Homogeneity in the distribution of matrix components in the hamster zona pellucida as revealed by backscattered electron imaging fracture-label.код для вставкиСкачать
THE ANATOMICAL RECORD 239:35-46 (1994) Homogeneity in the Distribution of Matrix Components in the Hamster Zona Pellucida as Revealed by Backscattered Electron Imaging Fracture-Label FREDERICK W.K. KAN, SYLVIA ZALZAL, EMMANUELLE ROUX, AND ANTONIO NANCI Department of Anatomy (F.W.K.K., E.R., A.N.), Faculty of Medicine and Department of Stomatology (S.Z., A.N.), Faculty of Dentistry, Uniuersite de Montreal, C.P. 6128, Succ. A, Montreal, Quebec, Canada ABSTRACT Background: Backscattered electron imaging fracture-labe1 (BEI-FL),an adaptation of the fracture-label method for scanning electron microscopy, offers the advantage of providing information about the distribution of antigenic and receptor sites with respect to the three-dimensional organization of tissues and cells over relatively large surfaces. Recently, using post-embedding cytochemistry on thin-sections of Lowicrylembedded oocytes, a homogenous distribution of glycoproteins in the zona pellucida (ZP) was demonstrated (Kan et al., 1989. Biol. Reprod., 40:585-598, Anat. Rec., 226:3747; Roux and Kan, 1991. Anat. Rec., 230:347-360). However, it can be argued that the chemical nature of resins and the physical conditions of tissue processing required for post-embedding cytochemistry may introduce changes in the tissue components and result in altered distribution of components. On the other hand, freeze-fracture exposes constituents in a minimally denaturing manner and, since no embedding media are used, binding sites are sterically available to the probe. We have, therefore, applied BEI-FL to examine the distribution of matrix glycoproteins in the Z P of hamster oocytes. Methods: Ovaries and cumulus masses obtained from superovulated female golden hamsters were fixed by immersion in 2.5%glutaraldehyde and processed for fracture-label. Tissues were labeled, respectively, with Wheat germ agglutinin (WGA)followed by ovomucoid-colloidalgold, Ricinus communis agglutinin I (RCA I)-colloidal gold or a monoclonal antibody against Hamster Oviductin-1 followed by protein A-gold, and then examined in the scaiming electron microscope. Results: Backscattered electron imaging revealed a homogenous distribution of WGA and RCA I binding sites throughout the cross-fractured matrix of the Z P of ovarian and postovulatory oocytes. Hamster Oviductin-1, an oviductal glycoprotein which is transferred to the Z P of oocytes during oviductal transit, was also found to be uniformly distributed throughout the Z P of postovulatory oocytes. Conclusions: Our results indicate that BEI-FL can be advantageously used to examine extracellular matrices and are consistent with the concept that glycoproteins are uniformly distributed throughout the Z P of the hamster Oocyte. 0 1994 Wiley-Liss, Inc. Key words: Fracture-label method, oviductin, zona pellucida, oocytes, cytochemistry, scanning electron microscopy, lectins The zona pellucida (ZP)is a n egg investment that mammalian spermatozoa have to traverse in order to initiate fertilization. Thus, this extracellular matrix surrounding oocytes occupies a unique Position within the female reproductive system for its consideration as a potential target for immunocontra0 1994 WILEY-LISS, INC Received September 9, 1993; accepted December 20, 1993, Address reprint requests to Dr. Frederick W.K. Kan, Department of Anatomy, Faculty of Medicine, Universite de Montreal, C.P. 6128, Succ. A, Montreal, Quebec, Canada, H3C 357. 36 F.W.K. KAN ET AL. ceptive therapy. Because of the important role played by the ZP in sperm adherence and penetration, two crucial events in fertilization, antibodies to specific ZP antigens have been isolated and purified in order to develop immunocontraceptive methods for the prevention of pregnancy (Gwatkin et al., 1977; Sacco, 1977, 1979,1981; Isojima et al., 1984; East et al., 1984,1985; Mori et al., 1983). Many of these studies utilizing antibodies to identify ZP antigens have provided additional information about the immunologic properties of this acellular structure. In the study of the morphological and lectin-binding properties of the mammalian ZP, several reports have indicated the asymmetric regional distribution of receptors among various species including the hamster (Fox and Shivers, 1975; Nicolson and co-workers, 1975; Lev6ill6 et al., 1987). In an earlier study carried out by Nicolson and co-workers (1975) on the hamster zonae, receptor sites