MOLECULAR REPRODUCTION AND DEVELOPMENT 3 2 5 1 - 6 1 (1992)
Human Zona Pellucida During In Vitro Fertilization: An Ultrastructural Study Using Saponin, Ruthenium Red, and Osmium-Thiocarbohydrazide GIUSEPPE FAMILIARI,’ STEFANIA A. NOTTOLA,’ GUIDO MACCHIARELLI,’ GIULIETTA MICARAF CESARE ARAGONAF AND PIETRO M. MOTTA’ ‘Department of H u m a n Anatomy and Assisted Reproduction Unit, University of Rome ‘Za Sapienza,” Rome, Italy ABSTRACT The human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one- to four-cells stages were studied by transmission electron microscopy (IEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin),and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by 0smium-thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1-0.4 k m in length and 10-14 nm thick as seen via TEM or 22-28 nm thick as seen via SEM (thedifference in thickness was due to the presence of the metal coating for SEM).The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in “large” and “tight”meshed networks. Mature oocytes and fertilized (polypronuclear)ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration,and selection of spermatozoa. 0 1992 Wiley-Liss, Inc.
Key Words: Oocyte, Polypronuclear ovum, Fertilization
INTRODUCTION During mammalian fertilization, the spermatozoon reaches the zona pellucida (ZP), a special egg coating made up of the glycoproteins ZPl,ZP2, and ZP3 (Was-
0 1992 WILEY-LISS, INC.
sarman, 1988,19901, binds to its surface, penetrates it, and reaches the oocyte membrane (Talbot, 1985; Yudin et al., 1988; Dietl, 1989). The ZP is known to play a crucial role in the regulation of sperm-egg interactions. In fact, in mammals, the ZP chemicophysical properties change drastically upon fertilization, (Wassarman, 1990), and a process called “zona reaction” occurs that contributes to inhibition of supernumerary spermatozoa penetration (Yanagimachi, 1988).Despite the many studies of these events, the ZP-sperm interactions are still not fully understood (Talbot, 1985; Yanagimachi, 1988). A recent review suggests that a three-dimensional approach to the study of these events may contribute to filling the gap in knowledge of the morphology (Familiari et al., 1991). However, to date only a few scanning electron microscopic (SEM) observations on the human ZP during fertilization have been made (Motta and Van Blerkom, 1974; Phillips and Shalgi, 1980a,b; Sundstrom, 1982; Familiari e t al., 1988, 198913). In fact, the material of the ZP, due to the presence of a large amount of surrounding extracellular amorphous material and due to the hydrophilic peculiarity of its components, is particularly hard to define through SEM observations without appropriate techniques to unmask and stabilize its components. To study the fine ultrastructure of the ZP, including its changes during fertilization, with particular regard to the three-dimensional arrangement of its components, the zona material has been investigated via SEM and transmission electron microscopy (TEM) in oocytes (at various developing stages) and polypronuclear ova (PO) (between one-cell and four-cell stages) deriving from a human in vitro fertilization and embryo transfer (IVF-ET) program. With this purpose, the specimens were treated with saponin, a detergent that permits the removal of the amorphous materials masking the real ZP structure. Then they were contrasted with ruthenium red (RR), a cationic dye that, together with 0smium and thiocarbohydrazide impregnation, stabilizes
Received November 19,1991; accepted January 2,1992. Address reprint requests to Prof. Giuseppe Familiari, Department of Anatomy, University of Rome “La Sapienza,” Via A. Borelli 50,00161 Rome, Italy.
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G. FAMILIAR1 ET AL. TABLE 1. Oocytes and Polypronuclear Ova Recovery and Fixation Stage at recovery Stage at fixation Oocytes Immature 13 Immaturea 8 Mature 31 Maturea 35c Atretic 6 Atretica 7d Polypronuclear ova Mature oocytes
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Fertilized ova (three pronuclei)b Two-cell polypronuclear ovab Three-cell polypronuclear ovab Four-cell polypronuclear ovab
4 1 3 2
aAfter 1-3 days of culture. bAfter 2-4 days of culture. “Five immature oocytes matured in vitro. mature oocyte degenerated in vitro. the structural glycoproteins of the ZP for high-resolution SEM observations (Familiari et al., 1987,1989a).
MATERIALS AND METHODS Protocol Eight immature, noninseminated oocytes, 35 inseminated but nonfertilized mature oocytes, seven atretic (postmature) inseminated but nonfertilized oocytes, and ten polypronuclear ova a t one-cell to four-cell stages were obtained from follicular aspirates from patients undergoing TVF-ET (Lopata, 1983) (Table l).All specimens were studied with the patients’ consent and represent material that normally would be discarded. All specimens were analyzed by phase-contrast light microscopy (LM) prior to electron microscopic procedures. The LM criteria for oocyte staging were the gen-
eral appearance of the cytoplasm and the presence of the germinal vesicle and polar body, according to a previously described classification (Aragona and Micara, 1987; Familiari et al., 1988). For ethical reasons, only polypronuclear ova displaying three or more pronuclei at the inverted bright-field microscopic level were studied. Thirty oocytes and six polypronuclear ova underwent chemical extraction a s described below. Twenty oocytes and four polypronuclear ova (control) were directly fixed in a glutaraldehyde solution and postfixed in osmium tetroxide (Familiari et al., 1988).
Extraction Procedure The specimens were washed in a cacodylate buffer solution (CBS) and incubated in a 0.02% saponin and
Fig. 1. Phase-contrast microscopy a: Mature preovulatory oocyte after insemination. b: Immature oocyte after 24 hr of culture. c: Atretic oocyte at insemination. d: Polypronuclearovum at three pronuclei stage after 22 hr of culture. e: Four-cell polypronuclear ovum 48 hr after insemination. x 160.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE
Fig. 2. Control samples. a:SEM. Mature oocyte. The zona pellucida appears spongy. Penetrating spermatozoa (arrows). x 900. b: TEM. Mature oocyte (MO). The zona pellucida (ZP) has a microgranular quasihomogeneous texture. Note the irregular margin (arrowheads). x 1,500. c: SEM. Atretic oocyte. The zona pellucida shows a completely smooth surface. ~ 7 0 0 d: . TEM. Early atretic oocyte (AO). The zona pellucida (ZP) has a microgranular appearance. Note its regular and
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compact outlined surface (arrowheads). x 1,500. e: SEM. Two-cell polypronuclear ovum. The zona pellucida shows a spongy surface. CC, cumulus corona cell. x 1,500. f: TEM. Two-cell polypronuclear ovum (PO). The zona pellucida (ZP) has a quasihomogeneous texture. An irregular margin is present similar to that of mature oocytes (arrowheads). X 1 , 5 0 0 .
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1.0%RR mixture, both in 0.1 M CBS a t pH 7.4 for 30 min. The specimens were then fixed in 3.0% glutaraldehyde plus 1.0% RR and 0.02% saponin in CBS 4°C. The following day, the specimens were washed in CBS containing 1.0% RR and 0.02% saponin, postfixed for 1h r in 1.0% OsO, in CBS containing 0.75% RR and 0.02% saponin (Familiari et al., 1989a), then again washed in CBS, and finally prepared for TEM or SEM.
