Perspectives

Biology of Mammalian Fertilization: Role of the Zona PellucidaJurrien DeanLaboratory ofCellular and Developmental Biology, National Institute ofDiabetes and Digestive and Kidney Diseases,National Institutes ofHealth, Bethesda, Maryland 20892

IntroductionIn mammals, a series of carefully orchestrated events culmi-nate in the fusion ofa sperm and egg to form a one-cell zygote,the obligatory precursor of all cells in the embryo. The overallrate of fertilization in humans has contributed to sustainedincreases in the world population and added urgency to theneed to develop new, effective contraceptive agents. For some,however, the success rate is considerably lower, and there aremillions of infertile couples in the United States. Dramatic ad-vances in reproductive biology have provided some relief forthese individuals through the development ofin vitro fertiliza-tion techniques. Further progress requires additional under-standing ofthe molecular basis for normal fertilization and ourwillingness to exploit this knowledge to facilitate desired preg-nancies or prevent unwanted ones. These joint desires to havegreater control over our reproductive destiny has led to an in-creased interest in the macromolecules involved in fertiliza-tion.

The zona pellucida, an extracellular matrix surroundingthe female gamete, contains the primary and secondary spermreceptors and plays a pivotal role in sperm-egg interactions.Although millions of sperm are deposited in the female repro-ductive tract, fewer than 100 approach the ovulated egg in theoviduct, and normally only one successfully penetratesthrough the zona pellucida to fuse with the plasma membraneof the ovum. Immediately following fertilization, the plasmamembrane and zona pellucida are modified to prevent poly-spermy and, perhaps, to provide additional resiliency to thezona so that it can protect the preimplantation embryo. Inter-actions between the sperm and egg appear to be relatively spe-cies-specific and research has focused on the male and femalemacromolecules involved. This perspective will concentrate onrecent advances in our knowledge of the zona pellucida genesand proteins of the mouse, an experimental animal that hasprovided much of our current understanding of the moleculardetails of fertilization.

Fertilization: a precisAfter ejaculation into the female reproductive tract, sperm un-dergo a poorly understood maturation process (capacitation)

Address reprint requests to Dr. Jurrien Dean, Laboratory of Cellularand Developmental Biology, National Institute of Diabetes and Diges-tive and Kidney Diseases, Building 6, Room B 1-26, National Institutesof Health, Bethesda, MD 20892.

Receivedforpublication 31 December 1991 and in revisedform 21January 1992.

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

before approaching the ovulated egg in the oviduct (Fig. 1 A).Motile sperm pass through the enveloping cumulus oophorus,which is composed of a glycosylaminoglycan matrix and cu-mulus cells. They then bind to the zona pellucida thatsurrounds the mammalian egg (1, 2). The mouse and humanzonae pellucidae are composed of three major glycoproteins,ZP1, ZP2, and ZP3. Solubilized zonae pellucidae from unfertil-ized mouse eggs (but not from two-cell embryos) can inhibitsperm binding to ovulated eggs (3). This sperm-receptor activ-ity of the zona has been ascribed to a class of 3.9-kD 0-linkedoligosaccharides on ZP3 (4). ZP2 has been implicated as a sec-ondary sperm receptor that binds sperm only after the induc-tion of the sperm acrosome reaction (5).

The acrosome is a membrane-bound organelle anteriorlylocated in the head of the sperm. It has been proposed that thebinding of sperm to ZP3 induces a signal transduction acrossthe sperm membrane by aggregating a sperm-specific 95-kDprotein with tyrosine kinase activity (6, 7). This leads to theacrosome reaction (8) in which the sperm plasma membranefuses with the outer acrosomal membrane, resulting in the exo-cytosis ofthe acrosomal contents (Fig. 1 B). The lytic enzymes(e.g., acrosin, glycosidases) that are released, as well as somethat remain associated with the inner acrosomal membrane,such as acrosin, appear to facilitate passage ofthe motile spermthrough the zona pellucida.

A number ofsperm surface macromolecules have been pro-posed as zona adhesion molecules, e.g., serine proteinases and,B-l1,4-galactosyltransferase. More recent candidates include a95,000-D protein with tyrosine kinase activity and a 56,000-Dprotein isolated by its ability to bind ZP3 (for review see [9]). Itis likely that more than one interaction is responsible for thebinding ofsperm to the zona, both initially and during penetra-tion. Some of the sperm ligands present on the surface of theinner acrosomal membrane (e.g., proacrosin and PH-20) maybecome available for zona binding only after the sperm acro-some reaction. These latter molecules may be particularly im-portant for successful penetration of the zona pellucida.

