, MD). Nonstained standards were purchased from Pharmacia LKB (Piscataway, NJ; see Figures 1 and 2). After SDSPAGE, proteins were transferred to nitrocellulose (Schleicher and Schuell. Keene, NH), according to the method of Towbin et al. (1979). Detection of ZP Binding Proteins Sperm recovered in TN buffer were incubated for 30 min at 37oC in TN containing 1.7 mM CaC12. Sperm were pelleted, resuspended in an equal volume of 2x SB. and applied to a 7.5% gel. After transfer of the proteins to nitrocellulose, the sheet was blocked with 3% non-fat milk in PBS containing IO mM NaNa. Three strips of nitrocellulose containing parallel sperm samples were incubated with 106 cpmlml of 1251labeled ZP protein, either whole ZP, ZP2, or ZP3. in PBS/l0 mM NaN3 for 3 hr at room temperature. After thorough washing in PBS containing 0.5% Nonidet P-40 and drying, the nitrocellulose strips were exposed to Kodak X-AR film with intensifying screens at -7oOC. lmmunodefecfion of RTyr Residues Sperm, recovered in TN buffer, were incubated using various conditions as indicated above. Sperm proteins were separated in 10% gels, and transferred to nitrocellulose sheets. The nonspecific reactivity of the sheet was blocked with 2% cold water fish skin gelatin in 50 mM Tris, 0.1 M NaCI. 0.5% Tween 20, and 10 mM NaN3 (TNT-G) for l-2 hr at room temperature. The nitrocellulose was then incubated for 3 hr at room temperature with rabbit anti-PTyr antibody in TN containing 0.5% Tween 20 (TNT). Samples were run in duplicate: one piece of nitrocellulose was incubated with anti-myr antibody and the other with the same antibody preparation that had been incubated previously with 40 mM o-phospho-DL-tyrosine for 1 hr. After washing (five times for 15 min each time) with TNT, the nitrocellulose sheets were incubated with 1n51-goat anti-rabbit IgG (lo6 cpmlml) for 1 hr at room temperature. Washed nitrocellulose sheets were dried and exposed to X-ray film with intensifying screens at -7OOC. To estimate relative levels of phosphorylation on tyrosine in different samples, the regions of the nitrocellulose blots containing the protein bands of interest were cut out and counted directly in a Beckman gamma counter. Dot Blot Assay for Detection of Phosphotyrosine Residues in Sperm Bound to ZP Solubilized ZP (10 ZPlpl) in TN buffer containing 1.7 mM CaCI, and 0.4% PVP wasapplied, as indicated in Table 1, to unblocked nitrocellulose with wells defined by a Bio-Rad dot blot apparatus. After incubation for 30 min, wells were washed with TN containing 1.7 mM CaC12

and 1% BSA for 30 min. After rinsing with TNT, 10 ~1 of 95 kd electroeluted sperm protein (see below) was applied as indicated in Table 1. After 30 min, the wells were washed with TNT and then with TN containing 1.7 mM CaC12 and 1% BSA, each for 30 min. Murine monoclonal anti-PTyr antibody (PY12,2 pg/ml; Glenney et al., 1988) suspended in TNT containing 0.1% BSA was applied to the nitrocellulose wells, as indicated in Table 1. After incubation overnight at 4OC, wells were washed with TNTcontaining 0.1% BSAfor 30 min. The dot blot apparatus was then disassembled and the entire nitrocellulose sheet was incubated with TNT-G containing 4% BSA and then processed for detection of !?Tyr residues by incubation with rabbit anti-mouse lg (1:lOO dilution of serum; Cappell Labs) and then with ‘%I-goat anti-rabbit IgG (lo6 cpmlml); each of these incubations was conducted for 45 min and was followed by thorough washing with TNT containing 0.1% BSA. Except for incubation with PY12, which occurred at 4oC, all other manipulations were conducted at room temperature. After the nitrocellulose sheet was dried, individual dots were counted directly in a gamma counter. Preparation of 95 kd Sperm Proteins Sperm (approximately 10Yml) were capacitated by incubation for 90 min in CM containing 100 WM Na3V04 at TPC in 5% COPS% air. Previous experiments indicated that the addition of 100 WM Na3V0, does not interfere with either capacitation or sperm binding to the ZP. A total of 3 x IO’ capacitated sperm were solubilized in SB and the proteins were separated on a 10% gel. Electrophoresis and all subsequent manipulations of the sperm proteins were conducted in the presence of 100 PM Na3V04. The 95 kd region was cut out of the gel, and the proteins were electroeluted. The preparation was dialyzed first against 8 M urea to remove the SDS and then against TN containing 1.7 mM CaC12 to remove the urea. Indirect lmmunofluorescence Sperm obtained at T = 0 in TN and after capacitation (with and without exposure to ZP proteins), were fixed in PBS containing 2% formaldehyde (Ultrapure-TEM Grade, Tousimis Research Corp., Rockville, MD) for 10 min. After centrifugation (1300 x g, for 15 min), sperm were incubated in PBS containing 50 mM glycine for 30 min, pelleted, and resuspended in PBS. Sperm were plated (5 x lo4 sperm/well) on E-well slides (Roboz Surgical Instrument Co., Inc.. Washington, DC). After drying at 3pC, slides were rinsed in PBS and then blocked with PBS containing 5% goat serum plus 2% BSAfor 30-60 min. Anti-PTyr antibody, either unblocked or blocked with o-phosphotyrosine, was added to the wells and incubated for 3 hr at room temperature in a moist chamber. After thorough washing in PBS, FITC-goat anti-rabbit IgG (1:200; Kierkegaard and Perry Laboratories, Inc., Gaithersburg, MD) was added and incubated as above for 1 hr. After washing as before, slides were rinsed in water and mounted in PBS containing 70% glycerol and 2.5% 1,4-diazobicyclic(2,2,2)octane (pH 8.6). Samples were maintained at 4’C until examination with phase contrast and epifluorescence optics. Unless indicated otherwise, all reagents were the highest grade possible and were purchased from either Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific Co. (Philadelphia, PA). Acknowledgments The authors are grateful to Drs. S. Earp, J. Glenney, P Maness, and B. McCune for their generous gifts of the anti-PTyr antibodies used in this study. We also wish to thank Dr. K. Burridge, who provided not only reagents for this work, but also constructive comments on the manuscript. Ms. A. Robinson skillfully assisted with many technical aspects of this work, including a constant supply of isolated zonae pellucidae for our use. We are also grateful to Dr. C. Carron and Ms. D. O’DellBunch for their thoughtful advice and constructive review of this manuscript. The work was supported by grants from the National Institutes of Health and the Andrew W. Mellon Foundation. L. L. received generous support from The Rotary Foundation of Rotary International. 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

January

30, 1989; revised

April 10, 1989.

Cell 1130

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