SEM of Extracted and Control Samples Twenty oocytes and three polypronuclear ova (extracted samples) as well as ten oocytes and two polypronuclear ova (control samples) were treated with thiocarbohydrazide (1.0%) and osmium tetroxide (1.0%) in distilled water (Kelley et al., 1973). The specimens were rinsed in distilled water and placed in a steel microchamber whose base and top were 400-mesh copper grids (Familiari et al., 1988).The cells were then dehydrated in acetone, critical-point dried, and mounted on aluminium stubs. TEM of Extracted and Control Samples Ten oocytes and three polyopronuclear ova (extracted samples) as well a s ten oocytes and two polypronuclear ova (control samples) were dehydrated in ethanol and embedded in a n epoxydic resin. Sectioning was performed with LKB and Reichert ultramicrotomes. Some ultrathin sections were stained with lead citrate (Reynolds, 1963), while others were examined without further staining. Observations Both the external and the internal surfaces of the ZP were studied via SEM. The ZP of some oocytes was fractured by means of a needle to expose the internal surface. SEM observations were made in Jeol S.800 and Hitachi S.4000 field emission scanning electron microscopes operating at 5-10 kV. TEM observations were made with a Zeiss EM9 S2 electron microscope. RESULTS Light Microscopy Mature oocytes showed a homogeneous and clear ooplasm as well as the presence of the first polar body. No pronuclei were found in these oocytes (Fig. la). Some of the immature oocytes had a clear cytoplasm and a n intact germinal vescicle, while others had undergone germinal vescicle breakdown but did not show the first polar body (Fig. lb). The atretic (postmature) oocytes displayed a dark, granular, retracted ooplasm and a large perivitelline space (Fig. lc). The one-cell polypronuclear ova contained three or more pronuclei (Fig. Id). Polypronuclear ova a t later developmental stages showed both a normal cleavage and blastomeres of normal size (Fig. le) as well a s a fragmented cleavage.
Electron Microscopy Control specimens. Mature oocytes. By SEM, the outer surface of the ZP was usually characterized by a spongy appearance, due to the presence of numerous fenestrations (Fig. 2a). In addition, the outer ZP showed a fine, downy surface (Fig. 3a). The inner surface of the ZP was clearly smooth (Fig. 3b) and, a t high magnification, showed a homogeneous microtrabecular structure (Fig. 3c). Via TEM, the ZP had a homogeneous microgranular appearance throughout its thickness and was characterized by a n irregular, rough outer margin (Fig. 2b). Zmmature and atretic oocytes. By SEM, in most of the immature and atretic oocytes, the outer surface of the ZP was smoother than t h a t of the mature oocytes. Fenestrations of this outer surface were rare or even absent in most samples (Fig. 2c). The inner surface of the ZP was smooth and similar to that of preovulatory mature oocytes. TEM showed the characteristic microgranular appearance of the ZP, but a regularly smooth outer margin was seen (Fig. 2d). Polypronuclear ova. By SEM, almost all the polypronuclear ova had a n intact ZP with a spongy, fenestrated outer surface (Fig. 2e) and a smooth inner surface, comparable to that seen in mature oocytes. TEM observations showed a microgranular texture similar to that observed in most of the mature oocytes (Fig. 20. Extracted specimens. Parallel SEM and TEM examinations of specimens treated with saponin and RR provided a valuable technical approach to the study of the ZP microarchitecture. However, i t should be noted that the addition of saponin to the fixative inevitably produced some alterations in the cytoplasmic morphology of the oocyte (Fig. 4a, b). TEM observations. TEM examination of the ZP in all types of oocytes and polypronuclear ova revealed a fine network of RR-positive filaments oriented in several directions and anastomosed with each other. These filaments measured 10-14 nm in thickness and 0.1-0.4 pm in length. Four types of arrangement of filaments were demonstrated in the different specimens studied (Fig. 4a-f). Type 1 was characterized by filaments arranged in a very tight meshed network. In type 2 the meshes of the filament network were less condensed than seen in Type 1. I n type 3 filaments were arranged in a loose or large meshed network. In type 4 the filaments were focally fused together to form a homogeneous structure without a reticular appearance. Mature oocytes. Most of mature oocytes had a ZP characterized by filaments arranged in a regular network. In the inner portion, this network was of type 1 (Fig. 4a, c), whereas the outer ZP portion presented a regular alternating pattern of zones with filaments arranged as in type 2 or type 3 (Fig. 4e). Zmmature and atretic oocytes. The immature and atretic oocyte Z P showed filaments arranged in the inner portion as in type 1 and in the outer portion as in type 2. Unlike the case in mature oocytes, the ZP outer portion of immature and atretic oocytes did not present areas with filaments arranged a s in type 3 (Fig. 40.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE
Fig. 3. Control samples. a: SEM. Mature oocyte. At high magnification the outer surface of the zona pellucida revealed a spongy organization. Note the microtrabecular appearance of branches (stars) and holes (asterisks). ( x 18,000).b,c:SEM. Mature oocyte. The inner surface of the zona pellucida has a very fine microtrabecular appearance. (b; x525 c; X24,OOO).
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Fig. 4. TEM. Extracted samples. a: Mature oocyte (MO).The zona pellucida (ZP)displays a regular network of filaments. Note the oocyte (MO) cellular changes due to saponin extraction, characterized by cytoplasm dissolution. Boxed area is enlarged in c. (x4,800). b: Fourcell polypronuclear ovum (PO). Areas of condensation are regularly distributed in the inner portion (arrows) of the zona pellucida (ZP). Note the ovum (PO) changes due to saponin extraction, characterized by cytoplasm dissolution. Boxed area is enlarged in d. (X4.800). c: Mature oocyte. High-power view of the boxed area in a. The fine network of filaments is associated with small granules a t the nodal
points of the reticulum (arrows). (x27,700). d Four-cell polypronuclear ovum. High-power view of boxed area in b. In the network of filaments, areas of major condensation are present (arrowheads). ( X 27,700). e: Mature oocyte. High-power view of the outer portion of the zona pellucida (ZP). A regular alternating pattern of zones with filaments arranged in tight (arrowheads) or loose (arrows) meshed network was present. ( x 27,700). E Atretic oocyte. High-power view of the outer portion of the zona (ZP). The filaments are arranged in a tight meshed network (arrowheads). x27,OOO.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE Polypronuclear ova. The ZP of polypronuclear ova was formed in the inner part mainly by filaments arranged as in type 1 and by numerous areas characterized by filament condensation as in type 4 (Fig. 4b,d). On the other hand, the outer part of the ZP displayed filaments arranged in a wider network, with a regular alternating of pattern type 2 and type 3, as seen in the outer ZP of matureoocytes (Fig. 4b). SEM examination. SEM allowed a detailed visualization of the filament arrangement of ZP in extracted specimens. The filaments measured 22-28 nm in thickness and 0.1-0.4 pm in length and, at high magnification, showed a globular structure (Figs. 5, 6). Remarkable differences of filament arrangement were seen in all types of specimens. Mature oocytes. The ZP outer surface in mature oocytes was characterized by filaments arranged in a regular pattern of alternating tight meshed and large meshed networks. The large meshed network had the same distribution of fenestrations as was seen on the spongy outer ZP of nonextracted samples. Occasionally, small, globular structures 80-120 nm in diameter were observed a t the points of intersection of the filaments of the large meshed networks (Fig. 5a,b). The ZP inner surface showed a homogeneous, fine network of filaments. In addition, numerous globular structures 80120 nm in diameter were seen a t the points of filament intersection (Fig. 6a,b). Immature or atretic oocytes. In these specimens, the outer ZP filaments were arranged in a homogeneous, tight meshed network (Fig. 5c). No areas of large meshed filaments, as were seen in the outer ZP of mature oocyte, were found. The inner ZP was characterized by the same type of filament arrangement as was observed in the inner surface of mature oocytes. Polypronuclear oua. The outer Z P of these specimens presented the same pattern of filament arrangement as was observed in mature oocytes. On the other hands, their inner ZP was different from the inner ZP of mature oocytes. In fact, the inner ZP surface of polypronuclear ova was characterized by the presence of areas of filaments fused together alternating with areas of filaments arranged as in the inner ZP of mature oocytes (Figs. 6c).
