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a Department of Reproduction, Obstetrics and Herd Health,
b Laboratory of Virology, and
c Department of Physiology, Biochemistry and Biometrics, Faculty of Veterinary Medicine, University of Ghent, B-9820 Merelbeke, Belgium
d Veterinary and Agrochemical Research Center, 1180 Brussels, Belgium
e Laboratory of Biochemistry and Molecular Cytology, Faculty of Agricultural and Applied Biological Sciences, University of Ghent, B-9000 Gent, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 µm2 of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 µm2) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively.
In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40–50 nm) and bovine herpesvirus-1 (BHV-1; 180–200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, bZP3-ß, and bZP4 [5]. Recently, it was revealed that bZP2 and bZP4 are fragments of bZP1 [5]. Each of these glycoproteins has specific functions. In cattle, sperm receptor activity is attributed to ZP3-. ZP2 serves as a secondary receptor for acrosome-reacted sperm. After sperm-egg fusion, cortical granule-associated enzymes convert ZP2 and ZP3 to ZP2f and ZP3f, respectively. These egg-induced modifications play a role in the block to polyspermy [6]. The glycoproteins determine the specific structure of the ZP [4,5,7]. When visualized by scanning electron microscopy (SEM), the mammalian ZP is found to be composed of a network dispersed with numerous pores [8] and with morphologically dissimilar internal and external surfaces. The external surface displays a fenestrated lattice-like appearance and the internal surface shows a regular rough appearance [9,10]. Reports concerning the properties of the ZP of bovine in vitro-produced (IVP) embryos are limited. Riddell et al. [11] demonstrated by SEM that the external surfaces of the ZP of immature oocytes are very irregular, with uneven distribution of numerous pores, crevices, and projections. Recently, the surface of the bovine ZP of immature oocytes, in vitro-maturing and in vitro-matured (IVM) oocytes, and in vitro-inseminated oocytes was studied by Suzuki and coworkers [12–14]. They found that the fine structure of the ZP of immature oocytes is characterized by a network with numerous wide meshes and deep holes. After maturation, this network becomes finer, and the meshes and holes seem to be less deep. No meshes have been found on the ZP of inseminated oocytes, probably due to fusion of several layers of the network.
Variations in the architecture of the ZP exist between species. When the porous surface is compared among species, the largest pores are observed in the ZP of the rabbit and the cat, the smallest in the cow. The pores are largest at the outer surface of the ZP but decrease in size centripetally [8,15]. In general, pore shape varies within and between the zonae of the species observed, but many zonae exhibit a more elliptical than circular shape. The arrangement of zona pores appears random in all species except the cat, where a definite trend toward concentric pore arrangement exists. Variation in network thickness has also been observed, with the broadest plate-like reticulum exhibited by the hamster zona. In the rabbit and the cat, this network is more complex and distinct, with a reduction of fiber width. The smallest size of the fibers composing the zona network is found in the cow and the opossum [8].
Since embryo transfer has become an expanding business, questions have arisen concerning the risk of virus transmission through embryos and the epidemiological complications of embryo transfer [16–18]. Two of the most important viral pathogens in cattle, herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV), are associated with reproductive failure, such as abortion and infertility; thus far, a limited number of investigations have been reported on the interactions of these viruses with IVP embryos. Recent studies suggest that the sanitary risks for exposure to BHV-1 and BVDV may be more important for embryos produced in vitro than in vivo, due to differences in the ZP of those produced in vitro, enabling easier adsorption of viruses [17,19]. In previous studies, we demonstrated that no virus replication nor signs of embryonic degeneration were detected in ZP-intact, IVP bovine embryos incubated with BHV-1 and BVDV [20,21]. From these findings, it was concluded that neither virus is able to cross the ZP. In other words, an intact zona of IVP embryos acts perfectly well as a protective barrier against BHV-1 and BVDV. Therefore, the aim of the present study was to characterize structural aspects of the ZP of IVP embryos at different stages of development to find an explanation for its barrier function against viral infection. The diameter of the outer pores and the continuity of the meshes towards the embryonic cells were examined by SEM and confocal laser scanning microscopy (CLSM), respectively.