for Wheat germ aggultinin and Ricinus communis agglutinin I were both shown to recognize moieties in the outer region of the ZP. A previous study using light microscope indirect immunofluorescence (Fox and Shivers, 1975) showed the localization of antisera, specific for oviductal and uterine antigens, to the peripheral region of the hamster ZP. The inner area was reported to be devoid of reaction. Using a similar procedure, an intense labeling of both the outer and inner regions of the hamster ZP were obtained when paraffin sections of hamster oocytes were immunostained with a polyclonal antiserum raised against hamster oviductal ZP (Leveil16 et al., 1987). Despite the above reports on the asymmetric distribution of ZP constituents in the hamster oocytes, recent studies carried out in our laboratory using a monoclonal antibody (Kan et al., 1988, 1989a) and a spectrum of lectins including WGA and RCA I (Kan et al., 1989b; Roux and Kan, 1991) indicated that the hamster ZP of postovulatory oocytes is constituted of a homogenous matrix of glycoproteins. For these studies, the high resolution protein A-gold (Bendayan, 1984) and lectin-gold (Roth, 1983, 1987) post-embedding labeling methods were applied to Lowicryl-embedded sections of hamster ovaries and cumulus masses. It is known that the chemical basis of resins and the physical conditions of the embedding procedure can introduce changes in tissue components which can be cytochemically demonstrated (Bendayan et al., 1987). We have recently combined backscattered electron imaging (BEI) in the scanning electron microscope (SEM) with fracture-label cytochemistry (Pinto da Silva et al., 1981) to visualize the in situ distribution of lectin-binding sites on freeze-fractured tissues and cells (Kan and Nanci, 1988).Cell surface antigens labeled by colloidal-gold particles can be visualized by SEM using the backscattered imaging mode (De Harven and Soligo, 1986; Hodges et al., 1987). Backscattered electron imaging-fracture-label (BEI-FL) has the advantage of revealing the distribution of labels in relation to the three-dimensional organization of structures within the cell and tissue. Most importantly, freeze-fracture avoids several steps of conventional tissue processing and exposes membrane and non-membrane constituents (e.g., the ZP) in a potentially less denaturing manner. Furthermore, freeze-fracture cytochemistry is essentially a preembedding technique, fractured membrane components or non-membranous constituents are directly accessible to antibodies, lectins, and colloidal gold markers. Therefore, in order to determine if zonation of matrix components occurs in the ZP of the hamster, we have used the BEI-FL to examine the distribution of lectin binding and antigenic sites in the ZP of hamster ovarian oocytes as well as oocytes collected from the oviduct after ovulation. MATERIALS AND METHODS Preparation of Ovarian Tissue and Collection of Superovulated Oocytes Sexually mature female golden hamsters (Mesocricetus aurutus, Charles River, St.-Constant, Quebec, Canada), 13-15 weeks old, were superovulated by intraperitoneal injection of 25 IU pregnant mare serum gonadotropin (PMSG, Equinex, Ayerst, Montreal, Quebec, Canada) and 25 IU human chorionic gonadotropin (hCG, A.P.L., Ayerst) 48 hours later (Fleming and Yanagimachi, 1980). The animals were sacrificed by cervical dislocation 17 hours after injection with hCG. The lower abdomens of the animals were incised, the ovaries and oviducts were rapidly removed, washed briefly in Dulbecco’s phosphate-buffered saline (PBS-D, pH 7.4; Gibco, Inc., Burlington, Ontario, Canada), and fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 hours at room temperature. Cumulus masses were collected in the following manner. Oviducts were excised and placed immediately in PBS-D. Under a dissecting microscope, the ampulla region of the oviduct was identified and its walls were opened with fine steel tweezers. The cumulus masses were collected in PBS-D and were fixed as described above. After fixation, the ovaries and cumulus masses were washed three times in 0.1 M cacodylate and kept overnight at 4°C in the same buffer. Preparation of Colloidal Gold Colloidal gold particles of approximately 15 nm diameter were prepared by the sodium citrate method as described by Frens (1973). Monoclonal Antibody Used The monoclonal antibody (MAb) (IgG,k) against acid-solubilized hamster ZP of oviductal oocytes was donated by Dr. Gilles Bleau (Maisonneuve-Rosemont Hospital Research Center, Montreal, Quebec). This antibody has been shown to recognize a glycoprotein of oviductal origin (Kan et al., 1989a) with a molecular mass of approximately 200 kDa (Robitaille et al., 1988). Tissue specificity of the MAb has been documented elsewhere (St.