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microgranular structure (Phillips and Shalgi, 1980b). In addition, recent SEM and TEM studies in mammals confirm that the ZP is formed by granulofilamentous materials randomly arranged in a sort of “three-dimensional lattice” (Talbot and DiCarlantonio, 1984; Yudin et al., 1988; Familiari et al., 1989a; McGregor et al., 1989). Our observations represent a new approach to provide a finer description of the ultrastructural architecture of the ZP surrounding human oocytes and polypronuclear ova during in vitro fertilization. Two main points emerge from these finding: 1) ultrastructural methodology and 2) fine architecture of ZP and its functional meaning.
Ultrastructural Methodology
We evaluated the human Z P filament architecture of specimens treated with the detergent saponin and the cationic dye RR. SEM observations associated with the use of nonionic detergents have previously been successfully applied to the study of whole cultured cells of various types. These studies have demonstrated that extraction of the soluble fraction of intracellular proteins and lipids leaves a residual framework of insoluble filamentous elements that can be studied by SEM (Isobe and Shimada, 1983;Mitsushima and Katsumoto, 1990). On this basis, we applied a similar technique to characterize filaments present in an extracellular environment. Indeed, the treatment of mouse ovary with detergents for soluble proteins associated with RR, which stains and stabilizes the structural glycoproteins and polyanionic carbohydrates and, thus, prevents their dissolution-alteration by aqueous fixatives, allowed the demonstration by SEM-TEM of the ZP filament organization (Familiari et al., 1989a). In the present study, the metal-sputtering coating for SEM observation was substituted with an osmium and thiocarbohydrazide treatment, as modified from Kelley et al. (1973). This resulted in a hardening method able to protect the zona filaments from a considerable shrinkage in drying, even by the critical-point method. As a consequence, the best preservation of zona filaments obtained allowed a more detailed visualization of DISCUSSION Z P architecture by field emission SEM. What is the artifactual grade caused by the use of The ultrastructure of the ZP has been studied extensively via SEM and TEM in various mammalian spe- RR? It is known that addition of cationic dyes such as cies (for recent reviews, see Dietl, 1989; Phillips, 1991). RR results in a molecular collapse of polyanionic Via SEM, in nonhuman mammals, the outer aspect of chains, causing these to appear as condensed granules ZP of developing oocytes and eggs after fertilization and (Hunziker and Schenk, 1984). In our case, using RR cleavage has a spongy appearance (Motta and Van plus saponin and osmium-thiocarbohydrazide, we obBlerkom, 1974; Baranska et al., 1976; Phillips and served thin filaments anastomosed to form a fine netShalgi, 1980a; Longo, 1981). SEM of the Z P of human work with an evident reduction of the collapse and oocytes at the time of fertilization showed a spongy or shrinkage of the structures examined with respect to less frequently also a smooth and compact appearance specimens conventionally treated for SEM. This is (Sundstrom, 1982;Familiari et al., 1988).Spongy zonae more evident if outer Z P surfaces are compared in conwere seen most often in mature oocytes and were asso- trol and extracted specimens. In fact, the almost empty ciated with a more likely fertilization of the egg. On the fenestrations observed in control ZP (Fig. 3a) were ocother hand, a smooth ZP was characteristic of imma- cupied by a characteristic reticulum of thin filaments ture or atretic human oocytes (Familiari et al., 1988). in treated ZP (Fig. 5a). In addition, the structure of As revealed via SEM, the inner surface of the ZP has a filaments we observed in human ZP was similar to that
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Fig. 5. SEM. Extracted samples. a.Mature oocyte. Outer surface of the zona. Many fenestrations are present in which the filaments form a large meshed network (asterisks).Note the tight meshed arrangement of filaments in correspondence to branches (stars).CCM, cumulus corona cell matrix.(x9,000).b: Mature oocyte. Outer surface of the zona pellucida at high magnification. Different patterns of filament aggregation can be seen. Note that filaments appear as a “bead on a
string” structures (arrowheads). (X50,OOO). c: Atretic oocyte. Outer surface of the zona. The filaments form generally a tight meshed network. Note areas in which filaments seem to loose their compact pattern (asterisks) without reaching the typical loose meshed structure observed in mature oocytes. CCM, cumulus corona cell matrix. ( X 7,000).
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE
Fig. 6. SEM. Extracted samples. a: Mature oocyte. Inner surface of the zona pellucida. Note the presence of a homogeneous fine network of filaments. The filaments converge to globular structures (arrowheads). (~22,000).b: Mature oocyte. Inner surface of the zona pellu-
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cida at high magnification. Filaments appear as globule-bearing structures (arrowheads). (x75,OOO). c: Fertilized ovum (three pronuclei). Inner surface of the zona. Note the presence of bulging areas of condensed, closely packed filaments (asterisks).(x20,OOO).
