Proc. Natl. Acad. Sci. USAVol. 89, pp. 11692-11695, December 1992Cell Biology

Regulation of mouse gamete interaction by a sperm tyrosine kinase(ferlization/sperm receptor/signal twanduction/zon bidn

LISETTE LEYTON, PASCALE LEGUEN, DONNA BUNCH*, AND PATRICIA M. SALINGtDepartments of Obstetrics and Gynecology and Cell Biology, Duke University Medical Center, Durham, NC 27710

Communicated by Neal L. First, September 11, 1992

ABSTRACT A 95-kDa mouse sperm protein has beenpreviously identified as a putative receptor involved in thesperm-egg interactions that lead to fertilization. The ligad forthis receptor is the zona pellucida glycoprotein ZP3. Thisconstituent of the oocyte-specific extracellular matrix mediatesnot only sperm binding to the zona but also triggers acrosomalexocytosis. The latter, also termed the acrosome reaction, is akey regulatory event upon which fertilization is absolutelydependent. Previously, we showed that the 95-kDa protein thatbinds ZP3 is a substrate for tyrosine kinase, and its phospho-tyrosine content increases after sperm-zona peflucida binding.Here, we show the presence of protein tyrosine kinase activityin sperm plasma membranes and in electroeluted 95-kDaprotein. The tyrosine kinase activity of the isolated protein isstimulated by solubilized zona pellucida and inhibited bytyrphostin RG-50864, a membrane-permeable tyrosine kinaseinhibitor. Furthermore, tyrphostin inhibits zona-triggered ac-rosomal exocytosis in a dose-dependent manner. These findingindicate that the 95-kDa protein participates in a criticalregulatory event of gamete interaction; moreover, our exper-iments suggest that sperm protein tyrosine kinase may be anexcellent target for the control of fertility.

The zona pellucida (zp) is an oocyte-specific extracellularmatrix and, in the mouse, is composed of three well-characterized glycoproteins, ZP1, ZP2, and ZP3 (1). ZP3binds to receptors located in the sperm plasma membrane andtriggers the acrosome reaction (AR), an exocytotic eventmandatory for fertilization (2, 3). Our earlier results showthat the AR is promoted by ZP3-induced receptor aggregation(4) and that ZP3, but not ZP2, specifically recognizes a mousesperm protein of 95 kDa. This 95-kDa protein, p95sPERm,exhibits phosphorylation on tyrosine residues; the level oftyrosine phosphorylation increases 4-fold when sperm areexposed to zp (5), indicating that a protein tyrosine kinase(PTK) is activated in response to ligand binding. Thus, thesefindings raise the possibility that the transducing machineryused by the sperm to convey extracellular signals to the cellinterior may be similar to that used by somatic cells possess-ing PTK receptors. For the latter type of receptor, ligandbinding to the extracellular domain stimulates receptor ag-gregation (6, 7), which activates an intrinsic tyrosine kinaseand leads to phosphorylation of the receptor as well as otherintracellular substrates (8, 9).

Since the activation ofPI7K receptors appears essential fortheir signaling action, specific inhibitors of PTK activityconstitute important research tools in studying the mecha-nisms responsible for the resulting pleiotropic responses (10).Given suspected similarities between p95sPERM and membersof the PTK receptor family, we investigated the effect of thePTK inhibitor tyrphostin RG-50864 (11) on isolated p95SPERM(electroeluted protein), on sperm membranes, and in livingcells. The results indicate that inhibition of zp-induced tyro-

sine phosphorylation of p95sPERm parallels inhibition of zp-induced ARs, suggesting that the activity of a sperm PTKregulates gamete interaction leading to fertilization.

MATERIALS AND METHODSCollection of Gametes. Mouse sperm were obtained from

the epididymides of mature CD-1 mice (Charles River Breed-ing Laboratories). Sperm were recovered in 20 mM Tris baseat pH 7.4/150 mM NaCl, washed once by centrifugation at100 x g for 10 min, and resuspended in the same buffercontaining 2 mM EGTA for noncapacitated sperm prepara-tions or in CM [Krebs-Ringer/bicarbonate medium, supple-mented with pyruvate, lactate, glucose, and bovine serumalbumin (BSA) (12)] for capacitated sperm preparations.Sperm in CM were capacitated by incubation for 60 min at370C in 5% C02/95% air.Mouse zp was isolated from ovarian follicles of 21-day-old

CD-1 mice by the procedure described elsewhere (13). Sol-ubilization ofthe zp proteins was accomplished by incubationfor 1 hr at 60WC in 20 mM Hepes (pH 7.4) containing 150 mMNaCl and 0.4% polyvinylpyrrolidone.