-Jacques and Bleau, 1988). Preparation of Lectin-Gold and Protein A-Gold complexes The lectins used in the present study and their respective specificity are the following: Wheat germ agglutinin [WGA: specific for N-acetylglucosamine and/or sialic acid (Bhavanandan and Katlic, 1979; Peters et al., 19791 and Ricinus communis agglutinin I [RCA I: specific for D-galactose (Olsnes et al., 1974; Irimura et al., 1975). Wheat germ agglutinin (WGA) labeling was revealed indirectly with ovomucoid-colloidal gold complex (Ovo-CG) (Geoghegan and Ackerman, 1977; Horisberger and Rosset, 1977). Ricinus cornrnu- GLYCOCOMPONENTS IN T H E HAMSTER ZONA PELLUCIDA nis agglutinin I-colloidal gold (RCA I-CG) complex was prepared as described by Roth (1983). The protein A-gold technique (Roth et al., 1978; Bendayan, 1984) was used for the detection of antigenic sites in the zona matrix of freeze-fractured postovulatory oocytes. Preparation of Samples for Fracture-Label Cytochemistty For fracture-label cytochemistry, ovaries and cumulus masses were embedded in a solution of 30% bovine serum albumin (BSA, Calbiochem, La Jolla, CA) in 0.01 M phosphate-buffered saline (PBS) which was crosslinked by adding glutaraldehyde to a final concentration of 1%(Kan and Pinto da Silva, 1986). The BSA gels containing ovaries and cumulus masses were trimmed into small cubes of approximately 1 x 1 x 2 mm in size, gradually impregnated with 30% glycerol in PBS, and then frozen in partially solidified Freon 22 cooled by liquid nitrogen. Freeze-Fractureand Cytochemical Labeling Frozen BSA gels containing the ovaries or cumulus masses were fractured under liquid nitrogen with a precooled scalpel blade. The fractured gels were then thawed in a solution containing 1%glutaraldehyde and 30% glycerol in PBS for 30 minutes, deglycerinated in 1 mM glycylglycine (Sigma Chemical Co., St. Louis, MO) in PBS containing 3%glycerol for 30 minutes, and then left in PBS alone for 1 hour at ambient temperature as previously described (Kan and Pinto da Silva, 1986). For WGA labeling, the fractured gels were incubated for 1hour with WGA (125 pg/ml PBS) followed by labeling with ovomucoid-colloidalgold complex (Ovo-CG) for 1hour. Control samples were labeled with WGA in the presence of 0.2 M N-acetylglucosamine and then with Ovo-CG as described above. For RCA I labeling, the gels were incubated for 1 hour with RCA I-CG complex. Control samples were incubated in the presence of 0.2 M D-galactose. The RCA I-CG complex was mixed with its competing sugar 2 hours prior to use. To study the distribution of Hamster Oviductin-1 glycoprotein in the ZP of postovulatory oocytes, fractured BSA gels containing the cumulus masses were first incubated for 10 minutes in 0.01 M PBS (pH 7.4) containing 1%ovalbumin and then labeled for 1 hour a t room temperature with monoclonal antibody. After washing three times in PBS to remove unbound antibody, the gels were incubated for 10 minutes in 0.01 M PBS containing 1%ovalbumin followed by labeling with protein-A gold complex for 30 minutes. To assess the specificity of the immunolabeling, control samples were incubated i) with a monoclonal antibody directed against a bacterial antigen followed by protein A-gold complex, or ii) with a supernatant from a culture of myeloma cells (SP2/0-Ag14)used in the preparation of the hybridoma followed by protein A-gold as described above. The above incubations were all carried out at room temperature. At the end of the labeling procedure, the labeled gels were rinsed three times with 0.0 1M PBS and fixed with 1%glutaraldehyde overnight a t 4°C. Some specimens were postfixed with 1%aqueous osmium tetroxide while others were left unosmicated. 