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The tight arrangement of ZP filaments observed in immature and atretic oocytes might affect the physiological binding of the spermatozoon to the surface of their zonae. In the mouse, the glycoproteins ZP2 and ZP3 forming the repeating unit of ZP filaments are respectively the secondary and primary sperm receptors (Wassarman, 1990). If this model also applies to humans, we can hypothesize that a tight filament arrangement could hide the ZP2 and ZP3 receptors and thus could make the ZP of immature or atretic oocytes unable to bind the sperm. The inner ZP. The mid and inner ZP showed a tight meshed network of filaments in all types of oocytes. In fertilized (polypronuclear) ova, these filaments were often fused together. This filament condensation could be related to the changes in the inner ZP during fertilization. In fact, the penetration of the spermatozoon into the oocyte modifies the properties of the ZP, a process called “zona reaction”, which plays a role in the mechaFine Architecture of Z P and Its nisms related to the block of polyspermy. Functional Meaning In mammals, the “zona reaction” is induced by enThe outer ZP. SEM as well as TEM correlated data zymes released from cortical granules into the ZP after allowed us to demonstrate that the outer ZP, in imma- fertilization (“cortical reaction”) (Wassarman, 1988; ture and atretic oocytes, shows reduction of the size of Yanagimachi, 1988; Vincent et al., 1990). In human the meshes of the filamentous network. This feature oocytes used for IVF-ET, the cortical granule content is corresponds to the “smooth” appearance of outer ZP released in the inner ZP and then gradually diffuses to described in previous studies (Sundstrom, 1982; Famil- the mid-ZP. The process is accompanied by morphologiiari et al., 1988). With mature oocytes and fertilized cal changes that begin in the inner ZP and then are (polypronuclear) ova, we observed numerous areas of seen in the mid-ZP portion. These ZP changes consist large meshed networks of filaments, which presented mainly of the presence of “blobs or striae of dense matethe same distribution a s the previously described rial,” which have been defined as “the morphological “holes” of the “spongy” human ZP (Sundstrom, 1982; expression of the zona reaction” (Sathananthan and Familiari et al., 1988).As was previously demonstrated Trounson, 1982a). Thus only the inner ZP seems actuin pig, the outer ZP, due to mechanical stretching dur- ally to play a role in the block to polyspermy. In fact, ing the deposition of zona material, is normally more supplementary reacted spermatozoa are rarely found in expanded than the inner ZP (Diet1 and Czuppon, 1984). this region, which is usually more compact and electron In addition, i t was shown in humans that a “smooth” dense than the outer zona, even in unfertilized oocytes outer ZP is characteristic of immature and atretic (Sathananthan and Trounson, 1982a,b). These data oocytes, whereas a “spongy” outer Z P was seen in ma- correlate well with the observations performed in our ture oocytes. These ultrastructural features were re- samples in which only the inner ZP showed ultrastruclated to the fertilizability of oocytes in that the tural changes. “smooth” ZP appeared to bind fewer spermatozoa than On the basis of biochemical studies, the mechanism the “spongy” ZP (Familiari et al., 1988). Furthermore, inducing the ZP changes associated with fertilization spermatozoa preferably reach the oocyte passing seems to involve the ZP glycoproteins ZP1 and ZP2. In through the “holes” of the spongy ZP (Tsuiki et al., the mouse it was shown that ZP2 undergoes proteolysis 1986). In humans, the maintaining of a loose filament following either fertilization or artificial activation network in outer Z P of mature oocytes might be due to (Moller and Wassarman, 1989). It has been suggested some “softening” factor produced by cumulus-corona that ZP2 proteolysis could cause a ZP structural rearcells (Tesarik et al., 19881, which actually show a n in- rangement eventually related to the formation of filatense secretory activation during fertilization (Nottola ment reciprocal interactions, which might make the ZP e t al., 1991). Therefore, the loose filament arrangement insoluble to sperm (Wassarman, 1990). Such a filament of outer ZP seems very suitable to increase the perme- rearrangement, hypothesized in the mouse, has been ability of this matrix to the spermatozoa. The evidence demonstrated in humans with our morphological data. of a maturation of Z P parallel to the oocyte maturation In addition, our morphological results correlate with was obtained in both the rat and the human. In fact it biochemical data in humans. In fact, the observation of was shown that the ZP gradually increases its perme- biochemical changes of the ZP1 induced by egg cortical ability to the sperm or its sperm-binding potential from granule secretion in in vitro-fertilized polypronuclear the time of germinal vesicle breakdown to completion of ova was related to the role of ZP in the block of oocyte maturation (Tesarik et al., 1988; Tesarik, 1990; polyspermy (Shabanovitz and O’Rand, 1988a,b). It Rufas and Shalgi, 1990; Oehninger e t al., 1991). should be pointed out that, during the IVF-ET procereported previously for the mouse ZP (Wassarman, 1988, 1990). In fact, by solubilization with elastase of mouse ZP, filaments (2-3 pm in length and 7-18 nm in thickness), structurally similar to that observed in situ in our human specimens, were shown on TEM (Wassarman, 1988,1990). However, the elastase solubilization does not preserve the architectural organization of the ZP but yields the visualization of only isolated filaments. Our method allows the study of the arrangement of the filaments. It should be noted that in our measurements we found filaments shorter than those described by Wassarman (1988, 1990). Actually, we measured the length of the filaments from end to end at the converging point between filaments or in correspondence to the point of their intersection, whereas the measurements in the previous study were obtained in entire, isolated filaments.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE dure, polyspermy may occur in oocytes, with the development of polypronuclear ova, independent from the activation of “zona reaction.” This is because a very large number of spermatozoa reach a n oocyte lacking the filter of the cumulus-corona cells, which are often lost prior to fertilization and which seem to cooperate in the selection of the sperm (Nottola et al., 1991).
ACKNOWLEDGMENTS This study was supported by funds from the “Ministero dell’universita e della Ricerca Scientifica e Tecnologica” (60% and 40%) and the “Consiglio Nazionale delle Ricerche” 1987-90. Thanks are due Mr. Antonio Familiari for the planning and construction of the steel microchamber for SEM preparation and for his cooperation in developing the ultrastructural methodology.
REFERENCES Aragona C, Micara G (1987): Morphological features of aspirated human oocytes in an IVF program. In Spera G, de Kretser DM (eds): “Morphological Basis of Human Reproductive Function.” New York; Plenum Press, Rome; Acta Medica, pp 135-142. Baranska W, Konwinski M, Kujawa M (1976): Fine structure of the zona pellucida of unfertilized egg cells and embryos. J Exp Zool 192:193-202. Dietl J (1989): “The Mammalian Egg Coat. Structure and Function.” Berlin; Springer-Verlag. Dietl J , Czuppon AB (1984): Ultrastructural studies of the porcine zona pellucida during the solubilization process by Li-3,5-diiodosalicylate. Gamete Res 9:45-54. Familiari G, Makabe S, Motta PM (1991): “Ultrastructure of the Ovary. Boston: “Kluwer Academic Publishers. Familiari G, Nottola SA, Familiari A, Motta PM (1989a): The threedimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse. Scanning and transmission electronmicroscopic observations using ruthenium red and detergents. Cell Tissue Res 257:247-253. Familiari G, Nottola SA, Micara G, Aragona C, Motta PM (1988): Is the sperm-binding capability of the zona pellucida linked to its surface structure? A scanning electron microscopic study of human in vitro fertilization. J In Vitro Fertil Embryo Transfer 5:136143. Familiari G, Nottola SA, Micara G, Aragona C, Motta PM (1989b): Human in vitro fertilization: The fine three-dimensional architecture of the zona pellucida. Prog Clin Biol Res 296:335-344. Familiari G, Nottola SA, Motta PM (1987):Focal cell contacts detected by ruthenium red, triton XlOO and saponin in the granulosa cell of mouse ovary. Tissue Cell 19:207-215. Hunziker EB, Schenk RK (1984): Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. 11. Intercellular matrix ultrastructure-Preservation of proteoglycans in their native state. J Cell Biol98:277-282. Isobe Y, Shimada Y (1983): Myofibrillogenesis in vitro as seen with the scanning electron microscope. Cell Tissue Res 231:481494. Kelley RO, Dekker RAF, Bluemink J G (1973): Ligand-mediated osmium binding: Its application in coating biological specimens for scanning electron microscopy. J Ultrastruct Res 45:254-258. Longo FJ (1981): Changes in the zonae pellucidae and plasmalemmae of aging mouse eggs. Biol Reprod 25:399-411. Lopata A (1983): Concepts in human in vitro fertilization and embryo transfer. Fertil Steril40:289-301. McGregor L, Flaherty SP, Breed WG (1989): Structure of the zona pellucida and cumulus oophorus in three species of native australian rodents. Gamete Res 23:279-287. Mitsushima A, Katsumoto T (1990): A preparation technique for observing cytoskeletons by high resolution scanning electron microscopy. J Submicrosc Cytol Pathol22:591-599. Moller CC, Wassarman PM (1989): Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs. Dev Biol 132:103-112.