Preparation of Sperm Proteins. A sperm plasma membranepreparation was obtained by demembranation of washednoncapacitated sperm using a vortex method described byBunch and Saling (14). Recovery of the membrane fractionwas performed by high-speed centrifugation in a 70.1 Ti rotor(Beckman; 247,000 x g, 40C, 35 min), and the membraneswere then resuspended in 20 mM Hepes, pH 7.4/150 mMNaCl/BSA (10 pg/ml)/100 pM Na3VO4. Capacitated spermwere not used for sperm membrane preparation because themembrane yield from these preparations is insufficient forpractical use.

Electroeluted p95SPERM was obtained as described (5).Briefly, sperm proteins from noncapacitated or capacitatedsperm were separated electrophoretically by SDS/PAGE(15). The 95-kDa region of the gel was excised; the proteinswere electroeluted, dialyzed against 8 M urea and thenagainst 20 mM Hepes, pH 7.4/150 mM NaCI, and finallyconcentrated in a Centricon-30 (Amicon). To prevent loss ofthe protein on the Amicon membrane, 10 ;lI of 1% polyvi-nylpyrrolidone was added once to the Centricon; this poly-mer is retained with the protein and will be contained in theprotein preparation hereafter. Proteinase inhibitors (1 mMbenzaniidine and 1 mM phenylmethylsulfonyl fluoride) and aphosphatase inhibitor (100 ,uM Na3VO4) were included in thebuffers used for electroelution, dialysis, and concentration ofthe protein.

Abbreviations: zp, zona pellucida; AR, acrosome reaction; PTK,protein tyrosine kinase; BSA, bovine serum albumin; CTC, chlor-tetracycline.*Present address: Reproductive/Developmental Toxicology Labo-ratory, Box 12233, National Institute of Environmental HealthScience, Research Triangle Park, NC 27709.tTo whom reprint requests should be addressed at: Box 3648, DukeUniversity Medical Center, Durham, NC 27710.

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Proc. Nadl. Acad. Sci. USA 89 (1992) 11693

For some experiments, electroeluted p95SPERM was labeledwith 125I-diiodinated Bolton-Hunter reagent (DuPont/NEN).Electroeluted p95sPERM prepared from 108 sperm (25 sAd) wasadded to the dried diiodinated ester (250 ACi of 125I-diiodinated Bolton-Hunter reagent; 4400 Ci/mmol; 1 Ci = 37GBq) and the reaction mixture was agitated for 15 min at 0C.The reaction was stopped by adding 0.5 ml of 0.2 M glycinein 20 mM Hepes, pH 7.4/150 mM NaCl for 5 min at 0C.lodinated protein was dialyzed against 20 mM Hepes, pH7.4/150 mM NaCi and concentrated in a Centricon-30.

