Three-dimensional structure of the zona pellucida at ovulation

Three-Dimensional Structure of the Zona Pellucidaat OvulationGIUSEPPE FAMILIARI,1* MICHELA RELUCENTI,1 ROSEMARIE HEYN,1 GIULIETTA MICARA,2

AND SILVIA CORRER1

1Laboratory of Electron Microscopy ‘‘Pietro M. Motta,’’ Department of Anatomy, Faculty of Medicine,University of Rome ‘‘La Sapienza,’’ 00161 Rome, Italy2Department of Gynecological Sciences, Perineonatology and Pediatrics, University of Rome ‘‘La Sapienza,’’ 00161 Rome, Italy

KEY WORDS oocyte; glycoproteins; extracellular matrix; fertilization; electron microscopy

ABSTRACT The mammalian zona pellucida (ZP) is an extracellular matrix surroundingoocytes and early embryos, which is critical for normal fertilization and preimplantation develop-ment. It is made up of three/four glycoproteins arranged in a delicate filamentous matrix. Scan-ning electron microscopy (SEM) studies have shown that ZP has a porous, net-like structure and/or nearly smooth and compact aspect. In this study, the fine 3-D structure of the human andmouse ZP is reviewed with the aim to integrate ultrastructural and molecular data, consideringthat the mouse is still used as a good model for human fertilization. By conventional SEM observa-tions, numerous evidences support that the spongy ZP appearance well correlates with matureoocytes. When observed through more sophisticated techniques at high resolution SEM, ZPshowed a delicate meshwork of thin interconnected filaments, in a regular alternating pattern ofwide and tight meshes. In mature oocytes, the wide meshes correspond to ‘‘pores’’ of the ‘‘spongy’’ZP, whereas the tight meshes correspond to the compact parts of the ZP surrounding the pores.In conclusion, the traditional ‘‘spongy’’ or ‘‘compact’’ appearance of the ZP at conventional SEMappears to be only the consequence of a prevalence of different arrangements of microfilament net-works, according to the maturation stage of the oocyte, and in agreement with the modern supra-molecular model of the ZP at the basis of egg–sperm recognition. Despite great differences in mo-lecular characterization of ZP glycoproteins between human and mouse ZP, there are no differen-ces in the 3-D organization of glycoproteic microfilaments in these species. Microsc. Res. Tech.69:415–426, 2006. VVC 2006 Wiley-Liss, Inc.

INTRODUCTION

The mammalian zona pellucida (ZP) consists of apeculiar extracellular matrix that surrounds the oocyteat ovulation and plays also an important role in theprocess of fertilization up to implantation of the fertil-ized egg (Familiari et al., 2006; Hoodbhoy and Dean,2004; Jungnickel et al., 2003; Motta et al., 1998, 2003;Talbot et al., 2003; Yanagimachi, 1981, 1994). The ul-trastructural and functional variation as well as thebiochemical and molecular composition of the ZP havebeen extensively studied during the last 40 years in dif-ferent mammalian species (Bleil and Wassarmann,1980a,b; Dunbar et al., 1994; Familiari et al., 1981;Greve and Wassarmann, 1985; Kang et al., 1979; Mottaet al., 1971; Sotelo and Porter, 1959; Stegner and War-temberg, 1961; Talbot and DiCarlantonio, 1984; Was-sarmann et al., 1984, 2005; Yudin et al., 1988; Zamboniand Mastroianni, 1966).

When observed by conventional scanning electronmicroscopy (SEM), mouse ZP shows different patternsin its outer and inner surfaces. The external surface ischaracterized by the presence of numerous fenestra-tions, giving it a somewhat spongy appearance (Mottaand Van Blerkom, 1975; Phillips and Shalgi, 1980a,b;Von Weymarn et al., 1980), whereas the internal sur-face is relatively smooth and compact (Phillips and

Shalgi, 1980a,b). Regional differences have also beenreported in the surface of hamster ZP (Keefe et al.,1997; Phillips and Shalgi, 1980a; Phillips et al., 1985).In a similar way, the ZP of canine, rabbit, and porcineoocytes appears as a fibrous and fenestrated network(Dudkiewicz and Williams, 1976; Funahashi et al.,2000, 2001; Strom-Holst et al., 2000; Suzuki et al.,2000).

A correlation between the morphology of the ZP sur-face and the degree of oocyte maturity has been stated inmammals (Calafell et al., 1992), including humans. Twobasic shapes of the ZP are recognized (Familiari et al.,1989b): a mesh-like zona in mature oocytes and a morecompact one in atretic or immature oocytes. Furtherinteresting results have been obtained using saponin,ruthenium red, osmium, and thiocarbohydrazide (Sap-RR-Os-Tc), a method that allows to observe the thin fila-ments composing the ZP, interwoven to form a fine

*Correspondence to: Giuseppe Familiari, MD, Laboratory of Electron Micros-copy ‘‘Pietro M. Motta,’’ Department of Anatomy, University of Rome ‘‘La Sapi-enza,’’ Via Alfonso Borelli 50, 00161 Rome, Italy.E-mail: [email protected]

Contract grant sponsor: Italian PRIN MURST grants 2004–2006.

Received 17 October 2005; accepted in revised form 16 December 2005

DOI 10.1002/jemt.20301

Published online 22May 2006 inWiley InterScience (www.interscience.wiley.com).

VVC 2006 WILEY-LISS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 69:415–426 (2006)

three-dimensional (3-D) network (Familiari et al., 1989b,1992b).