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Motta PM, Van Blerkom J (1974): A scanning electron microscopic study of the luteo-follicular complex. I. Follicle and oocyte. J Submicrosc Cytol6:297-310. Nottola SA, Familiari G , Micara G, Aragona C, Motta PM (1991):The ultrastructure of human cumulus-corona cells a t the time of fertilization and early embryogenesis. A scanning and transmission electron microscopic study in an in vitro fertilization program. Arch Histol Cytol54:145-161. Oehninger S, Veeck L, Franken D, Kruger TF, Acosta AA, Hodgen GD (1991): Human preovulatory oocytes have a higher sperm-binding ability than immature oocytes under hemizona assay conditions: Evidence supporting the concept of “zona maturation.” Fertil Steril 55: 1165-1170. Phillips DM (1991): Structure and function of the zona pellucida. In Familiari G, Makabe S, Motta PM (eds): “Ultrastructure of the Ovary.” Boston; Kluwer Academic Publishers, pp 63-72. Phillips DM, Shalgi RM (1980a): Surface architecture of the mouse and hamster zona pellucida and oocyte. J Ultrastruct Res 72:l-12. Phillips DM, Shalgi RM (1980b): Surface properties of the zona pellucida. J Exp Zool 213:1-8. Reynolds ES (1963):The use of lead citrate a t high pH as an electronopaque stain in electron microscopy. J Cell Biol 17:208-212. Rufas 0, Shalgi RM (1990): Maturation-associated changes in the rat zona pellucida. Mol Reprod Dev 26:32&330. Sathananthan AH, Trounson A 0 (1982a): Ultrastructure of cortical granule release and zona interaction in monospermic and polyspermic human ova fertilized in vitro. Gamete Res 6:225-234. Sathananthan AH, Trounson A 0 (1982b): Ultrastructural observations on cortical granules in human follicular oocytes cultured in vitro. Gamete Res 5:191-198. Shabanovitz RB, ORand MG (1988a): Molecular changes in the human zona pellucida associated with fertilization and human spermzona interactions. Ann NY Acad Sci 541:621-632. Shabanovitz RB, ORand MG (1988b): Characterization of the human zona pellucida from fertilized and unfertilized eggs. J Reprod Fertil 82:151-161. Sundstrom P (1982): Interaction between spermatozoa and ovum in vitro. In Hafez ESE, Kenemans P (eds.)“Atlas of Human Reproduction by Scanning Electron Microscopy.” Lancaster; MTP Press, pp 225-230. Talbot P (1985): Sperm penetration through oocyte investments in mammals. Am J Anat 174:331-346. Talbot P, DiCarlantonio G (1984): The oocyte cumulus complex: Ultrastructure of the extracellular components in hamsters and mice. Gamete Res 10:127-142. Tesarik J (1990): Zona pellucida penetrability of metaphase I and I1 human oocytes after aging and salt treatment. Fertil Steril54:346347. Tesarik J, Pilka L, Travnik P (1988): Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation. J Reprod Fertil83:487495. Tsuiki A, Hoshiai H, Takahashi K, Suzuki M, Hoshi K (1986):Spermegg interactions observed by scanning electron microscopy. Arch Androl 1 6 3 5 4 7 . Vincent C, Pickering SJ Johnson MH (1990): The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present. J Reprod Fertil89:253-259. Wassarman PM (1988): Zona pellucida glycoproteins. Ann Rev Biochem 57:415-442. Wassarman PM (1990): Zona pellucida glycoproteins: regulators of mammalian fertilization. In Evers, JHL Heineman MJ (eds):“From Ovulation to Implantation.” Amsterdam; Elsevier Science Publishers, pp 239-250. Yanagimachi R (1988):Mammalian Fertilization. In Knobil E, Neil1 J (eds): “The Physiology of Reproduction. Vol 1.”New York; Raven Press, pp 135-185. Yudin AI, Cherr GN, Katz DF (1988): Structure of the cumulus matrix and zona pellucida in the golden hamster: A new view of sperm interaction with oocyte-associated extracellular matrices. Cell Tissue Res 251:555-564.
Human Zona Pellucida During In Vitro Fertilization: An Ultrastructural Study Using Saponin, Ruthenium Red, and Osmium-Thiocarbohydrazide GIUSEPPE FAMILIARI,’ STEFANIA A. NOTTOLA,’ GUIDO MACCHIARELLI,’ GIULIETTA MICARAF CESARE ARAGONAF AND PIETRO M. MOTTA’ ‘Department of H u m a n Anatomy and Assisted Reproduction Unit, University of Rome ‘Za Sapienza,” Rome, Italy ABSTRACT The human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one- to four-cells stages were studied by transmission electron microscopy (IEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin),and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by 0smium-thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1-0.4 k m in length and 10-14 nm thick as seen via TEM or 22-28 nm thick as seen via SEM (thedifference in thickness was due to the presence of the metal coating for SEM).The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in “large” and “tight”meshed networks. Mature oocytes and fertilized (polypronuclear)ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration,and selection of spermatozoa. 0 1992 Wiley-Liss, Inc.
Key Words: Oocyte, Polypronuclear ovum, Fertilization
INTRODUCTION During mammalian fertilization, the spermatozoon reaches the zona pellucida (ZP), a special egg coating made up of the glycoproteins ZPl,ZP2, and ZP3 (Was-
0 1992 WILEY-LISS, INC.
sarman, 1988,19901, binds to its surface, penetrates it, and reaches the oocyte membrane (Talbot, 1985; Yudin et al., 1988; Dietl, 1989). The ZP is known to play a crucial role in the regulation of sperm-egg interactions. In fact, in mammals, the ZP chemicophysical properties change drastically upon fertilization, (Wassarman, 1990), and a process called “zona reaction” occurs that contributes to inhibition of supernumerary spermatozoa penetration (Yanagimachi, 1988).Despite the many studies of these events, the ZP-sperm interactions are still not fully understood (Talbot, 1985; Yanagimachi, 1988). A recent review suggests that a three-dimensional approach to the study of these events may contribute to filling the gap in knowledge of the morphology (Familiari et al., 1991). However, to date only a few scanning electron microscopic (SEM) observations on the human ZP during fertilization have been made (Motta and Van Blerkom, 1974; Phillips and Shalgi, 1980a,b; Sundstrom, 1982; Familiari e t al., 1988, 198913). In fact, the material of the ZP, due to the presence of a large amount of surrounding extracellular amorphous material and due to the hydrophilic peculiarity of its components, is particularly hard to define through SEM observations without appropriate techniques to unmask and stabilize its components. To study the fine ultrastructure of the ZP, including its changes during fertilization, with particular regard to the three-dimensional arrangement of its components, the zona material has been investigated via SEM and transmission electron microscopy (TEM) in oocytes (at various developing stages) and polypronuclear ova (PO) (between one-cell and four-cell stages) deriving from a human in vitro fertilization and embryo transfer (IVF-ET) program. With this purpose, the specimens were treated with saponin, a detergent that permits the removal of the amorphous materials masking the real ZP structure. Then they were contrasted with ruthenium red (RR), a cationic dye that, together with 0smium and thiocarbohydrazide impregnation, stabilizes
Received November 19,1991; accepted January 2,1992. Address reprint requests to Prof. Giuseppe Familiari, Department of Anatomy, University of Rome “La Sapienza,” Via A. Borelli 50,00161 Rome, Italy.
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G. FAMILIAR1 ET AL. TABLE 1. Oocytes and Polypronuclear Ova Recovery and Fixation Stage at recovery Stage at fixation Oocytes Immature 13 Immaturea 8 Mature 31 Maturea 35c Atretic 6 Atretica 7d Polypronuclear ova Mature oocytes
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Fertilized ova (three pronuclei)b Two-cell polypronuclear ovab Three-cell polypronuclear ovab Four-cell polypronuclear ovab
4 1 3 2
aAfter 1-3 days of culture. bAfter 2-4 days of culture. “Five immature oocytes matured in vitro. mature oocyte degenerated in vitro. the structural glycoproteins of the ZP for high-resolution SEM observations (Familiari et al., 1987,1989a).