Assay for Protein Kinase Activity. Protein kinase activitywas assessed by incubating sperm plasma membranes ob-tained from 2 x 106 sperm (2-3 ,ug of protein) or electroelutedp95SPERM from 2 x 107 sperm in a 50-jul reaction mixturecontaining 20 mM Hepes (pH 7.4), 150 mM NaCi, 2 mMCaCl2, 5 mM MnCl2, 1 jLM cAMP kinase inhibitor (16), 5 mMdithiothreitol, leupeptin (10 .g/ml), aprotinin (100 kallikreininhibitor units/ml), BSA (10 ,ug/ml), 100 juM Na3VO4, andATP. When radioactively labeled ATP was used, 30 jCi of[y-32P]ATP (4500 Ci/mmol; 2.2 ,uM ATP; ICN) was added tothe reaction mixture; otherwise, 1 mM unlabeled ATP wasused. To assess zp-stimulated phosphorylation of p95SPERM,either solubilized zp protein (120 jug/ml) or, as a control, BSA(120 ,ug/ml) was added to the reaction mixture containingelectroeluted p95SPERM (2 x 107 sperm). The reaction wasallowed to proceed for 20 min at 370C and then was termi-nated by the addition of 500 ul of ice-cold acetone. Precip-itated proteins were resuspended in SDS sample bufferwithout disulfide reducing agents and analyzed by SDS/PAGE. Electrophoretically separated proteins were trans-ferred (17) to poly(vinylidene difluoride) membrane (Immo-bilon, Millipore) and either exposed to Kodak x-ray film(when using radiolabeled ATP) or probed with murine anti-phosphotyrosine monoclonal antibody PY20 (ICN; ref. 18)for the immunodetection of tyrosine-phosphorylated pro-teins. Immunoblots were incubated with PY20 [1 j&g/ml in 20mM Tris base (pH 7.6) containing 0.15 M NaCl, 2% (vol/vol)gelatin, and 0.05% Tween 20] for 1 hr. After extensivewashing with the same buffer (without PY20), horseradishperoxidase-conjugated rabbit anti-mouse IgG (heavy andlight chains) (Kirkegaard and Perry Laboratories, Gaithers-burg, MD) was used as second antibody, which was thendetected using an enhanced chemiluminescence system(ECL, Amersham). In addition, both sperm membranes andelectroeluted p95sPERm were also assayed for kinase activityin the presence of tyrphostin RG-50864, the specific PTKinhibitor, at 80 jkM. In this case, sperm proteins wereincubated with tyrphostin for 5 min prior to the addition of zpproteins (or BSA) and ATP. Tyrphostin stock solutions were40mM in ethanol and stored at -700C. Aliquots were thawedonly once and diluted at the time of use.PTK Inhibitors and zp-Induced AR. Cauda epididymal

sperm were capacitated (60-min incubation in CM) in theabsence or in the presence of tyrphostin RG-50864 at theconcentrations indicated (0-150 ,ug/ml) and then exposed tosolubilized zp proteins (20 jsg/ml per 3 x 106 sperm) for 30min at 37TC. After the indicated incubations, acrosomalstatus was evaluated by the chlortetracycline (CTC) fluores-cence assay, which identifies three sperm patterns: (i) intactsperm (B pattern), (ii) sperm in a perifusion state (S pattern),and (iii) acrosome-reacted sperm (AR pattern) (12, 19, 20).Control sperm, incubated in CM alone, were examined inparallel using the CTC assay to quantitate the occurrence ofspontaneous ARs.

RESULTS AND DISCUSSIONWe have described (14) the preparation of mouse spermmembranes from mature noncapacitated sperm and demon-strated that this complex array of polypeptides exhibits zpreceptor activity. When this preparation was incubated with

[(y-32PJATP, examination of the resulting autoradiogram in-dicated that many membrane proteins were phosphorylated(Fig. 1A, lane -). Parallel incubation of the membranes withunlabeled ATP and subsequent probing with murine anti-phosphotyrosine monoclonal antibody PY20 (18) revealedthat p95sPERM is the only protein phosphorylated on tyrosineresidues (Fig. 1B, lane -). Examination of the proteinprofiles for replicates ofthese samples indicated that an equalamount of protein was precipitated with acetone under theconditions used (data not shown). When p95SPE" waselectroeluted from noncapacitated sperm and used to assesskinase activity, the only 32P-containing protein was the95-kDa p95sPERM (Fig. 1C, lane -) and it contained phos-photyrosine residues (Fig. iD, lane -) as detected withPY20. P1K activity, responsible for this phosphorylation,was inhibited by preincubation with 80 FAM tyrphostin, re-gardless of whether p95sPERM was assayed in situ in themembrane preparation or as isolated protein (Fig. 1 A-D,lanes +). Comparison of Fig. 1 A and B indicates that,although considerable kinase activity is blocked by tyrphos-tin, only one P1K substrate is detected in the preparation.Since tyrphostin RG-50864 has been described as a specificinhibitor for tyrosine kinases (10, 11), the results support thepossibility that multiple kinases under the regulation of aninitiating P11K are present in sperm membranes, as has beenreported in a variety of somatic cell systems controlled byknown PTK receptors (8, 21). The presence of (i) PTKactivity, (ii) tyrosine-phosphorylated 95-kDa p95sPFRm, and(iii) ZP3 receptor bioactivity in sperm membranes is consis-tent with our report (5) describing the presence of a 95-kDaprotein as the major substrate for tyrosine kinase and as aputative receptor for ZP3 in whole sperm.We have proposed (22) a working model suggesting that