Extensive studies on the chemical composition of theZP have shown it to be mainly composed of sulfated gly-coproteins (Bleil and Wassarman, 1980a,b; Wassarmanet al. 1984) with some species-specific differences (Avileset al., 1994; Green, 1997; Topper et al., 1997; Wassar-man, 1990a,b). Furthermore, the ZP contains receptorsfor the sperm that either restrict its binding from heter-ologous species to unfertilized eggs or prevents bindingof sperm from homologous species to fertilized eggs. Inaddition, binding of the sperm to the receptor causesthe sperm to undergo the acrosome reaction, a sort ofcellular exocytosis, so that the sperm-binding sites inthe ZP could be a target for immunocontraception (Epi-fano and Dean, 1994). Three subfamilies of genes arerecognized within the ZP family: ZPA or ZP2, ZPB orZP1, and ZPC or ZP3. ZPA genes encode the longestprotein sequences, whereas ZPC genes encode theshortest ones (Spargo and Hope, 2003). Bovine ZP iscomposed of only a few highly modified glycoproteins:bZP1, bZP2, bZP3-a, bZP3-b, and bZP4, of which bZP2and bZP4 are fragments of bZP1 (Topper et al., 1997).Each of these glycoproteins has a specific function. Thesperm receptor activity is attributed to ZP3-a, whereasZP2 serves as a secondary receptor for the acrosome-reacted sperm. After sperm–egg fusion, cortical gran-ule-associated enzymes convert ZP2 and ZP3 to ZP2fand ZP3f, respectively, causing a dramatic change in ZPsurface aspect (Suzuki et al., 1994, 1999; Vanrooseet al., 2000). Therefore, these egg-induced modificationsplay a role in the block to polyspermy (Kopf, 1990). TheZP of marsupials (Breed, 1996; Breed and Leigh, 1988;Hughes, 1977; Selwood, 2000), like that of eutherianmammals (Chamberlain, 1989), is composed of weaklyacidic sulfated glycoproteins with a filamentous ultra-structure. A number of recent studies have establishedthat ZPA (equivalent to mouse ZP2), ZPB (equivalent tomouse ZP1), and ZPC (equivalent to mouse ZP3) arepresent in marsupials (Haines et al., 1999; Mate andMcCartney, 1998; McCartney andMate, 1999).

Four ZP genes are expressed in human oocytes(huZP1, huZP2, huZP3, and huZP4) and preliminarydata obtained by Lefievre et al. (2004) show that thefour corresponding ZP proteins are present in thehuman ZP, as a fundamental difference with the mousemodel. In fact, the mouse ZP is composed of three majorglycoproteins: mZP1, mZP2, and mZP3 (Wassarman,2005). In the mouse, the inductor of the acrosome reac-tion and the sperm receptor is the glycoprotein ZP3, aconstituent of all mammalian egg ZP (Bleil and Was-sarman, 1980a, 1983; Wassarman, 1990a,b, 1995).

A great number of studies exploring the molecularmechanisms at the basis of fertilization have been doneon the mouse. Because of the difficulties related to thedirect studies in the human, the mouse model is stillconsidered a good one for mammalian fertilization.

With the aim to review and integrate ultrastructuraland molecular data on the ZP during the preovulatoryand the ovulatory periods in mammals, this work isdevoted to analyze and compare the microanatomy andmolecular organization of mouse and human ZP andthe possible relationship existent between these fea-tures and the functional properties of the ZP duringthe postovulatory period and fertilization.

MOUSE ZPUltrastructural Pattern in Growing and Atretic

Ovarian Follicles

Changes occurring in the fine structure of ZP ingrowing and atretic ovarian follicles have been studiedby us with SEM using lanthanum nitrate and RR(Familiari et al., 1981). In growing and atretic follicles,the ZP appears as a single layer of fine granular andfibrillar material that reacted intensely with RR onlyduring atresia. Therefore, the use of RR demonstratedthat mouse ZP is subjected to a process of biochemicalmodification that renders it diversely sensitive to thisstaining mainly during follicular atresia. In otherwords, the ZP appeared intensely stainable only inatretic follicles where it was composed of a dense globu-lar fibrous matrix, in contrast to growing follicles thatshow the ZP weakly reacting with RR. This reaction ofthe ZP to RR shares some similarity with observationscarried out in oocyte–follicular cell complexes afterovulation (Baranska et al., 1975). The marked affinityof ZP to RR in ovulated (Baranska et al., 1975) andatretic oocytes may be related to altered chemical fea-tures; in particular, it may reflect a different chemicalpermeability.

Our studies provided further details on the fine-struc-tural 3-D features of the ZP in growing and atretic ovar-ian follicles of mouse with the use of transmission elec-tron microscopy (TEM), SEM, RR, and detergents suchas Triton X-100 and saponin (Familiari et al., 1989a,1992a). These detergents are used for the extraction ofthe soluble fraction of the ZP proteins in an attempt toexpose the structural glycoproteins, which, in turn, canbe viewed as minute 3-D networks with TEM and SEM.The ZP of growing follicles appears to be composed ofinterconnected filaments that bind to globular struc-tures, thus building up a 3-D lattice. Figure 1e,obtained at very high magnification with conventionalSEM, and Figure 3c, obtained at the same magnifica-tion employing Sap-RR-Os-Tc technique, represent asingle branch of a spongy ZP in a mouse unfertilizedmature oocyte. A comparative view of these picturesoffers a real idea of the 3-D organization of ZP glycopro-teic filaments. In contrast to this, atretic follicles ofstages I, II, and III (as classified by Byskov, 1974)showed a ZP structure characterized by the presence ofclosely packed granules connected with short filamentsto form a close-mesh reticulum. This structural changeof the ZPmay represent one of the early events involvedin the onset of follicular atresia and may also lead todegradation of the ZP network as well as a subsequentincreased condensation (Familiari et al., 1989a).

