Biology of mammalian fertilization: role of the zona pellucida

Biology of mammalian fertilization: role of the

zona pellucida.

J Dean

J Clin Invest. 1992;89(4):1055-1059. https://doi.org/10.1172/JCI115684.

Research Article

Find the latest version:

http://jci.me/115684/pdf

Perspectives

Biology

of Mammalian Fertilization:

Role

of the

Zona

Pellucida

Jurrien Dean

Laboratory ofCellular andDevelopmental Biology,National InstituteofDiabetes andDigestiveandKidneyDiseases, NationalInstitutesofHealth,Bethesda, Maryland20892

Introduction

In mammals, aseries ofcarefully orchestratedevents culmi-nate inthefusion ofasperm andeggtoformaone-cell zygote, theobligatoryprecursor of all cells in theembryo.The overall rate of fertilization in humans has contributed tosustained

increasesin the world population and added urgencytothe

needtodevelopnew,effective

contraceptive

agents. For some, however,thesuccess rateis

considerably lower,

and thereare

millionsof infertile

couples

intheUnitedStates. Dramatic ad-vancesin reproductive biologyhave

provided

somerelief for

these individuals

through

the

development

of in vitro

fertiliza-tion techniques. Further progress requires additional

under-standing ofthemolecular basisfor normal fertilization andour

willingnesstoexploit thisknowledgetofacilitate desired

preg-nanciesorpreventunwantedones.These

joint

desirestohave greater controlover ourreproductive destinyhas ledto an

in-creased interest in the macromolecules involved in

fertiliza-tion.

The zona pellucida, an extracellular matrix

surrounding

thefemalegamete,contains the

primary

and

secondary

sperm receptors and playsa pivotal rolein sperm-egg interactions. Althoughmillions ofspermare

deposited

in thefemale

repro-ductivetract,fewerthan 100

approach

the ovulated egg in the

oviduct, and

normally only

one

successfully

penetrates

throughthezona

pellucida

tofusewith theplasmamembrane

oftheovum.

Immediately

following fertilization,

theplasma

membrane and zonapellucida aremodifiedto prevent

poly-spermy and, perhaps, to

provide

additional

resiliency

to the

zona sothatitcanprotect the

preimplantation embryo.

Inter-actionsbetween the sperm and egg appeartobe

relatively

spe-cies-specific and research has focusedonthemaleandfemale macromoleculesinvolved. This

perspective

willconcentrate on

recentadvances in ourknowledge ofthe zonapellucidagenes andproteins ofthe mouse, anexperimental animalthathas providedmuchofour currentunderstanding ofthemolecular

details offertilization.

Fertilization:

aprecis

Afterejaculation intothefemalereproductive tract, sperm

un-dergoapoorly understoodmaturation process(capacitation)

Addressreprint requests to Dr. Jurrien Dean, Laboratory of Cellular and DevelopmentalBiology,National Institute of Diabetes and Diges-tive andKidneyDiseases, Building 6, Room B 1-26, NationalInstitutes

of Health, Bethesda, MD 20892.

Receivedforpublication31 December 1991 and in revisedform 21 January1992.

The Journalof Clinical Investigation, Inc. Volume 89,April 1992, 1055-1059

before approaching the ovulated egg in the oviduct (Fig. 1 A). Motilesperm passthrough theenvelopingcumulusoophorus,

which is composed of aglycosylaminoglycan matrixand cu-mulus cells. They then bind to the zona pellucida that surroundsthe mammalianegg(1, 2).The mouse and human zonaepellucidaeare composed of threemajor glycoproteins,

ZP1,ZP2,andZP3.Solubilizedzonaepellucidae from

unfertil-izedmouse eggs (but not fromtwo-cellembryos) can inhibit spermbindingtoovulatedeggs(3).This sperm-receptor activ-ity of the zona has beenascribedto aclass of 3.9-kD 0-linked

oligosaccharidesonZP3(4).ZP2 has beenimplicatedas a

sec-ondarysperm receptor that binds spermonlyafter the induc-tion of the spermacrosomereaction (5).