MATERIALS AND METHODS Protocol Eight immature, noninseminated oocytes, 35 inseminated but nonfertilized mature oocytes, seven atretic (postmature) inseminated but nonfertilized oocytes, and ten polypronuclear ova a t one-cell to four-cell stages were obtained from follicular aspirates from patients undergoing TVF-ET (Lopata, 1983) (Table l).All specimens were studied with the patients’ consent and represent material that normally would be discarded. All specimens were analyzed by phase-contrast light microscopy (LM) prior to electron microscopic procedures. The LM criteria for oocyte staging were the gen-
eral appearance of the cytoplasm and the presence of the germinal vesicle and polar body, according to a previously described classification (Aragona and Micara, 1987; Familiari et al., 1988). For ethical reasons, only polypronuclear ova displaying three or more pronuclei at the inverted bright-field microscopic level were studied. Thirty oocytes and six polypronuclear ova underwent chemical extraction a s described below. Twenty oocytes and four polypronuclear ova (control) were directly fixed in a glutaraldehyde solution and postfixed in osmium tetroxide (Familiari et al., 1988).
Extraction Procedure The specimens were washed in a cacodylate buffer solution (CBS) and incubated in a 0.02% saponin and
Fig. 1. Phase-contrast microscopy a: Mature preovulatory oocyte after insemination. b: Immature oocyte after 24 hr of culture. c: Atretic oocyte at insemination. d: Polypronuclearovum at three pronuclei stage after 22 hr of culture. e: Four-cell polypronuclear ovum 48 hr after insemination. x 160.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE
Fig. 2. Control samples. a:SEM. Mature oocyte. The zona pellucida appears spongy. Penetrating spermatozoa (arrows). x 900. b: TEM. Mature oocyte (MO). The zona pellucida (ZP) has a microgranular quasihomogeneous texture. Note the irregular margin (arrowheads). x 1,500. c: SEM. Atretic oocyte. The zona pellucida shows a completely smooth surface. ~ 7 0 0 d: . TEM. Early atretic oocyte (AO). The zona pellucida (ZP) has a microgranular appearance. Note its regular and
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compact outlined surface (arrowheads). x 1,500. e: SEM. Two-cell polypronuclear ovum. The zona pellucida shows a spongy surface. CC, cumulus corona cell. x 1,500. f: TEM. Two-cell polypronuclear ovum (PO). The zona pellucida (ZP) has a quasihomogeneous texture. An irregular margin is present similar to that of mature oocytes (arrowheads). X 1 , 5 0 0 .
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1.0%RR mixture, both in 0.1 M CBS a t pH 7.4 for 30 min. The specimens were then fixed in 3.0% glutaraldehyde plus 1.0% RR and 0.02% saponin in CBS 4°C. The following day, the specimens were washed in CBS containing 1.0% RR and 0.02% saponin, postfixed for 1h r in 1.0% OsO, in CBS containing 0.75% RR and 0.02% saponin (Familiari et al., 1989a), then again washed in CBS, and finally prepared for TEM or SEM.
SEM of Extracted and Control Samples Twenty oocytes and three polypronuclear ova (extracted samples) as well as ten oocytes and two polypronuclear ova (control samples) were treated with thiocarbohydrazide (1.0%) and osmium tetroxide (1.0%) in distilled water (Kelley et al., 1973). The specimens were rinsed in distilled water and placed in a steel microchamber whose base and top were 400-mesh copper grids (Familiari et al., 1988).The cells were then dehydrated in acetone, critical-point dried, and mounted on aluminium stubs. TEM of Extracted and Control Samples Ten oocytes and three polyopronuclear ova (extracted samples) as well a s ten oocytes and two polypronuclear ova (control samples) were dehydrated in ethanol and embedded in a n epoxydic resin. Sectioning was performed with LKB and Reichert ultramicrotomes. Some ultrathin sections were stained with lead citrate (Reynolds, 1963), while others were examined without further staining. Observations Both the external and the internal surfaces of the ZP were studied via SEM. The ZP of some oocytes was fractured by means of a needle to expose the internal surface. SEM observations were made in Jeol S.800 and Hitachi S.4000 field emission scanning electron microscopes operating at 5-10 kV. TEM observations were made with a Zeiss EM9 S2 electron microscope. RESULTS Light Microscopy Mature oocytes showed a homogeneous and clear ooplasm as well as the presence of the first polar body. No pronuclei were found in these oocytes (Fig. la). Some of the immature oocytes had a clear cytoplasm and a n intact germinal vescicle, while others had undergone germinal vescicle breakdown but did not show the first polar body (Fig. lb). The atretic (postmature) oocytes displayed a dark, granular, retracted ooplasm and a large perivitelline space (Fig. lc). The one-cell polypronuclear ova contained three or more pronuclei (Fig. Id). Polypronuclear ova a t later developmental stages showed both a normal cleavage and blastomeres of normal size (Fig. le) as well a s a fragmented cleavage.
Electron Microscopy Control specimens. Mature oocytes. By SEM, the outer surface of the ZP was usually characterized by a spongy appearance, due to the presence of numerous fenestrations (Fig. 2a). In addition, the outer ZP showed a fine, downy surface (Fig. 3a). The inner surface of the ZP was clearly smooth (Fig. 3b) and, a t high magnification, showed a homogeneous microtrabecular structure (Fig. 3c). Via TEM, the ZP had a homogeneous microgranular appearance throughout its thickness and was characterized by a n irregular, rough outer margin (Fig. 2b). Zmmature and atretic oocytes. By SEM, in most of the immature and atretic oocytes, the outer surface of the ZP was smoother than t h a t of the mature oocytes. Fenestrations of this outer surface were rare or even absent in most samples (Fig. 2c). The inner surface of the ZP was smooth and similar to that of preovulatory mature oocytes. TEM showed the characteristic microgranular appearance of the ZP, but a regularly smooth outer margin was seen (Fig. 2d). Polypronuclear ova. By SEM, almost all the polypronuclear ova had a n intact ZP with a spongy, fenestrated outer surface (Fig. 2e) and a smooth inner surface, comparable to that seen in mature oocytes. TEM observations showed a microgranular texture similar to that observed in most of the mature oocytes (Fig. 20. Extracted specimens. Parallel SEM and TEM examinations of specimens treated with saponin and RR provided a valuable technical approach to the study of the ZP microarchitecture. However, i t should be noted that the addition of saponin to the fixative inevitably produced some alterations in the cytoplasmic morphology of the oocyte (Fig. 4a, b). TEM observations. TEM examination of the ZP in all types of oocytes and polypronuclear ova revealed a fine network of RR-positive filaments oriented in several directions and anastomosed with each other. These filaments measured 10-14 nm in thickness and 0.1-0.4 pm in length. Four types of arrangement of filaments were demonstrated in the different specimens studied (Fig. 4a-f). Type 1 was characterized by filaments arranged in a very tight meshed network. In type 2 the meshes of the filament network were less condensed than seen in Type 1. I n type 3 filaments were arranged in a loose or large meshed network. In type 4 the filaments were focally fused together to form a homogeneous structure without a reticular appearance. Mature oocytes. Most of mature oocytes had a ZP characterized by filaments arranged in a regular network. In the inner portion, this network was of type 1 (Fig. 4a, c), whereas the outer ZP portion presented a regular alternating pattern of zones with filaments arranged as in type 2 or type 3 (Fig. 4e). Zmmature and atretic oocytes. The immature and atretic oocyte Z P showed filaments arranged in the inner portion as in type 1 and in the outer portion as in type 2. Unlike the case in mature oocytes, the ZP outer portion of immature and atretic oocytes did not present areas with filaments arranged a s in type 3 (Fig. 40.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE
Fig. 3. Control samples. a: SEM. Mature oocyte. At high magnification the outer surface of the zona pellucida revealed a spongy organization. Note the microtrabecular appearance of branches (stars) and holes (asterisks). ( x 18,000).b,c:SEM. Mature oocyte. The inner surface of the zona pellucida has a very fine microtrabecular appearance. (b; x525 c; X24,OOO).