Ultrastructural and Histochemical ChangesPrior to Ovulation

Periovulatory follicles examined by SEM (Motta andVan Blerkom, 1975) have shown a quite irregular ZPsurface that contains numerous infoldings, channels,and crypts. Ultrastructural and histochemical changesin the ZP, during the final stages of oocyte maturationprior to ovulation, have also been described by Kauf-man et al. (1989). In particular, these authors eval-uated the ZP ultrastructure in preovulatory follicles ofimmature mice treated with exogenous gonadotro-phins. Structural changes have been observed during

416 G. FAMILIARI ET AL.

the final stages of oocyte maturation. There was a closerelation between the ultrastructural and histochemical(following PAS staining) features of the ZP. Significantdifferences regarding the location, density, and distri-bution of glycoconjugates were found within the ZPduring the final stages of oocyte maturation prior toand immediately following germinal vesicle break-down. Large antral follicles, with the oocyte containinga germinal vesicle surrounded by three to four layers ofcumulus cells, showed the ZP composed of three con-centric layers, the middle layer being the densest one.

In contrast to this, large Graafian follicles, clearly notdestined to ovulate, showed a relatively thin ZP thatconsists of two layers: an internal, densely stainingPAS-positive layer immediately adjacent to the oocyte,and a less densely stained outer layer occupying therest of the ZP. Significant changes in the structure andcomposition of the ZP were further observed in preovu-latory follicles within 4 h of hCG administration. Theclose association between the ZP layers observed withTEM and the concentric layers visualized after PASstaining has suggested that these layers reflect real dif-

Fig. 1. Conventional SEM. (a):Human unfertilized oocyte. Porous-net appearance of ZP (32,000).(b): Human unfertilized oocyte.Compact and smooth surfaced ZP(32,000). (c): Higher magnificationof (a). The spongy ZP structure isevident (34,000). (d): Higher magni-fication of (b). A dense and compactZP structure is shown (34,000). (e):Very high magnification of a mouseunfertilized oocyte showing a branchof the spongy structure of the ZP(350,000).

417ZONA PELLUCIDA STRUCTURE AND FUNCTION

ferences in density and distribution of glycoconjugatesduring the final stages of oocyte maturation, prior toand immediately following germinal vesicle break-down. It is likely that the changes observed in the pre-ovulatory oocyte are relevant for fertilization in thatthey may affect sperm–ZP binding and/or sperm–egginteractions.

Ultrastructural Features in Eggs Related toFertilization, Maturation, and Aging

The fine structure of the ZP in unfertilized eggs andembryos, in turn, has been extensively investigated byBaranska et al. (1975) with standard electron micros-copy and the use of RR. This technique allowed the vis-ualization of polyanionic molecules, since most of thesecorrespond to glycosaminoglycans present in the ZP.Under standard electron microscopy, the fine structureof the ZP appeared as a relatively homogeneous layer,while RR staining showed it to consist of coarse fibril-lar and granular material organized in distinct layers(Baranska et al., 1975). Unfertilized oocytes show athicker internal layer and a denser external one,whereas embryos have two additional layers: a coarsegrained intimal layer, beneath the internal one, and afine-grained peripheral layer, above the external one.These differences indicated that ultrastructuralchanges of the ZP occurred after fertilization (Baran-ska et al., 1975).

Time-dependent changes in the surface microarchi-tecture of both ZP and in vivo fertilized ova have beennoted (Jackowski and Dumont, 1979). In fact, matureunfertilized oocytes showed a highly textured ZP withdeep furrows that was transformed into a much smoother,ropy, porous, and less textured structure at fertilizationand thereafter.

Several studies have focused on age-dependentchanges (Dodson et al., 1989; Kaleta, 1979; Longo,1981; Meyer and Longo, 1979; Von Weymarn et al.,1980). Normal developing mice oocytes showed the dif-ferentiation of characteristic surface features; firstsigns consisting in a fibrous network-like structureinterspersed with pores were reported in oocytes from15-days-old mice (Von Weymarn et al., 1980). This sur-face microarchitecture becomes more prominent withadvancing age, beyond 30 days of age. Degeneratingoocytes, in turn, showed a disintegrating ZP (Von Wey-marn et al., 1980). Changes of the ZP related to oocyteaging have also been described using SEM and diges-tion with a-chymotrypsin (Longo, 1981). Differenceshave been revealed 1 and 24 h postovulation that indi-cated a reorganization of ZP with ovum senescence,resulting in alterations at the molecular and supramo-lecular levels. Considering that results obtained in vivoand in vitro are comparable, these structural changeswere likely to be independent of conditions within thefemale reproductive tract. The refractoriness of ZP ofaged mouse eggs to digestion with a-chymotrypsin sug-gested a possible mechanism to explain decrease inboth fertility and penetrability of senescent ova (Meyerand Longo, 1979). In fact, ZP being the primary site ofsperm recognition and attachment at fertilization, analteration in its structure may prevent fertilization inthat the sperm may be incapable of gaining access tothe surface of the ovum. These findings were furthersupported by Kaleta (1979) who demonstrated that

increased sensitivity to chymotrypsin digestion of theZP is directly correlated with an increase in fertiliza-tion of mouse eggs. The effect of ovum aging on in vitrofertilizability has been evaluated by Dodson et al.(1989). The data presented in this study clearly in-dicated that ZP hardening (technically defined asincreased time to enzymatically digest the ZP, andfunctionally defined as decreased sperm binding andpenetration) is not exclusively a fertilization-inducedphenomenon but may also result from either in vivo(intraoviductal) or in vitro ova aging. Furthermore, thesimilar sequence noted during ovum aging suggestedthat spontaneous cortical loss, ZP hardening, anddecreased sperm penetrability with aging are all com-parable phenomena.