Immediately after fertilization there are two major changesthat prevent polyspermy: a rapid electrical depolarization ofthe egg plasma membrane that blocks additional sperm in theperivitelline space from fusing with the egg (10), and biochemi-cal modifications of the zona pellucida. These latter changesoccur secondarily to the fusion ofcytoplasmic cortical granuleswith the egg plasma membrane, and the subsequent dischargeofthe granules' enzymatic contents into the perivitelline space.The release of proteinases and glycosidases modifies the zonapellucida (zona reaction), resulting in a block to additionalsperm binding and inhibition of zona-bound sperm penetra-tion (1, 10). Both ZP2 and ZP3 are modified by the zona reac-tion: ZP2 undergoes a proteolytic cleavage associated with the

Zona Pellucida 1055

A

Zona Pellucida

Sperm

SpermBinding

BAcrosome Intact Sperm

Tail Acrosome Reacted Sperm

block to polyspermy (11), and ZP3 loses both its ability toinduce the acrosome reaction and its sperm receptor activity(8, 12).

The zona pellucida proteinsThe zona pellucida proteins are synthesized, glycosylated, andsulfated in oocytes before their secretion to form the zona ma-trix that surrounds the growing oocyte, ovulated egg and earlyembryo.

Structure. Mouse ZP1 (185-200 kD) is a disulfide-linkeddimer which has yet to be characterized in molecular detail.The primary amino acid sequences of mouse ZP2 (120-140kD) and ZP3 (83 kD) have been deduced from the nucleic acidsequence oftheir cognate genes (see below). Although the pep-tide backbones ofthe human ZP2 and ZP3 are similar in lengthto those of their mouse homologues (see below), the maturehuman proteins are smaller, ZP1 (90-1 10 kD), ZP2 (64-76kD), and ZP3 (57-73 kD) (13), presumably due to differencesin glycosylation. Recent experiments with immunologicalprobes have identified two isoforms ofhuman ZP3, ZP3H andZP3L (14), but whether these represent different posttranscrip-tional modifications of a single ZP3 gene product or the pres-ence of an additional gene remains to be determined.

Biosynthesis. There is no zona pellucida surrounding rest-ing mouse oocytes, and zona protein synthesis is first detectedwhen oocytes enter their two week growth phase. Investigationsin a number of species have concluded that the zona pellucidaproteins are synthesized in oocytes and secreted into the zonamatrix, although some studies have also identified zona pro-teins in granulosa cells. More recently, transcripts ofthe singlecopy mouse Zp-2 (15) and Zp-3 (16) genes have been detectedonly in growing oocytes and the biosynthesis of the ZP2 andZP3 proteins is, likewise, restricted to oocytes (17, 18). At itspeak in 30-35-,um diameter oocytes, zona protein synthesisrepresents 7-8% of total protein synthesis. Zona productiondeclines in fully grown oocytes and is absent in ovulated eggs.Once incorporated into the extracellular matrix, the zona pel-lucida proteins have a long half-life (> 100 h) (19, 20).

Figure 1. Mammalian fertilization. (A) Thebinding of sperm to the zona pellucidasurrounding the ovulated egg induces thesperm acrosome reaction. The release oflytic enzymes from the acrosome and theforward motility of the sperm permit pene-tration ofthe zona pellucida. After fusionwith the egg's plasma membrane, the spermenters the cytoplasm and forms the malepronucleus of the one cell zygote. The fe-male pronucleus, formed at the same time,contains the female haploid genome. Fol-lowing fertilization, the zona pellucida isbiochemically modified to prevent addi-tional sperm from binding or penetratingthe zona. (B) The acrosome, a lysosomal-like structure on the anterior head ofsperm, contains an inner and outer mem-brane that fuse during the acrosome reac-tion. This results in the release of lytic en-zymes that are important for the penetra-tion of the zona pellucida by the sperm.