The acrosomeisamembrane-bound organelle anteriorly located inthe headofthe sperm. It hasbeen proposed that the

bindingof sperm to ZP3 induces asignaltransduction across the sperm membrane by aggregatinga sperm-specific 95-kD

protein with tyrosine kinase activity(6, 7). Thisleads to the

acrosomereaction (8) in whichthe spermplasma membrane fuses withthe outer acrosomalmembrane,resultingin the

exo-cytosis ofthe acrosomal contents(Fig.1 B). Thelyticenzymes

(e.g., acrosin, glycosidases) thatare released, aswellas some thatremainassociated withtheinneracrosomal membrane,

suchasacrosin,appear tofacilitatepassageofthemotilesperm

throughthe zonapellucida.

Anumberofspermsurfacemacromolecules have been

pro-posedas zonaadhesion molecules,e.g.,serine proteinasesand

,B-l1,4-galactosyltransferase.

More recentcandidatesinclude a

95,000-Dprotein with tyrosine kinase activityand a 56,000-D

proteinisolatedbyitsabilitytobindZP3(for review see [9]). It

islikely thatmorethan oneinteractionis responsible for the

binding ofsperm tothezona,bothinitiallyandduring

penetra-tion. Some of thespermligandspresent onthesurface of the

inneracrosomalmembrane (e.g., proacrosin and PH-20) may

becomeavailable forzonabindingonlyafterthe sperm

acro-somereaction.These latter molecules may be particularly im-portantfor successfulpenetration of the zona pellucida.

Immediatelyafterfertilizationthere aretwo major changes that prevent polyspermy: a rapid electrical depolarization of the eggplasmamembranethat blocks additional sperm in the

perivitellinespacefromfusingwith the egg(10),and biochemi-calmodifications of the zona pellucida. These latter changes occursecondarilyto thefusion ofcytoplasmic cortical granules with the egg plasma membrane, and the subsequent discharge

ofthegranules'enzymatic contents into the perivitelline space. The releaseof proteinases and glycosidasesmodifiesthe zona

A

Zona Pellucida

Sperm

Sperm Binding

B

Acrosome IntactSperm

Tail

Acrosome Reacted Sperm

block to polyspermy (11), and ZP3 loses both its ability to

inducethe acrosomereaction anditssperm receptor activity (8, 12).

The zona

pellucida

proteins

Thezonapellucida proteinsare

synthesized,

glycosylated,and

sulfatedin oocytesbefore their secretiontoform thezona

ma-trix that surrounds the

growing

oocyte,ovulatedegg andearly embryo.

Structure. Mouse ZP1 (185-200 kD)isadisulfide-linked dimer which has yettobe characterizedin moleculardetail. Theprimary amino acidsequences ofmouse ZP2 (120-140 kD) and ZP3 (83kD) havebeendeduced fromthenucleicacid sequence oftheircognate genes(see

below). Although

the

pep-tide backbones ofthe humanZP2 andZP3are_{similar in length}

to those of their mouse

homologues

(see

below),

the mature

humanproteins are smaller, ZP1

(90-1

10

kD),

ZP2 (64-76 kD),and ZP3 (57-73 kD) (13),

presumably

duetodifferences in glycosylation. Recent

experiments

with immunological

probes haveidentifiedtwoisoforms ofhuman

ZP3, ZP3H

and

ZP3L (14),but whether these represent different posttranscrip-tional modificationsofa

single

ZP3 gene

product

orthe

pres-enceofanadditionalgeneremainstobe determined.

Biosynthesis.

Thereisno zona

pellucida

surrounding

rest-ingmouseoocytes,andzona

protein synthesis

isfirst detected when oocytesentertheirtwoweekgrowthphase.

Investigations

inanumberof specieshave concluded that thezonapellucida proteinsaresynthesizedin oocytes and secretedintothezona

matrix, although somestudieshave also identifiedzona pro-teins ingranulosacells. More

recently,

transcripts

of thesingle

copymouseZp-2(15)andZp-3

(16)

genes have been detected

onlyingrowingoocytes and the

biosynthesis

of the ZP2 and ZP3proteins is, likewise,restrictedtooocytes(17, 18).Atits

peak in

_30-35-,um

diameter oocytes, zona

_{protein synthesis}

represents 7-8% of totalprotein synthesis. Zonaproduction

declines in

fully

grown oocytes and is absent in ovulated eggs. Once

incorporated

intothe extracellular

_matrix,

thezona

pel-lucida proteinshavealonghalf-life

(>

100

h)

(19, 20).