Nogues et al. (1988) reported four main ZP types inmouse oocytes (aged in vivo or in vitro from immature,young, and elderly females), termed A, B, C, and Dtypes, supporting previous findings of Longo (1980)regarding the polymorphism of ZP structure. Type Aoocytes were observed immediately after ovulation andonly in prepuberal or young females, a structure thatmay likely correspond to immature oocytes. Type Bwas the commonest type of ZP among the groupsobserved by these authors and its incidence decreasedwith advancing age. Type C was observed prior todegeneration and was similar to that observed byLongo (1980) in aged oocytes; this type of ZP increasedsignificantly after (in vivo or in vitro) oocyte aging andwith increasing female age. These latter data correlatewell with results obtained by Calafell et al. (1992) whoobserved a significant decrease in the rate of fertiliza-tion of mouse oocytes after 6 h of in vitro aging. Thisfact further supports the idea that there is a relation-ship between the characteristics of the ZP and theindex of fertilizability of the oocyte. A similar relation-ship can be established between the increase in type Czonae of aged females and findings of Catala et al.(1988) who observed a decrease in mouse fertilizationrate with advancing maternal age. Type D likely corre-sponded to degenerated oocytes, since the surface wasidentical to that observed in fragmented eggs. Interest-ingly, this latter type was similar to that observed inoocytes belonging to extremely immature female mice(Von Weymarn et al., 1980), a feature interpreted as astage preceding the structuration of the ZP surface.Therefore, stages previous to structuration or degener-ation showed morphological similarities.

Calafell et al. (1992), analyzing by SEM the ZP sur-face of immature and in vitro matured mouse oocytes,have suggested a further structural classification offive distinct ZP types. The Y-type corresponded tounstructured, amorphous ZP, with only a few pores ofsmall diameter (or none at all), completely coveredwith cellular debris originated from the ZP itself. Thistype is associated to very immature (GV) oocytes. TheZ-type ZP was more structured, with shallow pores ofsmall diameter and is associated to a more advanceddegree of oocyte maturation (metaphase II). The A/B-type ZP was characterized by a fibrous network withnumerous large pores, associated with fully cytoplas-mic and nuclear matured, freshly ovulated oocytes.The C-type showed a rough ZP surface with few poresand no cellular debris, associated to aged oocytes.Finally, the D-type ZP had a flat, unstructured, and


amorphous surface without cellular debris and is asso-ciated to degenerated oocytes (Calafell et al., 1992;Nogues et al., 1988). In addition, high fertilization lev-els have been reported in the metaphase I maturedgroup, as it may be expected considering the high ratesof A/B-type ZP (fully mature) observed in this group(Calafell et al., 1992). On the contrary, lower fertiliza-tion rates were found in the GV matured group, as itmay be expected considering that the Z-type ZP (stillimmature) predominated (Calafell et al., 1992). Theaforementioned data agreed with results obtained byus in human oocytes after in vitro fertilization pro-grams (Familiari et al., 1988). In this study, matureoocytes showed numerous spermatozoa attached to theZP, whereas immature oocytes evidenced a lower pres-ence of spermatozoa on the ZP surface.

Molecular Organization of ZP Glycoproteins

The molecular organization of ZP glycoproteins hasbeen extensively studied by Wassarman’s group (Was-sarman et al., 1996). ZP glycoproteins are synthesizedand secreted by growing oocytes during a two-to-threeweek period and not by granulosa cells (Bleil and Was-sarman, 1980b; Greve et al., 1982; Wassarman, 1990a).As oocytes increase in diameter, the ZP increases inthickness up to about 7 lm in a fully-grown mouseoocyte (about 80 lm in diameter). In particular, mouseZP contains about 3 ng of proteins, which, as alreadymentioned, correspond to the three glycoproteins calledmZP1 (about 200 kDa), mZP2 (about 120 kDa), andmZP3 (about 83 kDa) (Bleil and Wassarman, 1980a). Ithas been demonstrated that mZP2 and mZP3 areexpressed concomitantly and exclusively by growingoocytes (Epifano et al., 1995; Kinloch et al., 1995; Phil-pott et al., 1987; Roller et al., 1989).

Immunolabeling of mZP1, mZP2, and mZP3 wasdetected throughout the entire thickness of the ZP, irre-spective of the developmental stage of ovarian follicle.Double and triple immunolocalization studies revealedan asymmetric spatial distribution of the three ZP gly-coproteins in the zona matrix at various stages of follic-ular development (El-Mestrah et al., 2002).

Glycoproteins of the mouse ZP are organized in avery specific manner. The ZP is composed of long inter-connected filaments, which are polymers of mZP2 andmZP3 (Greve and Wassarman, 1985). An mZP2–mZP3dimer is located every 140 A or so along the filaments,thus imposing a structural periodicity that can be seenin electron micrographs of dissolved ZP. Filaments, inturn, are cross-linked by mZP1 to create a 3-D matrix.Thus, each of the ZP glycoproteins plays a structuralrole during ZP assembly. It has been suggested thatthe amino-terminal half of mZP3 is involved in dimerformation with mZP2 (Wassarman and Litscher, 1995).Furthermore, free-swimming sperm recognize andbind in a species-specific manner to particular serine/threonine-(O)-linked oligosaccharides located at theZP3 combining-site for the sperm (Florman and Was-sarman, 1985; Florman et al., 1984; Kinloch et al.,1995; Litscher and Wassarman, 1996; Miller et al.,1992; Rosiere and Wassarman, 1992; Wassarman,1995; Wassarman et al., 2005). In fact, oligosaccharideconstructs with defined structures can inhibit bindingof mouse sperm to unfertilized mouse eggs in vitro(Litscher et al., 1995). Indeed, results of experiments

with human ZP3 in transgenic mice (Rankin et al.,2003) are also entirely consistent with this proposal(Wassarman, 2005; Wassarman et al., 2005). Thus,structural differences among receptor oligosaccharidesmay account for the degree of species specificity of ga-mete interaction observed when mammalian eggs andsperm are cocultured in vitro (Gwatkin, 1977; Wassar-man, 2005; Wassarman and Litscher, 1995; Yanagima-chi, 1994). Following fertilization, free-swimming spermare unable to bind to mZP3 (Bleil and Wassarman,1980a,b). Conversion of mZP3 from an active to an inac-tive receptor during the so-called zona reaction helps toprevent polyspermic fertilization. However, the mecha-nism for inactivation of mZP3 is unclear, althoughinvolvement of cortical granule contents deposited intothe ZP shortly after fertilization is likely (Wassarman,2005).