37 Preparation of Peldri I1 Sublimated, Fracture-Labeled Cumulus Masses, and Ovaries for Backscattered Electron Imaging in the SEM The labeled samples were first dehydrated through a series of graded ethanols and then dried by the Peldri I1 sublimation method (Kan, 1990). Preliminary results indicated that gold labeling on osmicated samples could be detected by BE1 in the field emission scanning electron microscope. Following these initial observations, the labeled specimens were all osmicated prior to dehydration. The last change of pure ethanol was replaced by a mixture of Peldri I1 and pure ethanol (ratio 1:2) for 1hour a t room temperature. This was followed by 30 minutes a t room temperature in a fresh mixture (ratio 2:l). At the end of this period, the mixture was replaced by two changes of pure Peldri 11, each for 30 minutes. Since pure Peldri I1 solidifies a t 24"C, infiltration was carried out at 37°C. After the last change of pure Peldri 11, excess Peldri I1 was removed leaving just enough solution to cover the top of the specimens. The containers were then transferred to a glass Petri dish on top of a block of dry ice to allow the Peldri I1 to solidify rapidly. Sublimation was performed under vacuum in a Balzers BAF 400T freeze-fracture unit (Balzers AG, Balzers, Liechtenstein) at ambient temperature. The sublimation step lasted about 45 minutes and then the vacuum was broken by venting with nitrogen gas. The fractured faces of the specimens were identified under a dissecting microscope and the specimens were mounted on aluminum stubs with the fractureface up using conductive carbon paint (SPI Supplies, Westchester, PA). The specimens were rotary shadowed with a thin film of carbon in a Balzers MED 010 mini-high vacuum deposition system (Balzers AG, Balzers, Liechtenstein). They were then examined either with a JEOL JSM 840 scanning electron microscope fitted with a LaB, cathode and a JEOL BE1 circular solid-state detector or with a JEOL JSM 6300F field emission scanning electron microscope equipped with a similar detector. In either case, the microscope was operated at an accelerating voltage of 15 kV using an objective aperture of 75 pm and a specimen working distance of 8 mm. The normal signal polarity was used. RESULTS Ultrastructureof Freeze-FracturedHamster Ovarian Oocytes as Revealed by Scanning Electron Microscopy Secondary electron imaging (SEI) of fracture-label preparations of hamster ovaries in the SEM revealed the three-dimensional organization of cellular structures in cross-fractured ovarian oocytes a t different stages of development (Figs. 1 and 2). The granulosa cells surrounding the oocytes were frequently seen with their nuclei and cytoplasm exposed by the fracture process (Fig. 1).At high magnification, the crossfractured profile of the zona pellucida was identified as a distinct layer of extracellular matrix located between the centrally located oocyte and the surrounding multilayer of granulosa cells (Fig. 2). Inside the oocyte, cortical granules were observed close to the plasma membrane (Fig. 2). Lamellar structures, characteristic 38 F.W.K. KAN ET AL. Figures 1 and 2. Secondary electron images in the SEM of fracturelabel preparations of a portion of a hamster ovary. Cross-fractured ovarian follicle displays the zona pellucida (asterisks) in relation to the three dimensional architecture of the central oocyte and the surrounding granulosa cells (GO, some of which have their nuclear matrix (Nu) exposed (Fig. 1) x 3,250. Fig. 2: At high magnification, the zona pellucida (ZP) i s seen as a relatively smooth band of uniform thickness. The granulosa cells (GC) are intimately associated with the surface of the zona pellucida. Inside the oocyte, cortical granules (arrowheads) are shown resting on a meshwork of interlacing cytoskeleta1 elements. The lamellar structures are revealed as continuous sheets of membrane-like structures (arrows). x 15,600. of oocytes and found in abundance in the ooplasm, appeared as folded mcmbranc-likc shccts (Fig. 2). preparation at low magnification. Cross-fractured zonae pellucidae were then selected by SEI and viewed a t higher magnification (Fig. 3a). Backscattered electron imaging of RCA 1 binding sites in the zona pellucida revealed an intense labeling of this extracellular matrix by gold particles, seen as white particles with normal image signal polarity (Fig. 3b). A few gold parti- imaging Of I and wGA Backscapered Binding Sites in the Zona Pellucida of Ovarian Oocytes Freeze-fractured hamster ovarian oocytes labeled with RCA I-CG were first located on the fracture-label GLYCOCOMPONENTSIN THE HAMSTER ZONA PELLUCIDA 39 Figure 3. Electron micrographs of a freeze-fractured hamster ovarian follicle labeled with RCA I-CG (ax).a) Secondary electron image of the fracture-labeled follicle. X 2,100. b) Backscattered electron imaging of the region delimited by the box in (a) shows a uniform distribution of gold labeling throughout the zona pellucida (ZP). GC: granulosa cells, x 10,000,c ) At a higher magnification, individual gold particles are resolved. x 15,000. d) Control sample which was incubated with RCA I-CG in the presence of its blocking sugar D-galactose (0.2M) shows the absence of labeling