Figure

1. Mammalianfertilization.

(A)

The

binding

of spermtothezona

_pellucida

surrounding

theovulated_{egg induces the} spermacrosomereaction. The release of

lytic

enzymes fromtheacrosomeand the

forward

_motility

of thespermpermit pene-tration ofthezona

_pellucida.

After fusion with the

_{egg's plasma membrane,}

the_sperm

entersthe

cytoplasm

andformsthe male

pronucleusof theonecellzygote.The

fe-male

_pronucleus,

formedatthesame

_time,

contains the female

_haploid

genome.

Fol-lowing fertilization,

thezona

pellucida

is

biochemically

modifiedtoprevent addi-tionalspermfrom

_binding

or

_penetrating

thezona.

_(B)

Theacrosome,a

lysosomal-likestructureon_{the anterior head of} sperm,containsaninner andouter

mem-brane that fuse

_during

theacrosome

reac-tion. This results in the release of_lytic

en-zymesthatare

_important

_{for the}

penetra-tionofthezona

_{pellucida by}

the_sperm.

The

biosynthetic

pathways

of ZP2and ZP3 have been

in-vestigated using

radioactive precursors and in vitro culture.

The ZP2core

protein

hasa molecular

weight

of

76,373,

de-duced from its nucleic acid sequence

(15).

Six

_complex-type

N-linked side chains andanundeterminednumberof

0-linked

oligosaccharides

areattachedtothecoreproteinandthe

ma-ture 120-140-kD

glycoprotein

is secreted and

_incorporated

into the extracellularzona

pellucida

(21). The ZP3 protein is

first detected in the oocyteas a

44,000-D protein

towhich three

orfour

complex-type

N-linked

carbohydrate

sidechains are

added. The further attachment of0-linkedsugarsresultsin the

production

of the mature secreted 83-kD ZP3

glycoprotein

(18).

Theintracellulartransport patternsand mechanisms of

secretion for theseproteinshave yettobedelineated. Zona

pellucida

genes

Structure

of

themousegenes. Mouse

Zp-2

and

Zp-3,

each

sin-gle

copy genes, arelocatedonchromosomes 7 and

5,

respec-tively (22).

Mouse

Zp-2

contains 18exons

(Fig. 2)

that range in size from 45to 190

bp

andareseparated by17 introns(81to

1,490

bp).

The gene spans 12.1

kbp

of DNA. Zp-2 is

tran-scribed andprocessedintoa2,201-ntmRNA with very short 5'

(30 nt)

and 3'

(32 nt)

untranslatedregions.The detection ofa

2.4-kbtranscriptinoocyte RNA suggeststhat ZP2mRNA

con-tainsa

poly(A)

tail of 200nt.ZP2 mRNA hasa

single

open

readingframe initiatedat anATGthat encodesa

polypeptide

of713 amino acidswithamolecular weightof80,217. The first

34aminoacidsrepresentasignal peptidethatdirects secretion

and, following

cleavage,theresultantcorepolypeptide(76,373

D)

is incorporated into the extracellular matrix. The ZP2 aminoacidsequencecontainssevenpossibleN-linked

glycosyl-ation sites (Asn-X-Ser/Thr)andmore than 100potential 0-linkedglycosylationsites

(15).

The8.6-kbpmouseZp-3gene iscomprisedof eightexons

(Fig. 2)

rangingin sizefrom92to338bpand hasintrons whose

lengthsarebetween 125 and2,320 bp (23). The 1,317-nt ZP3 mRNAhas short 5'(29 nt)and 3'(16 nt) untranslatedregions.