Null Mice Lines for Specific ZP Proteins

Rankin et al. (1999, 2001) have studied abnormal ZPin mice lacking specific ZP proteins. Mice lacking ZP3but expressing ZPl and ZP2 do not form a ZP and areinfertile. Genetically altered mice lacking ZPl, in turn,form a ZP composed of ZP2 and ZP3; although mostfemales are fertile, litters are half their normal size. Todefine the structural abnormality of the ZP in ZP1 nullmice, oocytes have been isolated from mature folliclesand viewed by SEM. The ZP matrix was present in alloocytes examined but somewhat thinner than normal.In addition, the ZPl-null matrix showed large fenestra-tions up to the point that the egg plasma membranewas observed through these pores. It appears that ZP2and ZP3 were sufficient to form a ZP matrix aroundZPl-null eggs. Although most ZP1-null ovulated eggsshowed the ZP at light microscopy, their matrices wereoften thinner with a poorly defined peripheral borderwhen compared with that of normal eggs. These abnor-malities are further defined using TEM of expandedoocyte–cumulus complexes in the presence of RR toaccentuate the ZP matrix. Normal oocytes showed theZP matrix composed of stout filaments and small, elec-tron dense granules, most readily observed at the outerperiphery where the matrix is less compact. Moving to-ward the surface of the oocyte, the ZP became increas-ingly dense, thus obscuring structural details. Even ifZPl-null zonae were also composed of stout filamentsand small electron dense granules, these structureswere quite dispersed throughout the entire matrix,including the region nearest the oocyte. Mutant ZPappeared thinner than normal and its structural integ-rity was compromised because of a looser weave in thematrix network. This implied an increased fragilityand distortion of the ZP, which could lead to ectopicaccumulation of granulosa cells within the perivitellinespace. The enhanced fragility of the ZP in the absenceof ZP1 was consistent with a model of ZP structure inwhich the matrix is composed of filaments of heterodi-meric repeats of ZP2 and ZP3 cross-linked by dimers ofZPl (Green, 1997; Greve and Wassarman, 1985). How-ever, the ability of ZP2 and ZP3 to form a matrixaround ZPl-null eggs indicated that the biosynthesis ofZP2 and ZP3 and their ability to associate with eachother was independent from the presence of ZP1.

Rankin et al. (2001) have studied Zp2 null mouselines. They found that ZP1 and ZP3 proteins continued


to be synthesized and formed a thin zona matrix inearly follicle that was not sustained in preovulatory fol-licles. The abnormal zona matrix did not affect initialfolliculogenesis but significantly decreased both follicu-lar development up to the antral stage and the numberof ovulated oocytes.

Finally, Rankin et al. (2003) introduced a human ZPtransgene into mice that were homozygous nulls formZP3. Eggs obtained from these mice had a ZP com-posed of mouse ZP1–ZP2 and human ZP3. When eithermouse or human sperm were incubated with the eggsin vitro, it was found that mouse sperm, but not humansperm, could bind to the egg ZP. Similar results wereobtained when a human ZP2 transgene was introducedinto homozygous nulls for mouse ZP2 and when humanZP2–ZP3 transgenes were introduced into homozygousnulls for mouse ZP2–ZP3.

On the basis of these observations, Rankin et al.(2003) argued that no single mouse ZP protein is oblig-atory for taxon-specific sperm binding, and hypothe-sized that rather than a particular ZP glycoproteinserving as a sperm receptor, the supramolecular struc-ture of the ZP is necessary for sperm binding.

HUMAN ZPUltrastructural Features in Eggs Related to

Fertilization, Maturation, and Aging

Our group has extensively investigated by light mi-croscopy and SEM the ZP structure and the early inter-actions between human oocyte and spermatozoa(Familiari et al., 1988, 2001, 2006). Two basically dif-ferent ZP patterns were observed: a mesh-like, spongystructure having wide and/or tight meshes, and a com-pact, smooth surface (Figs. 1a–1e). The latter was com-monly seen in cultured oocytes belonging to immatureand atretic groups. These results have suggested thatthe spongy appearance of the ZP was mainly related tooocyte development and maturity. On the other hand,greater numbers of penetrating spermatozoa werenoted on oocytes showing the mesh-like ZP (Figs. 1aand 1c), whereas few, if any, sperm appeared flattenedagainst the ZP surface of oocytes showing the morecompact, smoother ZP (Figs. 1b and 1d). At the light ofthese results, we have hypothesized that the condensa-tion of the outer aspect of the ZP caused a disorienta-tion of sperm-binding sites, which would result inreduced sperm binding and penetration capacity, thusleading to impairment of in vitro oocyte fertilizability.

Studies of Tesarik et al. (1988) agreed with our obser-vations. In fact, they have reported that human oocytesat metaphase I of the first meiotic division, whenexposed in vitro to capacitated spermatozoa, were notpenetrated, whereas oocytes in metaphase II did. Thus,it has been suggested that the ZP of human oocytesapproaching meiotic maturity underwent some ma-turational changes, which rendered it more susceptibleto sperm penetration. Furthermore, marked differen-ces were noticed in the ultrastructure of the ZPbetween metaphase I and metaphase II oocytes fixedimmediately upon recovery. The ZP of metaphase Ioocytes appeared somewhat compact and homogene-ous, distinctly demarcated against the cumulus oopho-rus provided with scarce intercellular material. In con-trast to this, the ZP of metaphase II oocytes displayeda highly porous structure in its outer one third poorly

delineated toward the fully expanded cumulus. Fur-thermore, it showed wide slits filled with newly formedcumulus oophorus’ intercellular material onto its sur-face (Tesarik et al., 1988).