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Fig. 4. TEM. Extracted samples. a: Mature oocyte (MO).The zona pellucida (ZP)displays a regular network of filaments. Note the oocyte (MO) cellular changes due to saponin extraction, characterized by cytoplasm dissolution. Boxed area is enlarged in c. (x4,800). b: Fourcell polypronuclear ovum (PO). Areas of condensation are regularly distributed in the inner portion (arrows) of the zona pellucida (ZP). Note the ovum (PO) changes due to saponin extraction, characterized by cytoplasm dissolution. Boxed area is enlarged in d. (X4.800). c: Mature oocyte. High-power view of the boxed area in a. The fine network of filaments is associated with small granules a t the nodal
points of the reticulum (arrows). (x27,700). d Four-cell polypronuclear ovum. High-power view of boxed area in b. In the network of filaments, areas of major condensation are present (arrowheads). ( X 27,700). e: Mature oocyte. High-power view of the outer portion of the zona pellucida (ZP). A regular alternating pattern of zones with filaments arranged in tight (arrowheads) or loose (arrows) meshed network was present. ( x 27,700). E Atretic oocyte. High-power view of the outer portion of the zona (ZP). The filaments are arranged in a tight meshed network (arrowheads). x27,OOO.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE Polypronuclear ova. The ZP of polypronuclear ova was formed in the inner part mainly by filaments arranged as in type 1 and by numerous areas characterized by filament condensation as in type 4 (Fig. 4b,d). On the other hand, the outer part of the ZP displayed filaments arranged in a wider network, with a regular alternating of pattern type 2 and type 3, as seen in the outer ZP of matureoocytes (Fig. 4b). SEM examination. SEM allowed a detailed visualization of the filament arrangement of ZP in extracted specimens. The filaments measured 22-28 nm in thickness and 0.1-0.4 pm in length and, at high magnification, showed a globular structure (Figs. 5, 6). Remarkable differences of filament arrangement were seen in all types of specimens. Mature oocytes. The ZP outer surface in mature oocytes was characterized by filaments arranged in a regular pattern of alternating tight meshed and large meshed networks. The large meshed network had the same distribution of fenestrations as was seen on the spongy outer ZP of nonextracted samples. Occasionally, small, globular structures 80-120 nm in diameter were observed a t the points of intersection of the filaments of the large meshed networks (Fig. 5a,b). The ZP inner surface showed a homogeneous, fine network of filaments. In addition, numerous globular structures 80120 nm in diameter were seen a t the points of filament intersection (Fig. 6a,b). Immature or atretic oocytes. In these specimens, the outer ZP filaments were arranged in a homogeneous, tight meshed network (Fig. 5c). No areas of large meshed filaments, as were seen in the outer ZP of mature oocyte, were found. The inner ZP was characterized by the same type of filament arrangement as was observed in the inner surface of mature oocytes. Polypronuclear oua. The outer Z P of these specimens presented the same pattern of filament arrangement as was observed in mature oocytes. On the other hands, their inner ZP was different from the inner ZP of mature oocytes. In fact, the inner ZP surface of polypronuclear ova was characterized by the presence of areas of filaments fused together alternating with areas of filaments arranged as in the inner ZP of mature oocytes (Figs. 6c).
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microgranular structure (Phillips and Shalgi, 1980b). In addition, recent SEM and TEM studies in mammals confirm that the ZP is formed by granulofilamentous materials randomly arranged in a sort of “three-dimensional lattice” (Talbot and DiCarlantonio, 1984; Yudin et al., 1988; Familiari et al., 1989a; McGregor et al., 1989). Our observations represent a new approach to provide a finer description of the ultrastructural architecture of the ZP surrounding human oocytes and polypronuclear ova during in vitro fertilization. Two main points emerge from these finding: 1) ultrastructural methodology and 2) fine architecture of ZP and its functional meaning.
Ultrastructural Methodology
We evaluated the human Z P filament architecture of specimens treated with the detergent saponin and the cationic dye RR. SEM observations associated with the use of nonionic detergents have previously been successfully applied to the study of whole cultured cells of various types. These studies have demonstrated that extraction of the soluble fraction of intracellular proteins and lipids leaves a residual framework of insoluble filamentous elements that can be studied by SEM (Isobe and Shimada, 1983;Mitsushima and Katsumoto, 1990). On this basis, we applied a similar technique to characterize filaments present in an extracellular environment. Indeed, the treatment of mouse ovary with detergents for soluble proteins associated with RR, which stains and stabilizes the structural glycoproteins and polyanionic carbohydrates and, thus, prevents their dissolution-alteration by aqueous fixatives, allowed the demonstration by SEM-TEM of the ZP filament organization (Familiari et al., 1989a). In the present study, the metal-sputtering coating for SEM observation was substituted with an osmium and thiocarbohydrazide treatment, as modified from Kelley et al. (1973). This resulted in a hardening method able to protect the zona filaments from a considerable shrinkage in drying, even by the critical-point method. As a consequence, the best preservation of zona filaments obtained allowed a more detailed visualization of DISCUSSION Z P architecture by field emission SEM. What is the artifactual grade caused by the use of The ultrastructure of the ZP has been studied extensively via SEM and TEM in various mammalian spe- RR? It is known that addition of cationic dyes such as cies (for recent reviews, see Dietl, 1989; Phillips, 1991). RR results in a molecular collapse of polyanionic Via SEM, in nonhuman mammals, the outer aspect of chains, causing these to appear as condensed granules ZP of developing oocytes and eggs after fertilization and (Hunziker and Schenk, 1984). In our case, using RR cleavage has a spongy appearance (Motta and Van plus saponin and osmium-thiocarbohydrazide, we obBlerkom, 1974; Baranska et al., 1976; Phillips and served thin filaments anastomosed to form a fine netShalgi, 1980a; Longo, 1981). SEM of the Z P of human work with an evident reduction of the collapse and oocytes at the time of fertilization showed a spongy or shrinkage of the structures examined with respect to less frequently also a smooth and compact appearance specimens conventionally treated for SEM. This is (Sundstrom, 1982;Familiari et al., 1988).Spongy zonae more evident if outer Z P surfaces are compared in conwere seen most often in mature oocytes and were asso- trol and extracted specimens. In fact, the almost empty ciated with a more likely fertilization of the egg. On the fenestrations observed in control ZP (Fig. 3a) were ocother hand, a smooth ZP was characteristic of imma- cupied by a characteristic reticulum of thin filaments ture or atretic human oocytes (Familiari et al., 1988). in treated ZP (Fig. 5a). In addition, the structure of As revealed via SEM, the inner surface of the ZP has a filaments we observed in human ZP was similar to that
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Fig. 5. SEM. Extracted samples. a.Mature oocyte. Outer surface of the zona. Many fenestrations are present in which the filaments form a large meshed network (asterisks).Note the tight meshed arrangement of filaments in correspondence to branches (stars).CCM, cumulus corona cell matrix.(x9,000).b: Mature oocyte. Outer surface of the zona pellucida at high magnification. Different patterns of filament aggregation can be seen. Note that filaments appear as a “bead on a
string” structures (arrowheads). (X50,OOO). c: Atretic oocyte. Outer surface of the zona. The filaments form generally a tight meshed network. Note areas in which filaments seem to loose their compact pattern (asterisks) without reaching the typical loose meshed structure observed in mature oocytes. CCM, cumulus corona cell matrix. ( X 7,000).