A

Mouse

Zp-2

Gene

111213141516 12 3 4 5 6 7 89 _{10 \\// \117} 18

Human ZP2Gene

1 2 3 4 5 67 89 10 11 1213141,5617 1819

HH]HHHE+Hl11 I oil I I 1

5 11 1 1 Al

_~~~~~11

I Nil I I I I

B

MouseZp-3Gene

1 2 34 5 67

5' *

1 1

11 I

i

HumanZP3 Gene

5,

2 3 4 5

1 I ad

1 l Al

embedded within the consensus AATAAA polyadenylation signal. Short untranslated regions are characteristic of both ZP2(mouseandhuman)and ZP3(mouse, human,and ham-ster) mRNAs. It isnotclear whatrole,ifany,these short

un-translatedregions playingeneexpression,norwhethertheyare

important forprocessing ZP2 andZP3transcripts. The poly-peptidededucedfromthe

single

open

reading

frame ofmouse

ZP3 mRNAhasamolecularweight of 46,307andconsists of

424amino acids (24).Thereleaseofapredicted 22-amino acid signal peptide wouldresult in the secretion ofa43,943-D

pro-tein, consistent withthe

reported

44-kDZP3core

protein (18).

Althoughthereisno overall

similarity

inthe amino acid

sequencesofZP2 andZP3, they shareatleastone common

structural motif.Each hasavery

hydrophobic region consisting

of23 and 26aminoacids,

respectively,

nearthe

carboxyl

ter-minus(15, 24).Hydrophobic domains liketheseare

typically

presentinmembrane-spanningdomains ofproteins. The hy-drophobic regionsin ZP2and ZP3mayplayanimportantrole in theintracellulartrafficking ofthese secretedproteinsorin

their interactionsin theextracellularmatrix.Thecarboxyl

ter-minal hydrophobic domain is present in mouse (ZP2 and ZP3), human (ZP2 andZP3), hamster(ZP3),and arecently described rabbitzonaprotein (15, 24-27;andLiang,L.F., and J.Dean,manuscriptinpreparation).

Conservation

of

the zonagenes. Thegenes encodingZP2 andZP3areconservedamongmammals.Using

cross-hybrid-izationwithmouse nucleic acidsequences as acriterion, the

degreeofconservation ofZp-3 isvariable with the rat,dog,

cow, andhumanzonagenesbeingmorerelatedto mouse than thepigandrabbitgenes(28).The human homologue ofZp-2 andthe human and hamster homologues ofZp-3 have been

characterized. The human ZP2gene(Fig. 2) iscomposedof 19

exons(onemorethan themouse) whose nucleic acidsequence

is 70% thesame asthatof itsmouse counterpart(Liang,L. F.,

andJ.Dean,manuscript in preparation). Theencodedproteins

are60%identical,although the human protein has745amino acids compared with 713 amino acids in mouse ZP2. The mouse, human, and hamster ZP3 genescontain eight exons

2 KB

Figure 2.Genomic loci ofhuman and mouse

Zp-2andZp-3.(A)ComparisonofmouseZp-2 (18 exons)andhumanZP2(19 exons)genes

with transcription units of12.1 and 13kbp, respectively (15;andLiang,L.F., andJ.Dean, manuscriptinpreparation). (B) Alignment of

67 8 mouseZp-3 (8.6 kbp) and human ZP3 genes

I (18.3 kbp), eachof whichcontaineightexons | | | 3 (23, 25).

each. Thecodingsequencesofthemouseand humangenesare

74%thesame,andtheyencode 424 amino acidpeptidesthat

are67%identical (25).The hamstergeneencodesa422-amino

acidproteinthat is81% identicalto mouseZP3(26).The ZP3

mRNAinmouseoocytesis1.5kb(includinga200-ntpoly(A) tail)andisindistinguishableinsize fromthatofratandrabbit

ZP3 mRNAs (24). Takentogether, these datasuggestthat the overallstructureofthezonagenesandtheirgeneproductsare

conservedamongmammals.

Expression ofthe zona genes

NeitherZP2norZP3transcriptsarepresentinrestingmouse oocytes (10-15

rim).