The biosynthetic pathways of ZP2 and ZP3 have been in-vestigated using radioactive precursors and in vitro culture.The ZP2 core protein has a molecular weight of 76,373, de-duced from its nucleic acid sequence (15). Six complex-typeN-linked side chains and an undetermined number of0-linkedoligosaccharides are attached to the core protein and the ma-ture 120-140-kD glycoprotein is secreted and incorporatedinto the extracellular zona pellucida (21). The ZP3 protein isfirst detected in the oocyte as a 44,000-D protein to which threeor four complex-type N-linked carbohydrate side chains areadded. The further attachment of0-linked sugars results in theproduction of the mature secreted 83-kD ZP3 glycoprotein(18). The intracellular transport patterns and mechanisms ofsecretion for these proteins have yet to be delineated.

Zona pellucida genesStructure ofthe mouse genes. Mouse Zp-2 and Zp-3, each sin-gle copy genes, are located on chromosomes 7 and 5, respec-tively (22). Mouse Zp-2 contains 18 exons (Fig. 2) that range insize from 45 to 190 bp and are separated by 17 introns (81 to1,490 bp). The gene spans 12.1 kbp of DNA. Zp-2 is tran-scribed and processed into a 2,201-nt mRNA with very short 5'(30 nt) and 3' (32 nt) untranslated regions. The detection of a2.4-kb transcript in oocyte RNA suggests that ZP2mRNA con-tains a poly(A) tail of 200 nt. ZP2 mRNA has a single openreading frame initiated at an ATG that encodes a polypeptideof713 amino acids with a molecular weight of80,217. The first34 amino acids represent a signal peptide that directs secretionand, following cleavage, the resultant core polypeptide (76,373D) is incorporated into the extracellular matrix. The ZP2amino acid sequence contains seven possible N-linked glycosyl-ation sites (Asn-X-Ser/Thr) and more than 100 potential 0-linked glycosylation sites (15).

The 8.6-kbp mouse Zp-3 gene is comprised of eight exons(Fig. 2) ranging in size from 92 to 338 bp and has introns whoselengths are between 125 and 2,320 bp (23). The 1,317-nt ZP3mRNA has short 5' (29 nt) and 3' (16 nt) untranslated regions.The latter is so abbreviated that the TAA termination codon is

1056 J. Dean

AMouse Zp-2 Gene

1112 1314 151612 3 4 5 6 7 89 10 \\// \117 18

Human ZP2 Gene1 2 3 4 5 67 89 10 11 121314 1,56 17 1819HH]HHHE+Hl11 I oil I I 1511 1 1 Al ~~~~~11I Nil I I I I

BMouse Zp-3 Gene

1 2 34 5 67

5' * 1 1 11iI

Human ZP3 Gene

5,2 3 4 5

1 I ad1 l Al

embedded within the consensus AATAAA polyadenylationsignal. Short untranslated regions are characteristic of bothZP2 (mouse and human) and ZP3 (mouse, human, and ham-ster) mRNAs. It is not clear what role, if any, these short un-translated regions play in gene expression, nor whether they areimportant for processing ZP2 and ZP3 transcripts. The poly-peptide deduced from the single open reading frame of mouseZP3 mRNA has a molecular weight of 46,307 and consists of424 amino acids (24). The release ofa predicted 22-amino acidsignal peptide would result in the secretion of a 43,943-D pro-tein, consistent with the reported 44-kD ZP3 core protein (18).

Although there is no overall similarity in the amino acidsequences of ZP2 and ZP3, they share at least one commonstructural motif. Each has a very hydrophobic region consistingof 23 and 26 amino acids, respectively, near the carboxyl ter-minus (15, 24). Hydrophobic domains like these are typicallypresent in membrane-spanning domains of proteins. The hy-drophobic regions in ZP2 and ZP3 may play an important rolein the intracellular trafficking of these secreted proteins or intheir interactions in the extracellular matrix. The carboxyl ter-minal hydrophobic domain is present in mouse (ZP2 andZP3), human (ZP2 and ZP3), hamster (ZP3), and a recentlydescribed rabbit zona protein (15, 24-27; and Liang, L. F., andJ. Dean, manuscript in preparation).