over the zona pellucida (ZP). A few gold particles over the zona matrix represents background levels of labeling. X 5,500. cles were found associated with what appeared to be the plasma membrane of the neighboring granulosa cells (Fig. 3b). At a higher magnification (Fig. 3c), the gold particles were found to be homogenously distributed throughout the zona matrix. The labeling was abolished when freeze-fractured samples were incubated with RCA I-CG in the presence of its blocking sugar D-galactose, indicating the specificity of the labeling (Fig. 3d). Similar fracture-label preparations of hamster ovaries were also incubated with WGA followed by labeling with ovomucoid-gold. Secondary electron imaging was again used to reveal the disposition of the zona pellucida in relation to the three-dimensional architec- 40 F.W.K. KAN ET AL. Figure 4. a) Secondary electron image of a portion of a freeze-fractured hamster ovarian follicle after labeling of WGA-binding sites. b) Using BEI, a uniform distribution of gold labeling is seen throughout the zona matrix. Two cortical granules (arrows) immediately below the zona pellucida (ZP) are also labeled with gold particles. GC: granulosa cells; Nu: nucleus. x 7,500. ture of the surrounding granulosa cells exposed in cross-fractured profiles (Fig. 4a). Backscattered electron imaging of the same region showed an intense labeling of the zona pellucida (Fig. 4b). However, the inner region of the zona pellucida close to the oocyte appeared to be more intensely labeled than the outer region (Fig. 4b). ticles labeling the zona matrix could be resolved (Fig. 8b) and some of them were found in linear arrays (Fig. 8b). Similar to the labeling of the zona pellucida by RCA I and WGA, labeling with anti-Oviductin-1 antibody in postovulatory oocytes was also found to be homogenous in this extracellular matrix (Fig. 9a). Over the zona pellucida, there were small regions which were free of Backscattered Electron Imaging of Lectin-Binding and Antigenic Sites in Postovulatory Oocytes Oviductal oocytes in cumulus masses were also processed for freeze-fracture and then labeled, respectively, with RCA I-CG, WGA-OVO-CG,and a monoFigure 5. Low magnification SEM survey of a fracture-label prepaclonal antibody against Hamster Oviductin-1. Low ration of a cumulus mass labeled with RCA I-CG. The cumulus mass magnification SEI of fracture-label preparations of shown here, consists of a postovulatory oocyte (arrowhead) and nupostovulatory oocytes served to reveal the disposition merous cumulus cells (arrows), embedded in a matrix of BSA crossof the oocytes within the cumulus mass (Fig. 5). At linked with glutaraldehyde to form a gel. C: carbon paint. x 100. higher magnification, the inner and outer boundary of Figure 6. Secondary electron image of a cross-fractured oocyte. The the zona pellucida surrounding the oocyte could be de- white arrows indicate the thickness of the zona pellucida. Several lineated in cross-fractured oocytes (Fig. 6). Some of the cavities (arrows) at the immediate surrounding of the oocyte correneighboring cumulus cells were fractured in such a spond to the concave exoplasmic membrane half of freeze-fractured manner that only the exoplasmic leaflets of their cumulus cells. x 1,500. plasma membranes were left behind (Fig. 6). ComparFigure 7. a) Secondary electron image of a portion of a postovulatory ison of the secondary electron image (Fig. 7a) with the hamster oocyte shown in Fig. 6 displaying the zona pellucida (ZP) and corresponding backscattered electron image (Fig. 7b) the superficial region of the oocyte. In this preparation, the plasma membrane (PM) of the oocyte is separated from the structural eleprovided more details about the spatial distribution of ments of the ooplasm (at right) as a result of the fracture process. the RCA I labeling. In postovulatory hamster oocytes, Several cortical granules (arrowheads) are seen adjacent to the cytoRCA I labeline aDDeared to be uniformly distributed ulasmic side of the ulasma membrane. x 10.000. b) Corresuondina backscattered electron imaging reveals a uniform labeling of ihe zona throughout the zona matrix (Fig. 7b,c). (ZP) by RCA I-CG. The cortical granules are also labeled A similar labeling patternwas found in postovu~a-pellucida (arrowheads). x 10,000. c) At high magnification, the gold particles tory oocytes fractured and labeled with WGA-oVo-CG are seen to be uniformly distributed throughout the zona pellucida (Fig. 8a). At higher magnification, individual gold par- (ZP). ~21,000. - GLYCOCOMPONENTS IN T H E HAMSTER