ZP3 binding to the extracellular domain of p95sPERm inducesreceptor aggregation, thereby triggering the chain of eventsthat stimulates the intrinsic P1K activity of p95SPERm and

A200- V7

92.5-

68-

45_

29-

Bc

S-p95D

FIG. 1. Effect of tyrphostin RG-50864 on the tyrosine phosphor-ylation of sperm membrane proteins and electroeluted p95SPERm. (Aand B) Sperm membranes were prepared from washed noncapaci-tated cauda epididymal mouse sperm (14). Each lane contains 2-3 pgof sperm membrane protein recovered from 2 x 106 sperm. (C andD) p95sPERm was isolated by electroelution from noncapacitatedcauda epididymal sperm. Each lane contains p95SP" isolated from2 x 107 cells. Membrane proteins or isolated p95SPERM were incu-bated without (lanes -) or with (lanes +) 80 pM tyrphostin for 5 minand the kinase assay was initiated by addition of [y32P]ATP (A andC) or unlabeled ATP (B and D). After acetone precipitation, theproteins were solubilized in SDS-sample buffer and separated bySDS/PAGE. The gel was transferred to Immobilon membranes andthe blots were either exposed to x-ray films (A and C) or probed withmonoclonal antibody PY20 (B and D). Molecular mass standards(kDa) are indicated to the left of A. df, Dye front.

Cell Biology: Leyton et al.

Proc. Nat!. Acad. Sci. USA 89 (1992)

leads eventually to the AR. If this model were correct, wewould predict that (i) ligand should stimulate phosphoryla-tion of p95sPERM directly and (ii) specific PTK inhibitorsshould inhibit the phosphorylation of p95sPERm and the AR,which is the anticipated cellular response to p95SPERM acti-vation. To explore the validity of these predictions, we firstexamined p95sPEPm with respect to zp-stimulated phosphor-ylation (Fig. 2). Since zp-triggered acrosomal exocytosisoccurs only after capacitation, a final maturational processthat alters the surface characteristics and increases the flu-idity ofsperm plasma membranes (for review, see ref. 23), weutilized capacitated sperm for this experiment. Moreover, toavoid ambiguity due to other kinase activity that may beassociated with sperm membranes, we isolated p95sPERm byelectroelution from SDS gels and employed the isolatedprotein in the kinase assay. We have shown (5) that, incapacitated sperm, anti-phosphotyrosine antibodies recog-nize three proteins of 95, 75, and 52 kDa. To obtain electro-eluted p95sPERm from capacitated sperm, only the 95-kDaregion was cut out of the gel; therefore, even though theisolated p95sPERM may not represent a completely purifiedprotein, the presence of the other phosphotyrosine-containing proteins in the preparation was excluded.

In the absence of zp proteins, p95sPERM isolated fromcapacitated sperm exhibited incorporation of 32p from[t-32P]ATP (Fig. 2, lane 1) that was reduced substantially bypreexposure to tyrphostin (Fig. 2, lane 2). Incubation of thep95sPERM preparation with zp proteins increased the level ofphosphorylation significantly (Fig. 2, lane 3); such ligand-stimulated kinase activity was reduced essentially to unstim-ulated levels by prior incubation with tyrphostin (Fig. 2, lane4), confirming tyrosine phosphorylation. When only solubi-lized zp, without any sperm protein, was used in the kinaseassay, no 32p incorporation into protein was detected (data

1 2 3 4

20i -

92.5- _

68-

.0

c100c- !