Windt et al. (2001) performed an ultrastructuralevaluation of recurrent and in vitro maturation resist-ant metaphase I-arrested oocytes and suggested thatthe ‘‘narrow and fibrous’’ appearance of the ZP is char-acteristic of the immature stage.

Further supporting results to our hypothesis havebeen reported by us comparing, the ZP between cul-tured human early embryos and blastocysts and unfer-tilized oocytes (Familiari et al., 1989b; Nottola et al.,2005). In fact, at SEM examination, almost all polypro-nuclear embryos were surrounded by an intact ZP hav-ing a spongy surface similar to that observed in a greatnumber of preovulatory oocytes before fertilization(Familiari et al., 1989b). In our studies on ZP coveringpolypronuclear embryos, we observed amorphousmaterial emerging from the inner zona and meltingthe pores, while Nikas et al. (1994) noted the same inZP of fertilized ova at pronuclear stage. This micro-structural difference may be due to different prepara-tory techniques.

Furthermore, SEM data belonging to human blasto-cysts showed that the ZP ultrastructure in healthyblastocysts was characterized by numerous large fenes-trations, formed by networked filaments, rather simi-lar to that of healthy mature oocytes and early embryos(Nottola et al., 2005).

Other supporting data arose from the study ofhuman oocytes using thiocarbohydrazide and osmiumtetroxide as chemical stabilizers of the ZP during speci-men preparation for SEM study (Familiari et al.,1992a,b, 2001). Human oocytes and polypronuclearembryos derived from assisted reproduction trialsobserved without sputtering coating at high resolutionSEM (H-SEM) revealed similar pictures concerningthe distribution of ZP patterns such as spongy struc-ture or smooth surface and their relationships withmature, immature, or atretic oocytes (Figs. 2a–2e).

Appearance of ZP External Surface andSperm Binding

The structural organization of ZP surface of treatedimmature (prophase/metaphase I) oocytes comparedwell with that of mature (metaphase II) oocytes (Famil-iari et al., 1988; Tesarik et al., 1988) using TEM andSEM. The dramatic increase in sperm binding totreated ZP together with the ultrastructural changesallowed us to suggest that the modified ZP surface wasable to enhance sperm binding. Consequently, ourresults confirmed the proposal that sperm binding wasclosely correlated to structural features of the ZP sur-face (Familiari et al., 1988). Contrasting results havebeen presented by Magerkurth et al. (1999), who haveanalyzed by SEM the morphology and the sperm-bind-ing patterns of the human ZP. These authors reportedthat oocytes had an extremely heterogeneous morphol-ogy of the ZP surface, within and among patients. Fur-thermore, Magerkurth et al. (1999) have described fourdifferent types of ZP morphology, considering four cate-gories of oocytes (mature, immature, fertilized, andunfertilized oocytes), from a porous, net-like structureto a nearly smooth and compact surface. According to


these authors, no correlation could be establishedbetween ZP type and oocyte maturity or fertilizabilityand, therefore, they suggested that the heterogeneousmorphology of the ZP plays no important role insperm–oocyte interaction. Nevertheless, recent obser-vations of our group on the microanatomy of the ZPsurface in inseminated but unfertilized mature humanoocytes derived from assisted reproduction trials(Familiari et al., 2001) have supported our previousresults (Familiari et al., 1988, 1992a,b). Following acomparative analysis of traditional SEM techniques(gold coating and conductive staining methods) andSap-RR-Os-Tc method, the main aspect of the ZP bytraditional SEM consisted mostly (78.5%) in a porous,net-like structure. After the Sap RR-Os-Tc method,most oocytes (86.1%) showed the ZP consisting of alter-

nating tight and large meshed networks (Familiariet al., 2001). Therefore, the well-standardized proce-dures, the stabilizing action of the conductive stainingon the ZP material, and the results obtained with theSap-RR-Os-Tc method strongly emphasize that ZPchanges occurring in oocytes of various groups are notartifacts but genuine features, very likely related totheir actual maturation status.

Medium Culture pH and Changes inZP Structure

The influence of elevated pH levels on the structuraland functional characteristics of human ZP has beeninvestigated by Henkel et al. (1995). Fresh and salt-stored immature oocytes were randomly incubated ineither synthetic human tubal fluid medium (untreated

Fig. 2. H-SEM Os-Tc withoutgold coating. (a): Human fertilizedovum. The ZP shows a spongy sur-face (31,500). (b): Human atreticoocyte. The ZP appears completelycompact and with a smooth surface(31,500). (c): High magnification of(a) showing the spongy structure ofthe zona (318,000). (d): High mag-nification of (b) showing the compactand dense structure of the zona(318,000). (e): Very high magnifica-tion of a mature oocyte with a sper-matozoon tail on the left (350,000).


zonae) or in a chemically defined medium (treatedzonae). Sperm-binding experiments using hemizonaassay conditions exhibited a 10-fold increased bindingof sperm to treated oocytes when compared with un-treated oocytes. Recordings during incubation showedhigher pH levels in treated versus untreated ZP. Thesurface of treated ZP showed a spongy appearance,whereas that of untreated ZP appeared compact andsmooth. Moreover, treated ZP related to a markedincrease of sperm binding, suggesting that altered ZPsurfaces enhance sperm binding. The ZP of both freshand salt-stored oocytes showed fine granular matricesof light to medium electron density. ZP of oocytes incu-bated in standard medium showed a compact, granu-lar, and medium electron dense shape with smooth andhomogeneous surfaces, whereas treated ZP, on the con-trary, showed numerous openings on their surfacesand a marked spongy appearance. In addition, acidicZP solvents may significantly have improved spermbinding to immature ZP in sperm–oocyte coincubationif compared with control samples supplemented withbasic ZP solvents, which have exhibited sperm-bindingranges corresponding with those reported for prophaseand metaphase I oocyte (Henkel et al., 1995). Themarked spongy appearance of the surface belonging totreated ZP was likely to be the effect of the particularculture medium on the fine structure of ZP matrix,thus causing a manifold increased surface. Thesestructural changes were possibly due to the slightlyincreased pH levels in the medium.