ZONA PELLUCIDA Figs. 5-7 41 42 F.W.K. KAN ET AL. Figure 8. a) Backscattered electron image of a freeze-fractured postovulatory hamster oocyte incubated with WGA followed by labeling with ovomucoid-gold complex. A uniform distribution of gold particles is seen throughout the entire thickness of the zona pellucida (ZP).A cumulus cell (CC) in the neighborhood, however, is unlabeled. x 8,000. b) At high magnification, individual gold particles can be resolved. Some of the gold particles are in linear arrays (arrowheads). x 100,000. gold particles (Fig. 9b,c). These gold particles-free ar- oocytes before and after ovulation using several lectins eas corresponded to the porous regions of the zona ma- (Helix pomatia lectin, Wheat germ agglutinin, Limas trix (the outer one third of the ZP has been reported to flavus agglutinin, and Ricinus communis agglutinin I) be more porous than the rest of the zona matrix; Talbot, also reveal a uniform distribution of lectin-binding 1984). At high magnification, gold particles were seen sites in the zona matrix of postovulatory oocytes (Kan to be associated with three-dimensional finger-like et al., 1989b; Roux and Kan, 1991). The results obstructures (Fig. 9d) corresponding to the darkly stained tained from these labeling experiments validate our thick filamentous structures seen on thin-sections la- previous proposal of a homogenous distribution of glybeled by the post-embedding cyctochemical technique (Fig. 10) prepared as in Kan et al. (1989a). Control experiments for establishing the specificity of the monoclonal antibody labeling showed a negative reacFigure 9. a) Backscattered electron image of a postovulatory hamtion over the ZP (Fig. 11). ster oocyte fractured and incubated with a monoclonal antibody ~ DISCUSSION Immunogold labeling on tissue sections of the zona pellucida (ZP)of hamster postovulatory oocytes using a monoclonal antibody directed against a high molecular weight glycoprotein of oviductal origin reveals a uniform distribution of antigenic sites throughout the matrix of the ZP (Kan et al., 1989a). Prior treatment of the cumulus masses with hyaluronidase to disperse the cumulus cells does not affect the uniform labeling of the ZP by the monoclonal antibody (Kan et al., 1989a), suggesting that the hamster ZP does not contain hyaluronic acid. Lectin cytochemical studies on hamster against the Hamster Oviductin-1 followed by labeling with protein A-gold complex showing an intense and uniform labeling of the zona pellucida (ZP). x 8,000. b) The outer region of the zona pellucida (ZP) appears to be more porous (as indicated by the unlabeled spaces) than c) the inner region of the zona pellucida (ZP) matrix. However, the gold labeling is found to be uniform except where there are spaces in the matrix. ~35,000.d) At high magnification, the gold particles detected in the zona matrix are associated specifically with finger-like projections (arrow). x 100,000. Figure 10. Ultrathin section of a Lowicryl-embedded hamster postovulatory oocyte prepared as in Kan et al. (1989a) and labeled with the monoclonal antibody followed by protein A-gold complex. Immunogold labeling is associated with electron dense material in the matrix of the zona pellucida probably corresponding to the finger-like structures revealed by backscattered electron imaging. x 85,000. GLYCOCOMPONENTS IN THE HAMSTER ZONA PELLUCIDA Figs. 9-10 43 44 F.W.K. KAN ET AL. Figure 1 1. Control sample of a freeze-fractured cumulus mass incubated with a supernatant of cultured myeloma cells and then labeled with protein A-gold complex. a) Secondary electron image showing a cumulus cell (CC) in contact with the surface of the zona pellucida (ZP)of a postovulatory oocyte. b) Complementary BE1 shows the absence of gold particles over the zona pellucida. x 6,000. coconjugatesin the ZP (Kan et al., 1988).In the present study using a different approach, we have provided, further evidence demonstrating the homogenous distribution of antigenic and lectin-binding sites throughout the zona matrix of hamster postovulatory oocytes. The BEI-FL technique not only offers the possibility of examining the three-dimensional architecture of the ZP in relation to the other structures inside the ooplasm and the surrounding granulosa cells, but also allows for the localization of antigenic and lectin-binding sites in cross-fractured oocytes with a high degree of sensitivity. We have previously shown that Hamster Oviductin-1 glycoprotein is absent in the ovary, is secreted by non-ciliated secretory cells in the oviduct, and is