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE
Fig. 6. SEM. Extracted samples. a: Mature oocyte. Inner surface of the zona pellucida. Note the presence of a homogeneous fine network of filaments. The filaments converge to globular structures (arrowheads). (~22,000).b: Mature oocyte. Inner surface of the zona pellu-
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cida at high magnification. Filaments appear as globule-bearing structures (arrowheads). (x75,OOO). c: Fertilized ovum (three pronuclei). Inner surface of the zona. Note the presence of bulging areas of condensed, closely packed filaments (asterisks).(x20,OOO).
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The tight arrangement of ZP filaments observed in immature and atretic oocytes might affect the physiological binding of the spermatozoon to the surface of their zonae. In the mouse, the glycoproteins ZP2 and ZP3 forming the repeating unit of ZP filaments are respectively the secondary and primary sperm receptors (Wassarman, 1990). If this model also applies to humans, we can hypothesize that a tight filament arrangement could hide the ZP2 and ZP3 receptors and thus could make the ZP of immature or atretic oocytes unable to bind the sperm. The inner ZP. The mid and inner ZP showed a tight meshed network of filaments in all types of oocytes. In fertilized (polypronuclear) ova, these filaments were often fused together. This filament condensation could be related to the changes in the inner ZP during fertilization. In fact, the penetration of the spermatozoon into the oocyte modifies the properties of the ZP, a process called “zona reaction”, which plays a role in the mechaFine Architecture of Z P and Its nisms related to the block of polyspermy. Functional Meaning In mammals, the “zona reaction” is induced by enThe outer ZP. SEM as well as TEM correlated data zymes released from cortical granules into the ZP after allowed us to demonstrate that the outer ZP, in imma- fertilization (“cortical reaction”) (Wassarman, 1988; ture and atretic oocytes, shows reduction of the size of Yanagimachi, 1988; Vincent et al., 1990). In human the meshes of the filamentous network. This feature oocytes used for IVF-ET, the cortical granule content is corresponds to the “smooth” appearance of outer ZP released in the inner ZP and then gradually diffuses to described in previous studies (Sundstrom, 1982; Famil- the mid-ZP. The process is accompanied by morphologiiari et al., 1988). With mature oocytes and fertilized cal changes that begin in the inner ZP and then are (polypronuclear) ova, we observed numerous areas of seen in the mid-ZP portion. These ZP changes consist large meshed networks of filaments, which presented mainly of the presence of “blobs or striae of dense matethe same distribution a s the previously described rial,” which have been defined as “the morphological “holes” of the “spongy” human ZP (Sundstrom, 1982; expression of the zona reaction” (Sathananthan and Familiari et al., 1988).As was previously demonstrated Trounson, 1982a). Thus only the inner ZP seems actuin pig, the outer ZP, due to mechanical stretching dur- ally to play a role in the block to polyspermy. In fact, ing the deposition of zona material, is normally more supplementary reacted spermatozoa are rarely found in expanded than the inner ZP (Diet1 and Czuppon, 1984). this region, which is usually more compact and electron In addition, i t was shown in humans that a “smooth” dense than the outer zona, even in unfertilized oocytes outer ZP is characteristic of immature and atretic (Sathananthan and Trounson, 1982a,b). These data oocytes, whereas a “spongy” outer Z P was seen in ma- correlate well with the observations performed in our ture oocytes. These ultrastructural features were re- samples in which only the inner ZP showed ultrastruclated to the fertilizability of oocytes in that the tural changes. “smooth” ZP appeared to bind fewer spermatozoa than On the basis of biochemical studies, the mechanism the “spongy” ZP (Familiari et al., 1988). Furthermore, inducing the ZP changes associated with fertilization spermatozoa preferably reach the oocyte passing seems to involve the ZP glycoproteins ZP1 and ZP2. In through the “holes” of the spongy ZP (Tsuiki et al., the mouse it was shown that ZP2 undergoes proteolysis 1986). In humans, the maintaining of a loose filament following either fertilization or artificial activation network in outer Z P of mature oocytes might be due to (Moller and Wassarman, 1989). It has been suggested some “softening” factor produced by cumulus-corona that ZP2 proteolysis could cause a ZP structural rearcells (Tesarik et al., 19881, which actually show a n in- rangement eventually related to the formation of filatense secretory activation during fertilization (Nottola ment reciprocal interactions, which might make the ZP e t al., 1991). Therefore, the loose filament arrangement insoluble to sperm (Wassarman, 1990). Such a filament of outer ZP seems very suitable to increase the perme- rearrangement, hypothesized in the mouse, has been ability of this matrix to the spermatozoa. The evidence demonstrated in humans with our morphological data. of a maturation of Z P parallel to the oocyte maturation In addition, our morphological results correlate with was obtained in both the rat and the human. In fact it biochemical data in humans. In fact, the observation of was shown that the ZP gradually increases its perme- biochemical changes of the ZP1 induced by egg cortical ability to the sperm or its sperm-binding potential from granule secretion in in vitro-fertilized polypronuclear the time of germinal vesicle breakdown to completion of ova was related to the role of ZP in the block of oocyte maturation (Tesarik et al., 1988; Tesarik, 1990; polyspermy (Shabanovitz and O’Rand, 1988a,b). It Rufas and Shalgi, 1990; Oehninger e t al., 1991). should be pointed out that, during the IVF-ET procereported previously for the mouse ZP (Wassarman, 1988, 1990). In fact, by solubilization with elastase of mouse ZP, filaments (2-3 pm in length and 7-18 nm in thickness), structurally similar to that observed in situ in our human specimens, were shown on TEM (Wassarman, 1988,1990). However, the elastase solubilization does not preserve the architectural organization of the ZP but yields the visualization of only isolated filaments. Our method allows the study of the arrangement of the filaments. It should be noted that in our measurements we found filaments shorter than those described by Wassarman (1988, 1990). Actually, we measured the length of the filaments from end to end at the converging point between filaments or in correspondence to the point of their intersection, whereas the measurements in the previous study were obtained in entire, isolated filaments.
HUMAN ZONA PELLUCIDA ULTRASTRUCTURE dure, polyspermy may occur in oocytes, with the development of polypronuclear ova, independent from the activation of “zona reaction.” This is because a very large number of spermatozoa reach a n oocyte lacking the filter of the cumulus-corona cells, which are often lost prior to fertilization and which seem to cooperate in the selection of the sperm (Nottola et al., 1991).
ACKNOWLEDGMENTS This study was supported by funds from the “Ministero dell’universita e della Ricerca Scientifica e Tecnologica” (60% and 40%) and the “Consiglio Nazionale delle Ricerche” 1987-90. Thanks are due Mr. Antonio Familiari for the planning and construction of the steel microchamber for SEM preparation and for his cooperation in developing the ultrastructural methodology.
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