3D Ultrastructure of the ZP FilamentousGlycoproteins at Ovulation and

During Fertilization

As shown by our group (Familiari et al., 1992a,b,2001), variously arranged networks of filaments com-posing the outer and inner surfaces of the ZP in humanoocytes and polypronuclear embryos from assistedreproduction trials were clearly observed by H-SEMand the Sap-RR-Os-Tc method (Figs. 3a–3d).

Filaments are very similar to those observed in themouse ZP as described earlier (Familiari et al., 1989a,1992a) and may also correspond to the negativelystained mZP2–mZP3 filaments resembling ‘‘beads-on-a-string’’ interconnected by mZP1 in a 3-D matrix asalso shown in the mouse ZP (Greeve and Wassarman,1985).

Human ZP filaments were straight or curved, 0.1–0.4 lm in length and 10–14 nm in thickness, as seen byTEM, 22–28 nm thick as seen by H-SEM. This differ-ence in thickness is due to the chemical coating consid-ered for H-SEM sample preparation. The filamentarrangement was remarkably different between theinner and outer surfaces of the ZP and among the vari-ous stages studied. Those present in the outer surfaceof the ZP were basically arranged in large and tightmeshed networks, whereas filaments of mature oocytesand fertilized (polypronuclear) ova had a regular alter-nating pattern of wide and tight meshed networks.Immature and atretic oocytes displayed almost exclu-sively a tight meshed network of filaments.

Therefore, comparing traditional SEM images withH-SEM pictures of Sap-RR-Os-Tc-treated samples(Figs. 1a and 3a), the outer surface of mature oocyteconsisted in filaments arranged in a multilayered net-

work that appears compact in the zones delimiting themeshes of the network, whereas it appears loose in themeshes holes. The latter seemed empty when observedwith conventional SEM; they were, in turn, made up ofa loose filament arrangement when observed with Sap-RR-Os-Tc treatment.

The inner surface of the ZP belonging to unfertilizedoocytes at any stage was arranged in repetitive struc-tures characterized by numerous short and straight fil-aments that anastomosed with each other, sometimesforming small, rounded structures at the intersections.After fertilization, the inner surface of the ZP evi-denced numerous areas wherein filaments fused to-gether. On the contrary, the outer ZP presented thesame filament pattern as observed in human matureoocytes, suggesting that cortical reaction gives rise tomodifications in the inner layer of the ZP consisting inregularly disposed areas of very closely packed fila-ments. These results well correlate with our traditionalSEM observations (Familiari et al., 1989b), but are incontrast with those of Nikas studies (Nikas et al., 1994)due to different preparatory techniques.

These data clearly reveal that oocyte maturation andfertilization in humans are accompanied by 3-D modifi-cations in the arrangement of ZP filaments, which maybe relevant in the processes of binding, penetration,and selection of spermatozoa as well as the block ofpolyspermy.

Molecular Organization of ZP Glycoproteins

Four ZP genes are expressed in human oocytes,called huZP1, huZP2, hZP3, and huZP4 (Conner et al.,2005; Lefievre et al., 2004). Data obtained by theseauthors show that these four corresponding ZP pro-teins are present in the human ZP, as a fundamentaldifference with the mouse model. Mass spectrometrysuggests that huZP4 levels in the human are equiva-lent to those of huZP3 and huZP2, whereas the huZP1is a rather minor component (Lefievre et al., 2004). Asalready mentioned, Rankin et al. (2003) have shownthat humanized mouse zonae expressing human ZP2and ZP3 can bind mouse sperm but are unable to bindhuman sperm, possibly related to a requirement forspecies-specific glycosilation (Wassarman, 2005). Assupposed by Conner et al. (2005), this result may alter-natively reflect human sperm having evolved to bind toa ZP consisting of four ZP proteins rather than three.It should also be considered that ZP4 itself is requiredfor a direct interaction as part of the sperm receptor onthe ZP like other mammalian species in which the ZP4is supposed to have sperm-binding activity (Govindet al., 2000; Topper et al., 1997; Yurevicz et al., 1998).

In addition, a recent study reveals variable glycosila-tion of the human ZP throughout its thickness, withpronounced differences between the most external andinternal regions of this matrix. This heterogeneous car-bohydrate composition could be responsible for the dif-ferent sperm-binding ability detected between theouter and inner regions of the ZP (Jimenez-Movillaet al., 2004). Finally, human ZP glycoproteins expresssome carbohydrate sequences not previously detectedin other species (Jimenez-Movilla et al., 2004).


CONCLUDING REMARKS

Many researches have been built up on the structureand properties of the ZP in various species. Neverthe-less, some questions still remain to be clarified. Forexample, all the results presented up until now do notdefine univocally the 3-D ZP structure. In fact, it is stillnot well understood whether variations of ZP ultra-structure are related or not to oocyte maturation stageor whether the various phenotypes observed corre-spond to artifacts. To explain the 3-D morphologicaldifferences, it should be pointed out that a glycoproteinmatrix such as the ZP, due to its extremely hydration

state, is very difficult to be preserved during ordinaryelectron microscopy procedures. In particular, strongsolutions of ethanol or glutaraldehyde may causeshrinkage of ZP gel structure, thus generating relevantartifacts. Therefore, to correctly interpret and comparethe morphology of oocytes treated differently (as differ-ent experimental designs are not carried out simulta-neously), particular care should be given in treating ZPfor SEM examination and standardized procedures;this will certainly reduce or even avoid ultrastructuralartifacts. An example of misunderstanding caused bythese problems in the histochemical field is the idea