However, once oocyte growth com-mences, increasingamounts ofZP2and ZP3 transcriptsare

detected. Maximumlevelsofthetranscripts (1,000 fg ofZP2 and 400fg of ZP3)are presentinoocytesthatare 50,um in diameter, representing 1 and 0.4%, respectively, of total

poly(A)+RNA.The molarratio ofZP2 and ZP3transcripts is

- 2: 1, andthis ratio is maintainedduring the growth phase of

oocytedevelopment (15, 16).Astheoocytereachesits full size (75-80

_jAm),

theamountofZP2and ZP3transcripts declines, and, in ovulatedeggs, theamountofthesetwotranscripts is

<5%of its peaklevel(15).ZP2 andZP3mRNAs inovulated

eggs are 200 nt shorter than

they

are in

growing

oocytes. This decrease corresponds to the estimated length of their

poly(A)tails(15)andisverysimilartothedecreaseobserved

for

_f3-actin

anda-tubulinmRNAsinmouse eggs(29).Thishas

ledtothehypothesisthat thezona mRNAs,likeother maternal messages, undergorapiddeadenylation and degradation

dur-ing meiotic maturationand ovulation.

Mechanismof

oocyte-specific

expression. The expression of mouseZp-2 and Zp-3 is restrictedto growing oocytes and is not

detectedin othermousetissues(15, 16,24,28). Thecoordinate

transcription of the zona genes is restrictedtothe two-week

growth phase of oogenesis and serves both as a molecular

markerforoocyte growthand differentiation, and as a

mecha-nisms that restrict gene expression to the female germline, and, although severalpreviously characterized transcription factors

are presentinoocytes,theirtarget geneshave yet to be

deter-mined.

Iftheregulation ofzona geneexpression isunder the

con-trolofshared regulatory factors, the DNA-binding sites ofthese factorsmaybeevolutionarily conservedamongmammals. A

comparison ofthe first 300 bpupstreamofthe initiationsite of

mouse and humanZP2 genes reveals that they are 70% identi-cal.Thereisa TATAAboxatposition -31 bpand aCCAAT

box atposition -65 bpinthemousegene;theseelementsare present atcomparable positionsinthe human gene (15;and Liang, L. F., and J. Dean, manuscript in preparation). In con-trast,comparison ofthe5'flankingsequencesofthe mouse and human ZP3 genesindicatesno longstretchesofsequence

iden-tity. The TATAA box at-29 bp in the mouse Zp-3 is also present in the human homologue. Neither ZP3 gene has an

identifiableCCAAT box (23, 25).

Interestingly, close examination ofthe first 300 bp up-streamof fourzonagenes(mouse Zp-2 and Zp-3;human ZP2

and ZP3) revealed five short (4-12 bp), conservedDNA se-quences(elements I,IIA,IIB,III, and IV), locatedat compara-ble distances from the transcription initiation site (30). We have demonstrated that one ofthese sequences, element IV

(positioned

200

bp

upstream fromthe TATAA

box),

is

nec-essary and sufficient for reporter gene expression driven by mouse Zp-2 and Zp-3 promoters microinjected into oocytes

(30).Oligonucleotides containing elementIVfrom eitherZp-2 orZp-3 formDNA-proteincomplexesofidenticalmobility in

gel retardation assaysusingextractsofoocytesbut not other

tissues(Fig. 3).

Thecenterof elementIVfrom both the humanand mouse

Zp-2 and Zp-3genescontainsthemotifCANNTG which has beenidentifiedas aconsensussequence(31) forthebinding of basichelix-loop-helix (HLH) transcription factors(32). These

factors generally bind DNA ashomo- or heterodimers. The HLHdomain isrequired forthe protein-protein interactions involved indimerization, while the adjacent basic domain

in-teracts directly with DNA. HLH

transcription

factors have been showntobeinvolved in theregulation oftranscription of

severaltissuespecificgenes

(for review,

see[33]).

Thetranscription factor-DNA complex identifiedin these

experiments maycontain one or more

previously

character-ized basic HLH proteins, or may represent the first known

example ofan

oocyte-specific

transcription

factor. The isola-tionandbiochemicalcharacterization ofthezonagene activa-tion factors willnotonly

provide

informationabouttheir

struc-tures,but will

permit

investigation

of their

putative

functions

as regulators ofzona pellucida gene

transcription

in mouse

oocytes. The concept ofa commontranscription factor(s)

bind-ingtoelementIVofthe promoterregion ofthemouseZp-2

andZp-3genes thatparticipatesin thecoordinateand tissue-specific expression ofthezonapellucidagenes isanappealing hypothesis whichcan now be tested. Therelativelylow level of reporter geneactivity in thesestudies (30)aswellasin

trans-genicmice(34)suggests that additionalregulatoryfactors may

beimportant forinvivo levels ofzonageneactivity.