CZ)CL

o 60 r

w 40 _i-oD _Co 4Q

EE 20 _

0_ZPT

Fluorescence Pattern*B KS 7-AR

+

FIG. 3. Effect of tyrphostin RG-50864 on the zp-stimulated AR.Cauda epididymal sperm (3 x 106 sperm per ml) were capacitated (5)in the presence (+) or absence (-) of80gM tyrphostin (T). Aliquotsfrom each sample were incubated for 30 min in the absence (-) orpresence (+) of solubilized zp proteins (ZP; 20 4g/ml). The AR wasmonitored using the CTC fluorescence assay (12, 19, 20). Threesperm populations are identified with this method: sperm with intactplasma membranes and acrosomes (B pattern), sperm in a perifusionstate (S pattern), and fully acrosome-reacted sperm (AR pattern).Replicate experiments were conducted for each set of incubationconditions and at least 100 sperm were scored for each condition perexperiment. Data represent the mean obtained from five experi-ments. Sample standard deviation for all values did not exceed 8%.

not shown). For the results shown in Fig. 2, the same amountofsperm protein was used in each sample; however, to verifythat the same amount of protein was precipitated by ice-coldacetone under all conditions tested, in a parallel experiment,isolated p95sPERM was radiolabeled with 15Ii-iodinated Bol-ton-Hunter reagent before use in the kinase assay. For thisexperiment, unlabeled ATP was used. The iodinated proteinswere detected by both autoradiography and by direct ycounting. Both methods indicated that equivalent amounts ofsperm protein were precipitated in all cases. For example, theacetone-precipitated cpm were 3.5 x 10' (lane 1), 3.7 x 10'(lane 2), 3.7 x 105 (lane 3), and 3.8 x 1io (lane 4).We then examined the effect of these same ligand and

inhibitor treatments with respect to the zp-stimulated cellularresponse of sperm, the AR. The histogram shown in Fig. 3

60

5045-

ar 40* 30

df- 20

10zP - + +

Tyrph +

FIG. 2. Effect of tyrphostin RG-50864 on zp-stimulated phos-phorylation of p95 P '. Cauda epididymal sperm (3 x 106 spermper ml) were capacitated as described (5). Sperm proteins wereseparated electrophoretically on an 8% polyacrylamide gel, the95-kDa region of the gel was cut out, and the proteins were electro-eluted to obtain isolated p95sPERm. Kinase activity was assessed with(+) or without (-) prior incubation with 80 AM tyrphostin (Tyrph)and in the presence (+) or absence (-) ofsolubilized zp proteins (ZP;120 pg/ml). Each lane contains p95sPERm isolated from 2 x 107sperm. The resulting autoradiogram was obtained after exposing32P-labeled proteins to x-ray film for 1 hr with intensifying screens at4eC. Molecular mass markers (kDa) are indicated to the left.

W0 40 80 120Tyrphostin, ,uM

160 200

FiG. 4. Effect of increasing tyrphostin RG-50864 concentrationon the zp-stimulated AR. Sperm (3 x 106 sperm per ml) werecapacitated in the absence or in the presence of tyrphostin at theconcentrations indicated, solubilized zp (20 pg/ml) was added, andthe preparation was incubated for 30 min. ARs were monitored usingthe CTC fluorescence assay (12, 19, 20); the percent AR representsthe mean ± sample standard deviation calculated from valuesobtained in three replicate experiments. In the absence of solubilizedzp, control samples incubated in CM alone or in CM plus 100 ,uMtyrphostin displayed spontaneous ARs of 7 ± 1%.

+

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Proc. Natl. Acad. Sci. USA 89 (1992) 11695

Table 1. Effect of tyrphostin RG-50864 on sperm binding to zpTotal no. Bound sperm,of eggs no. per egg

Control sperm 92 20 ± 7Tyrphostin-treated sperm 94 28 ± 11Cauda epididymal mouse sperm (3 x 106 sperm per ml) were

capacitated in CM (5) without or with 80 ,AM tyrphostin RG-50864.Cumulus masses were obtained from the oviducts of superovulatedfemale mice and incubated inCM containing 0.05% hyaluronidase for5-10 min for recovery of cumulus-free zp-intact eggs. Capacitatedsperm were added to eggs at a final concentration of 2 x 105 spermper ml and incubated at 370C. After 15 min, eggs with adherent spermwere recovered using a wide-bore (two egg diameters) micropipetteand washed to remove loosely associated sperm. Eggs with boundsperm were fixed in 2.5% (vol/vol) glutaraldehyde, mounted onslides, and viewed with phase-contrast optics, and the number ofsperm bound to each egg was counted. These data summarize theresults (mean ± SD) obtained in three replicate experiments.