Fig. 3. Sap-RR-Os-Tc method.(a): Outer surface of the ZP of ahuman mature oocyte. Many fenes-trations are present in which the fil-aments form a large meshed net-work (39,000). (b): Outer surface ofthe ZP of a human atretic oocyte.The filaments form a tight meshednetwork (39,000). (c): Mouse ma-ture oocyte. Very high magnifica-tion of filaments arranged in finenetworks in a branch of the spongystructure of the ZP (350,000).(d): Higher magnification of theouter surface of the ZP of a humanmature oocyte. Different patternsof filament aggregation can beseen. Note that filaments appearas a beads-on-a-string structure(350,000).


that ZP proteins are expressed and secreted by theoocyte in conjunction with surrounding granulosa cells.In fact, Eberspaecher et al. (2001) have reported thatsamples fixed with formalin and immunohistochemi-cally stained reveal that only ZP belonging to oocytesshows protein synthesis and secretion, while samplesfixed with Bouin’s reagent also show staining on granu-losa cells. Results obtained in humans, mice, and mon-keys demonstrate that ZP proteins are expressed andassembled exclusively by the oocyte and not by granu-losa cells. Previous results of ZP protein synthetic ac-tivity by granulosa cells correspond to improper fixa-tion of the tissues, leading to a misunderstanding ofthe real ZP microarchitecture.

Thus, another question arises: is the morphology of theZP depending on the maturity stage of the oocyte? An an-swer could be found out just considering the role that theZP plays in that structure and function develop side byside. ZP is the sperm-binding site and it has to be crossedby the sperm so as to achieve fertilization. Consideringthe question from an evolutionist’s point of view, it is rea-sonable to hypothesize a ZP structure model as a 3-Dmeshwork, which can well accomplish those tasks. Awide texture may not only avoid the exposition ofadequate sperm-binding sites but may also favor an eas-ier digestion with acrosomic enzymes. A tight texture, inturn, may hamper sperms binding and would make enzy-matic digestion more difficult. Such a theoretical modelhas practical evidence not only when morphological andfunctional properties are examined but also when consid-ering genetic and biochemical assays. In fact, experi-ments using knock-out mice for the three genes codifyingZP proteins demonstrate that the three proteins form a3-D network with ZP2–ZP3 heterodimers bound by ZP1(Rankin et al., 1999, 2001). It has also been shown thatthe glycoconjugate distribution changes during oocytematuration (Rankin et al., 1999, 2001).

When evaluating the sperm-binding ability of the ZPat different pH conditions, the percentage of bindingincreases as the structure becomes less tight. There-fore, ZP binding properties and morphology are regu-lated by changes in the pH. On the other hand, disap-pearance of cortical granules and consequent zonahardening are observed not only in fertilized oocytesbut also in unfertilized aged oocytes as a physiologicalaging process (Henkel et al., 1995).

Some authors believe that there is no correlationbetween ZP morphological changes and maturationstage of the oocyte, and that spongy or compact ZPaspects found in oocytes belonging to groups of differ-ent ages is a ‘‘random,’’ casual finding, independentfrom maturation stage of the oocyte (Magerkurth et al.,1999). On the contrary, as previously stated (Familiariet al., 1992b, 2001, 2006), we consider the above resultsas evidence for a real correlation existing among ZPstructure, function, and oocyte age (or maturationstage) as extensively reported in both mouse andhuman as well as in other mammalian species. We thusemphasize the concept that a modern view of the ZPsurface is a 3-D network of crossing filaments, and thespongy or compact appearance is the consequence of adifferent microfilaments’ network arrangement in thedifferent oocyte maturation stages.

Further, molecular support to our ultrastructuraldata arises from the last supramolecular model at the

basis of egg–sperm recognition (Hoodbhoy and Dean,2004; Rankin et al., 2003). Rankin et al. (2003) pro-posed a model in which the ZP is composed, at least, ofZP2 and ZP3 forming a 3-D matrix around ovulatedeggs to which sperm will bind. Following fertilizationand cortical granule exocytosis, the cleavage of ZP2modifies the 3-D structure of the ZP matrix withoutloss of zona components, so that sperm can no longerbind (Baranska et al., 1975; Familiari et al., 1992b,2001; Funahashi et al., 2001; Jackowsky and Dumont,1979). On the basis of the earlier evidence, it is ratherlogical to hypothesize that the 3-D network of ZP fila-ments is not randomly arranged but rather finalized toaccomplish fertilization of the mature oocyte.

Other recent data (Sun et al., 2005) are consistentwith our observations concerning 3-D compaction of ZPfilaments in the inner layer of the ZP in the postfertili-zation period (Familiari et al., 1992b, 2006). Sun et al.(2005) investigated protein structure changes in themouse ZP related to fertilization with a microroboticsystem. Their experimental studies quantitatively des-cribe a stiffness increase seen in the mouse ZP afterfertilization, providing an understanding of ZP proteinstructure modification, as related to an increase in thenumber of cross-links of protein ZP1 between ZP2 andZP3 at the basis of ZP hardening and related to stiff-ness increase of the ZP.

Last question arises: is the mouse a good model forhuman fertilization? As extensively shown, there aregreat similarities in the ultrastructural organizationbetween mouse and human ZP at both SEM and TEMexamination, as well as in the microarrangement of theZP filaments, and ZP morphological modificationsrelated to development, aging, and fertilization.

On the other hand, there are great differences in theZP glycoproteins, since mouse ZP is made of three gly-coproteins, whereas human ZP is made of four glyco-proteins. At present, glycoprotein differences do notaffect structural morphology and further studies areneeded to clarify the relationships between ultrastruc-tural and molecular organization.

ACKNOWLEDGMENTS

Thanks are due to Mr. Gianfranco Franchitto,Mr. Ezio Battaglione, and Mr. Antonio Familiari fortheir skillful technical assistance and their cooperationin developing the ultrastructural methodology.

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Three-dimensional structure of the zona pellucida at ovulation

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