1. Abbreviationusedinthispaper:HLH, helix-loop-helix.

Ovary Follicle Cells Testes

-Self Mut -Self Mut

!

i|u

MwtJ

'W.E,

-Self Mut ES B K L S T

--&&~~~~S

M

:

Figure3. Ovary-specific binding ofatranscription factortoa zona promoterelement. Gelmobility shiftassaysusing radioactively

la-belledoligonucleotides containing element IV frommouseZp-2.

Arrowindicates the oocyte-specific DNA-protein complexpresentin the directbindingassayswithbothZp-2andZp-3element IV

oligo-nucleotides (30).Ovary, lane 1,mouse[32P]Zp-2oligonucleotide

containing element IV reacted with 2-3ggof ovarianextract;lane 2,

aslaneI but competed with 50-fold molarexcessof unlabeledmouse

Zp-2oligonucleotide; lane 3,aslane 2 but competed withaZp-2

oligonucleotide containinga6-bp mutation in element IV. Follicle

cells,asabove but with 2-3ggfollicle cellprotein. Testes,asabove

but with 2-3jigtestesprotein. Tissues, lanes 1-6, mouse[32P]Zp-2

oligonucleotide reacted with 2-3jsg extractof embryonicstemcells (lane 1), brain (lane 2), kidney (lane 3), liver (lane 4), spleen (lane 5), andthymus (lane 6).

Conclusions

The elucidation of the primarystructureof thezonaproteins

hasprovideda meansfor determiningtheprotein and

carbohy-drate domains thatarecriticalfor species-specific fertilization.

Geneticdefectsin thezona genes areexpectedtoperturb

fe-male(butnotmale)gametematuration, folliculogenesis,and

fertilization. The establishment of animal models with null

mutationsinthezona genes(using embryonicstemcell

tech-nology)willdefinethe phenotypes of such genetic defectsand

facilitate the search for their humancoupterparts.The

oocyte-specific zona pellucida promoters can be used to establish transgenicmouselinesexpressing humanzonapellucidagenes.

Ifthe human ZP3 (or ZP2)transgeneproduct is incorporated

into the mouse zona pellucida, the transgenic animal could

provideamuch neededsourceofoocytesforassaying human

spermfunction ininfertility clinics.

Thezonapellucidahas also beenrecognizedas atarget for

immunocontraception. Passive administration of antibodies that bindtothezonapellucidapreventspermpenetration (35)

and vaccination of mice with "self"zonapeptideselicits

anti-bodies that bindtothezonapellucidatocauselong-term,

re-versible contraception (36). However, vaccinationwith zona

pellucida peptidescanalso induceaTcell-mediated

oophori-tisinsusceptible strainsofmice(37), whichhaveprovideda

model for investigatingthe pathogenesis of human

re-sponsetodifferent regionsof the zonaproteinsisimperative in the rational design of contraceptive vaccines that will selec-tively induceantibodiesleadingtoinfertility,but not causeT

cell-mediated ovarian damage.

References

1.Hartmann, J. F., R. B. Gwatkin, and C. F. Hutchison. 1972.Earlycontact

interactionsbetween mammaliangametes invitro: evidencethat the vitellus

influencesadherencebetweensperm and zonapellucida.Proc.Nail. Acad.Sci.

USA.69:2767-2769.

2.Saling, P. M., J.Sowinski,and B. T.Storey.1979. Anultrastructuralstudy ofepididymalmouse spermatozoabindingto zonaepellucidaeinvitro:

sequen-tialrelationshipto the acrosomereaction.J.Exp. Zool.209:229-238. 3.Bleil,J.D., and P. M.Wassarman. 1980. Mammaliansperm-egg interac-tion:identificationof aglycoproteinin mouse egg zonaepellucidaepossessing receptoractivityforsperm.Cell. 20:873-882.