displays the distribution of the sperm population in eachsample according to acrosomal status, determined using theCTC assay (12, 19, 20). In Fig. 3, the CTC patterns observedindicated that, in the absence ofzp [bar ZP(-) and T(-)], themajority of sperm displayed intact acrosomes (B pattern,solid bar). In contrast, treatment of the same sample with zpat 20 pg/ml [bar ZP(+) and T(-)] induced the AR in -55%of the population (AR pattern, open bar). Tyrphostin pre-treatment of the sample abolished the stimulatory activity ofthe zp proteins, and the sperm behaved essentially the sameas control samples: 74% of the sperm were acrosome-intact[B pattern, bar ZP(+) and T(+)]. The effect of tyrphostin onthe zp-stimulated response is concentration-dependent (Fig.4) and could not be attributed to interference with sperm-zpinteraction, since capacitated sperm that were pretreatedwith tyrphostin bound to zp as well as control nontreatedsperm (Table 1). Our analysis also suggests that tyrphostindoes not interfere with the capacitation process since, whenit is added to capacitated sperm (at 100 ,uM) immediatelybefore zp addition, tyrphostin inhibits ARs almost as well aswhen it was added prior to capacitation: 16 + 5% AR vs. 8± 2% AR, respectively.

Similar to these results with tyrphostin, we found thatpreexposure of sperm to another membrane-permeable PTKinhibitor, genistein, also blocked zp-induced ARs. We ob-served that, at concentrations as low as 20 ,tg/ml, genisteinblocked 100% of the zp-stimulated ARs (data not shown).Both tyrphostin and genistein share specificity in terms ofPTK inhibition without effect on serine/threonine kinases(24, 25), but their specificities diverge beyond that aspect.Our finding that both reagents were potent inhibitors of thezp-triggered AR strengthens the conclusion that phosphory-lation on tyrosine residues is the relevant reaction for theseevents of gamete interaction.

In the present experiments, we found that the PTK activityof electroeluted p95SPERM is stimulated by its ligand, zp, andinhibited by tyrphostin, a PTK inhibitor. Both types ofmodulation were observed with isolated p95SPERm, consis-tent with the idea that ligand and inhibitor mediate theirrespective effects directly on p95sPERM. These modificationsin the PTK activity ofthe p95sPERM preparation paralleled thephysiological response of sperm cells to zp and tyrphostin:stimulation led to acrosomal exocytosis, whereas inhibitionblocked the response. Thus, these data suggest that a recep-tor-effector signaling system similar to that in somatic cellsresponsive to various hormones and growth factors may alsoexist in mammalian sperm. An understanding of the molec-ular mechanisms by which p95sPERM might control the oc-

currence of the AR will be essential for the purposefulregulation of the fertilization process.

In general, the binding of a ligand to its respective PTKreceptor elicits an array of cellular events, including phos-phorylation of the receptor and other cellular substrates, ionfluxes, inositol phospholipid hydrolysis, and altered geneexpression (26). However, discerning the mechanism bywhich these individual effects are integrated to achieveoverall cellularresponse remains unresolved. We believe thatspermatozoa may represent a valuable system in this regard,since the transcriptional activity of the genome ceases in thehighly condensed spermatozoon nucleus (27). Therefore,ligand-stimulated activation in this system is likely to beachieved by preexisting cellular components or their precur-sors rather than via altered gene expression.We are grateful to Dr. A. B. Schreiber for providing tyrphostin

RG-50864 and to Ms. A. Robinson for expert technical assistance.Drs. D. Burks, K. Burridge, M. Caron, and A. Quest providedconstructive criticism that improved the manuscript greatly. Thisresearch was supported by grants from the National Institutes ofHealth. Fellowship support was generously provided for P.L. byEuropean Molecular Biology Organization and The Lalor Founda-tions and for L.L. by the Rockefeller Foundation and the Andrew W.Mellon Foundation. P.M.S. was supported by a Research CareerDevelopment Award from the National Institutes of Health.

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