Conservation of the zona genes. The genes encoding ZP2and ZP3 are conserved among mammals. Using cross-hybrid-ization with mouse nucleic acid sequences as a criterion, thedegree of conservation of Zp-3 is variable with the rat, dog,cow, and human zona genes being more related to mouse thanthe pig and rabbit genes (28). The human homologue of Zp-2and the human and hamster homologues of Zp-3 have beencharacterized. The human ZP2 gene (Fig. 2) is composed of 19exons (one more than the mouse) whose nucleic acid sequenceis 70% the same as that of its mouse counterpart (Liang, L. F.,and J. Dean, manuscript in preparation). The encoded proteinsare 60% identical, although the human protein has 745 aminoacids compared with 713 amino acids in mouse ZP2. Themouse, human, and hamster ZP3 genes contain eight exons

2 KB

Figure 2. Genomic loci of human and mouseZp-2 and Zp-3. (A) Comparison of mouse Zp-2(18 exons) and human ZP2 (19 exons) geneswith transcription units of 12.1 and 13 kbp,respectively (15; and Liang, L. F., and J. Dean,manuscript in preparation). (B) Alignment of

6 7 8 mouse Zp-3 (8.6 kbp) and human ZP3 genesI ( 18.3 kbp), each ofwhich contain eight exons

| | | 3 (23, 25).

each. The coding sequences ofthe mouse and human genes are74% the same, and they encode 424 amino acid peptides thatare 67% identical (25). The hamster gene encodes a 422-aminoacid protein that is 81% identical to mouse ZP3 (26). The ZP3mRNA in mouse oocytes is 1.5 kb (including a 200-nt poly(A)tail) and is indistinguishable in size from that of rat and rabbitZP3 mRNAs (24). Taken together, these data suggest that theoverall structure of the zona genes and their gene products areconserved among mammals.

Expression ofthe zona genesNeither ZP2 nor ZP3 transcripts are present in resting mouseoocytes (10-15 rim). However, once oocyte growth com-mences, increasing amounts of ZP2 and ZP3 transcripts aredetected. Maximum levels of the transcripts (1,000 fg of ZP2and 400 fg of ZP3) are present in oocytes that are 50 ,um indiameter, representing 1 and 0.4%, respectively, of totalpoly(A)+ RNA. The molar ratio ofZP2 and ZP3 transcripts is- 2: 1, and this ratio is maintained during the growth phase ofoocyte development (15, 16). As the oocyte reaches its full size(75-80 jAm), the amount ofZP2 and ZP3 transcripts declines,and, in ovulated eggs, the amount of these two transcripts is< 5% of its peak level (15). ZP2 and ZP3 mRNAs in ovulatedeggs are 200 nt shorter than they are in growing oocytes.This decrease corresponds to the estimated length of theirpoly(A) tails (15) and is very similar to the decrease observedfor f3-actin and a-tubulin mRNAs in mouse eggs (29). This hasled to the hypothesis that the zona mRNAs, like other maternalmessages, undergo rapid deadenylation and degradation dur-ing meiotic maturation and ovulation.

Mechanism ofoocyte-specific expression. The expression ofmouse Zp-2 and Zp-3 is restricted to growing oocytes and is notdetected in other mouse tissues (15, 16,24,28). The coordinatetranscription of the zona genes is restricted to the two-weekgrowth phase of oogenesis and serves both as a molecularmarker for oocyte growth and differentiation, and as a para-digm for investigating molecular mechanisms of gene activa-tion in the oocyte. Virtually nothing is known about the mecha-

Zona Pellucida 1057

nisms that restrict gene expression to the female germline, and,although several previously characterized transcription factorsare present in oocytes, their target genes have yet to be deter-mined.

If the regulation of zona gene expression is under the con-trol ofshared regulatory factors, the DNA-binding sites ofthesefactors may be evolutionarily conserved among mammals. Acomparison ofthe first 300 bp upstream ofthe initiation site ofmouse and human ZP2 genes reveals that they are 70% identi-cal. There is a TATAA box at position -31 bp and a CCAATbox at position -65 bp in the mouse gene; these elements arepresent at comparable positions in the human gene (15; andLiang, L. F., and J. Dean, manuscript in preparation). In con-trast, comparison ofthe 5' flanking sequences ofthe mouse andhuman ZP3 genes indicates no long stretches ofsequence iden-tity. The TATAA box at -29 bp in the mouse Zp-3 is alsopresent in the human homologue. Neither ZP3 gene has anidentifiable CCAAT box (23, 25).