4.Florman, H. M., and P. M.Wassarman.1985. 0-linked oligosaccharidesof mouseegg ZP3 accountfor itssperm receptoractivity.Cell. 41:313-324.

5.Bleil, J. D., J. M.Greve,and P. M. Wassarman. 1988.Identificationof a secondary sperm receptor inthemouse egg zonapellucida:role in maintenance

ofbinding of acrosome-reactedsperm to eggs. Dev.Biod128:376-385. 6. Leyton, L., and P.Saling. 1989. Evidence that aggregation ofmouse sperm receptors by ZP3triggersthe acrosomereaction.J.Cell Bid. 108:2163-2168.

7.Leyton, L., and P.Saling. 1989. 95kd spermproteins bind ZP3and serve as

tyrosine kinasesubstrates in response to zonabinding. Cell. 57:1123-1130.

8.Bleil,J.D.,and P. M. Wassarman. 1983. Sperm-egg interactionsin the mouse: sequenceofevents andinduction ofthe acrosomereaction byazona

pellucidaglycoprotein.Dev.Biol. 95:317-324.

9.Saling,P. M.1989.Mammaliansperminteractionwith extracellular

matri-cesof the egg.OxfRev.Reprod.Biol. 11:339-388.

10. Sato, K. 1979.Polyspermy-preventing mechanismsin mouse eggs ferti-lizedinvitro.J.Exp.Zool. 210:353-359.

11.Bleil,J.D.,C.F.Beall, andP.M. Wassarman.1981.Mammalian sperm-egginteraction: fertilization ofmouse eggstriggers modification ofthe majorzona

pellucida glycoprotein,ZP2. Dev.Biol. 86:189-197.

12.Bleil,J.D., and P. M. Wassarman. 1988. Galactoseatthenonreducing terminus of0-linked oligosaccharides ofmouse egg zonapellucidaglycoprotein

ZP3is essential for the glycoprotein'ssperm receptoractivity.Proc.Natl. Acad.

Sci. USA.85:6778-6782.

13.Shabanowitz,R.B., and M.G. O'Rand. 1988. Characterization of the

human zonapellucida fromfertilizedandunfertilizedeggs.J.Reprod. Fertil. 82:151-161.

14. Shabanowitz,R. B. 1990.Mouseantibodiestohuman zonapellucida. evidencethat human ZP3is stronglyimmunogenicandcontainstwodistinct isomer chains. Biol. Reprod. 43:260-270.

15.Liang, L.-F., S.M.Chamow,and J. Dean.1990. Oocyte-specific

expres-sion ofmouseZp-2: developmental regulation ofthe zonapellucidagenes.Mol. Cell. Biol. 10:1507-1515.

16.Philpott, C. C.,M.J.Ringuette,and J. Dean. 1987. Oocyte-specific

ex-pression and developmental regulation of ZP3,the sperm receptorofthe mouse zonapellucida.Dev.Biol. 121:568-575.

17. Bleil,J. D., and P. M. Wassarman. 1980.Synthesisof zona pellucida

proteins by denudedandfollicle-enclosedmouse oocytes during culture in vitro. Proc.Natl. Acad.Sci. USA. 77:1029-1033.

18.Salzmann,G. S., J. M. Greve, R. J. Roller, and P. M.Wassarman.1983.

Biosynthesisof the sperm receptor during oogenesis in the mouse. EMBO (Eur.

Mol. Biol.Organ.) J. 2:1451-1456.

19.Bleil,J.D.,and P. M.Wassarman. 1980. Structure and functionofthe

zonapellucida:identification andcharacterizationof theproteinsof themouse

oocyte'szonapellucida.Dev.Biol.76:185-202.

20.Shimizu, S.,M.Tsuji,and J.Dean. 1983. In vitrobiosynthesisofthree sulfatedglycoproteinsofmurinezonaepellucidae byoocytes grown in follicle culture. J. Biol.Chem. 258:5858-5863.

21.Greve,J.M.,G. S.Salzmann,R. J.Roller,and P.M. Wassarman. 1982.