transferred to the ZP of oocytes during their passage through the oviduct (Kan et al., 1989a). However, when the monoclonal antibody is applied to freeze-fractured postovulatory oocytes in the cumulus masses, a uniform distribution of antigenic sites is observed in the ZP of postovulatory oocytes. The present data further support the notion that matrix components are distributed homogenously in the hamster ZP. In an early study of hamster eggs, Fox and Shiver (19751, using indirect immunofluorescence, reported a peripheral localization of oviductal and uterine antigens in the ZP. An electron microscopic study carried out by direct incubation of hamster eggs with ferritin-conjugated lectins similarly showed the localization of WGA and RCA I binding sites in the outer region of the ZP (Nicolson et al., 1975). Discrepancy between these studies and the present data may be attributed to technical limitations such as limit in resolution and restricted accessibility of lectin binding sites to the probes. Backscattered electron imaging fracture-label is essentially a pre-embedding labeling technique. Since upon fracture the zona matrix components are directly exposed to labeling probes, antigenic and lectin-binding sites are sterically accessible to the labeling molecules thus avoiding masking of binding sites by the embedding medium, a problem inherent to post-embedding techniques. The BEI-FL results confirm the uniform distribution of antigenic and lectin-binding sites in the ZP of hamster postovulatory oocytes as previously seen on thin-sections of Lowicryl-embedded samples (Kan et al., 1989a,b; Roux and Kan, 1991). However, Abe and Oikawa (1990) have reported, using a similar postembedding labeling technique, that there are topographical differences in distribution of ZP-0, an oviductal glycoprotein in the ZP of golden hamster eggs, and that the golden hamster egg can be morphologically divided into three regions. While it is possible that this discrepancy in labeling pattern may be due to a difference in the epitopes recognized by the different monoclonal antibodies used in these studies (an unlikely situation since studies with a spectrum of lectins also revealed uniform labeling of the golden hamster egg ZP), the present data does not support the proposal that the ZP of the golden hamster egg is ultrastructurally divisible into three distinct regions (Abe and Oikawa, 1990). Although the outer one third of the ZP of the golden hamster may be more porous, the matrix of the ZP appears to be structurally similar throughout its entire thickness. Recently, colloidal gold immunolabeling of an oviduct-specific glycoprotein in the ZP of ovulated and early embryos in the baboon (Boice et al., 1990) and gilts (Buhi et al., 1993) also demonstrated a uniform distribution of gold particles in the ZP. Most recently, we have demonstrated that antigenic sites, detected by immunocytochemistry with the monoclonal antibody against Hamster Oviductin-1, remain uniformly distributed throughout the zona pellucida of GLYCOCOMPONENTS IN THE HAMSTER ZONA PELLUCIDA 2-cell, 4-cell, and 8-cell stage hamster embryos (Kan et al., 1993). In the present study, colloidal gold particles of approximately 15 nm were used as markers. One may argue that the use of smaller gold particles could give a different result than that obtained with 15 nm gold since it has been suggested that large gold particles may fail to reveal the specific topographical distribution of cell surface macromolecules, which can be demonstrated by the use of small gold particles (Kan and Pinto da Silva, 1989). In an early postembedding immunolabeling study (Kan et al., 1989a), however, a uniform labeling of the ZP was obtained-when thin sections of Lowicryl-embedded postovulatory oocytes were labeled with gold particles of either 15or 11nm in diameter. In conclusion, the present results, taken together with previous data indicate that the Z P of hamster postovulatory oocytes consists of an extracellular matrix of glycocomponents which are not distributed in zones within the ZP but are uniformly distributed throughout its thickness. ACKNOWLEDGMENTS The authors wish to thank Mr. Jean LBveille for photographic work. This work was supported by grants from the Medical Research Council of Canada to F.W.K. Kan and A. Nanci. LITERATURE CITED Abe, H. and T. Oikawa 1990 Ultrastructural evidence for an association between an oviductal glycoprotein and the zona pellucida of the golden hamster egg. J. Exp. Zool., 256:210-221. 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