Interestingly, close examination of the first 300 bp up-stream of four zona genes (mouse Zp-2 and Zp-3; human ZP2and ZP3) revealed five short (4-12 bp), conserved DNA se-quences (elements I, IIA, IIB, III, and IV), located at compara-ble distances from the transcription initiation site (30). Wehave demonstrated that one of these sequences, element IV(positioned 200 bp upstream from the TATAA box), is nec-essary and sufficient for reporter gene expression driven bymouse Zp-2 and Zp-3 promoters microinjected into oocytes(30). Oligonucleotides containing element IV from either Zp-2or Zp-3 form DNA-protein complexes of identical mobility ingel retardation assays using extracts of oocytes but not othertissues (Fig. 3).

The center ofelement IV from both the human and mouseZp-2 and Zp-3 genes contains the motifCANNTG which hasbeen identified as a consensus sequence (31) for the binding ofbasic helix-loop-helix (HLH) transcription factors (32). Thesefactors generally bind DNA as homo- or heterodimers. TheHLH domain is required for the protein-protein interactionsinvolved in dimerization, while the adjacent basic domain in-teracts directly with DNA. HLH transcription factors havebeen shown to be involved in the regulation oftranscription ofseveral tissue specific genes (for review, see [33]).

The transcription factor-DNA complex identified in theseexperiments may contain one or more previously character-ized basic HLH proteins, or may represent the first knownexample of an oocyte-specific transcription factor. The isola-tion and biochemical characterization ofthe zona gene activa-tion factors will not only provide information about their struc-tures, but will permit investigation of their putative functionsas regulators of zona pellucida gene transcription in mouseoocytes. The concept ofa common transcription factor(s) bind-ing to element IV of the promoter region of the mouse Zp-2and Zp-3 genes that participates in the coordinate and tissue-specific expression of the zona pellucida genes is an appealinghypothesis which can now be tested. The relatively low level ofreporter gene activity in these studies (30) as well as in trans-genic mice (34) suggests that additional regulatory factors maybe important for in vivo levels of zona gene activity.

1. Abbreviation used in this paper: 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:Figure 3. Ovary-specific binding of a transcription factor to a zona

promoter element. Gel mobility shift assays using radioactively la-belled oligonucleotides containing element IV from mouse Zp-2.Arrow indicates the oocyte-specific DNA-protein complex present inthe direct binding assays with both Zp-2 and Zp-3 element IV oligo-nucleotides (30). Ovary, lane 1, mouse [32P]Zp-2 oligonucleotidecontaining element IV reacted with 2-3 gg of ovarian extract; lane 2,as lane I but competed with 50-fold molar excess ofunlabeled mouseZp-2 oligonucleotide; lane 3, as lane 2 but competed with a Zp-2oligonucleotide containing a 6-bp mutation in element IV. Folliclecells, as above but with 2-3 gg follicle cell protein. Testes, as abovebut with 2-3 jig testes protein. Tissues, lanes 1-6, mouse [32P]Zp-2oligonucleotide reacted with 2-3 jsg extract ofembryonic stem cells(lane 1), brain (lane 2), kidney (lane 3), liver (lane 4), spleen (lane 5),and thymus (lane 6).

ConclusionsThe elucidation of the primary structure of the zona proteinshas provided a means for determining the protein and carbohy-drate domains that are critical for species-specific fertilization.Genetic defects in the zona genes are expected to perturb fe-male (but not male) gamete maturation, folliculogenesis, andfertilization. The establishment of animal models with nullmutations in the zona genes (using embryonic stem cell tech-nology) will define the phenotypes of such genetic defects andfacilitate the search for their human coupterparts. The oocyte-specific zona pellucida promoters can be used to establishtransgenic mouse lines expressing human zona pellucida genes.If the human ZP3 (or ZP2) transgene product is incorporatedinto the mouse zona pellucida, the transgenic animal couldprovide a much needed source of oocytes for assaying humansperm function in infertility clinics.

The zona pellucida has also been recognized as a target forimmunocontraception. Passive administration of antibodiesthat bind to the zona pellucida prevent sperm penetration (35)and vaccination of mice with "self" zona peptides elicits anti-bodies that bind to the zona pellucida to cause long-term, re-

versible contraception (36). However, vaccination with zona

pellucida peptides can also induce a T cell-mediated oophori-tis in susceptible strains of mice (37), which have provided a

model for investigating the pathogenesis of human autoim-mune oophoritis. The precise definition of immunological re-

1058 J. Dean

sponse to different regions ofthe zona proteins is imperative inthe rational design of contraceptive vaccines that will selec-tively induce antibodies leading to infertility, but not cause Tcell-mediated ovarian damage.

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