Biosynthesisof themajorzonapellucida glycoproteinsecretedbyoocytesduring mammalianoogenesis.Cell.31:749-759.

22.Lunsford,R.D.,N. A.Jenkins,C. A.Kozak,L.-F.Liang,C. M.Silan, N.G.Copeland,andJ. Dean. 1990.GenomicmappingofmurineZp-2andZp-3,

twooocyte-specificlociencodingzonapellucida proteins. Genomics.6:184-187. 23.Chamberlin,M.E.,and J. Dean. 1989. Genomicorganizationofasex specificgene: theprimarysperm receptor of the mouse zonapellucida.Dev.Biol.

131:207-214.

24.Ringuette,M.J.,M. E.Chamberlin,A.W.Baur,D. A.Sobieski,and J. Dean. 1988. MolecularanalysisofcDNAcodingforZP3,aspermbinding pro-tein of themouse zonapellucida.Dev.Biol. 127:287-295.

25.Chamberlin,M.E.,and J. Dean. 1990. Human homolog of themouse

sperm receptor. Proc. Natl. Acad. Sci. USA.87:6014-6018.

26.Kinloch,R.A.,B.Ruiz-Seiler,and P.M. Wassarman. 1990. Genomic organization andpolypeptideprimary structure of zona pellucidaglycoprotein hZP3,thehamster sperm receptor. Dev. Biol. 142:414-421.

27.Schwoebel, E.,S.Prasad,T. M.Timmons,R.Cook,H.Kimura,E.-M.

Niu,P.Cheung,S.Skinner,S. E. Avery, B.Wilkins,and B. S. Dunbar. 1991. Isolation and characterization of afull-lengthcDNA encoding the 55 kDa rabbit zonapellucida protein. J. Biol. Chem. 266:7214-7219.

28.Ringuette,M.J.,D. A. Sobieski, S. M. Chamow, and J. Dean. 1986.

Oocyte-specificgeneexpression: molecular characterization of a cDNAcoding

forZP-3,the sperm receptor of the mouse zona pellucida. Proc.Nail.Acad. Sci. USA. 83:4341-4345.

29.Paynton, B.V.,R.Rempel, and R. Bachvarova. 1988. Changes in state of

adenylationand time course of degradation of maternal mRNAs during oocyte maturation andearlyembryonic development in the mouse. Dev. Biol. 129:304-314.

30.Millar, S. E., E. Lader, L.-F. Liang, and J. Dean. 1991.Oocyte-specific

factors bindaconserved upstream sequencerequiredfor mouse zonapellucida promoter activity. Mol. Cell. Biol. 12:6197-6204.

31.Lassar, A. B., J. N. Buskin, D. Lockshon, R. L. Davis, S. Apone, S. D.

Hauschka,and H.Weintraub. 1989.MyoDis a sequence-specific DNAbinding

proteinrequiring aregionof myc homology to bind to the muscle creatine kinase enhancer. Cell. 58:823-831.

32.Murre, C.,P.S.McCaw,and D. Baltimore.1989.Anew DNA binding and dimerization motif inimmunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 56:777-783.

33.Blackwell, T. K., and H. Weintraub. 1990.Differencesandsimilaritiesin

DNA-binding preferences ofmyoD andE2Aproteincomplexes revealed by bind-ing site selection. Science (Wash. DC).250:1104-1110.

34. Lira, S. A., R. A.Kinloch,S.Mortillo,and P. M.Wassarman. 1990.An upstream region of the mouse ZP3 gene directs expression offireflyluciferase specifically to growing oocytes intransgenicmice. Proc. Natl. Acad. Sci. USA. 87:7215-7219.

35.East, I. J., B. J. Gulyas, and J. Dean. 1985.Monoclonal antibodiesto the murine zonapellucida proteinwith sperm receptoractivity: effectson fertiliza-tion and earlydevelopment.Dev. Biol. 109:268-273.

36.Millar, S. E., S. M. Chamow, A. W. Baur, C. Oliver, F. Robey, and J. Dean. 1989. Vaccination with asyntheticzonapellucidapeptideproduces

long-termcontraceptioninfemalemice. Science (Wash. DC). 246:935-938.