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro embryo production Bovine embryos were produced by standard in vitro fertilization (IVF) procedures. Ovaries from healthy animals with normal reproductive tracts were collected at slaughter and transported to the laboratory in warm physiological saline containing 25 µg/ml kanamycin sulfate at 30°C, within 4 h after collection. Ovaries were processed according to methods described previously [22]. Briefly, oocytes were aspirated from 2–6-mm follicles and matured for 24 h in modified bicarbonate-buffered TCM 199 medium (Gibco BRL, Merelbeke, Belgium) supplemented with 20% heat-inactivated estrous cow serum, 0.2 mM sodium pyruvate (Sigma, Bornem, Belgium), 50 µg/ml gentamicin sulphate medium (Gibco BRL), and 0.04 mM glutamine (Sigma). Then, the cumulus-oocytes complexes were matured at 38.5°C in 5% CO2 in air at 100% humidity for 24 h. A fourth of the matured oocytes were vortexed to remove the cumulus cells. Residual cumulus cells were removed by pipetting. The remaining mature oocytes were inseminated with a final sperm concentration of 106 sperm/ml in 500 µl IVF-TALP (Tyrode's solution supplemented with albumin, lactate, and pyruvate) supplemented with 6 mg/ml fatty acid-free BSA, 20 µg/ml D-penicillamine, 10 µM hypotaurine, 1 µM epinephrine, and 25 µg/ml heparin (all from Sigma) for 24 h at 38.5°C in 5% CO2 in air with maximal humidity. For each experiment, three 0.25-ml straws filled with semen from a bull free of BHV-1 and BVDV were used. Frozen-thawed sperm was separated over a Percoll gradient (45–90%; Pharmacia, Uppsala, Sweden). Maturation and fertilization media were not covered with paraffin oil. After insemination, oocytes were vortexed to remove excess sperm and cumulus cells. Groups of 25 inseminated oocytes were transferred into 50-µl droplets of oviduct coculture overlaid with paraffin oil (Merck-Belgolabo, Overijse, Belgium). The culture medium was Ménézo-B2 (INRA, Paris, France) supplemented with 2.5 µg/ml fungizone (Gibco), 10% FCS, and bovine oviduct epithelial cells (BOEC). Coincubation took place at 38.5°C in 5% CO2 in air with maximal humidity.
Preparation of oocyte/embryos for SEM Mature oocytes without their cumulus investments, zygotes (1 day post fertilization), 8-cell stage embryos (3 days post fertilization), and morulae (6 days post fertilization) were fixed for SEM as follows: the ZP-intact oocytes/embryos were washed 3 times in distilled water. The oocytes/embryos were then fixed for 1 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Agar Scientific Ltd., Stansted, UK), pH 7.2, at 4°C. After fixation, the oocytes/embryos were washed twice for 5 min in 2% cacodylate buffer (Agar Scientific) at 4°C; after washing, they were postfixed in 1% osmium tetroxide in distilled water for 2 h at 20°C. Subsequently, the oocytes/embryos were rinsed for 10 min in distilled water and then were dehydrated in a series of increasing concentrations of ethanol and acetone. Following dehydration, the oocytes/embryos were dried in a Bal-Tec CPD 03 critical-point dryer using liquid CO2 as the transitional fluid. After drying, they were coated with 20-nm gold by a Balzers sputtering device (Balzer, Liechtenstein). SEM observations were made with a Philips (Eindhoven, The Netherlands) 501 scanning electron microscope at an accelerating voltage of 30 kV. Since the preparation of oocytes and embryos might result in some fixation artifacts, a cautious interpretation of the results is desirable.
Preparation of oocytes/embryos for CLSM The continuity of the meshes towards the embryonic cells was examined by CLSM. For this purpose, fluorescent microspheres (Fluospheres, Molecular Probes Europe, Leiden, The Netherlands) were used: one type (crimson red fluorescence with excitation/emission maxima of 365/415) with a size of 200 nm, and another type (blue and yellow-green fluorescence with excitation/emission maxima of 625/645) with a diameter of 40 nm. These beads have the same physical dimensions as BVDV (40–55 nm) and BHV-1 (180–200 nm), respectively. The permeability of the ZP to these microspheres was investigated by the following experimental design: ZP-intact cumulus-free embryos were put in a drop of medium on a microscopic slide and evaluated with an inverted microscope equipped with two positioning manipulators and with two 3-dimensional remote-control micromanipulators. Two holding pipettes with an outer diameter of 110 µm and an opening (inner diameter) of 25 µm were used. One pipette held the embryo; the other pipette was placed on the surface of the ZP and a mixture of the microspheres was deposited there with positive pressure. The localization of the microspheres in the ZP was visually demonstrated by a Bio-Rad MRC 1024 Laser Scanning Confocal Microscope (Bio-Rad House, Hertfordshire, UK) linked to a Nikon Diaphot 300 microscope (Nikon Corp., Tokyo, Japan) and interfaced to a Compaq Prosignia 300 (Compaq Computer Corp., Houston, TX). Krypton-argon laser light was used to excite crimson red fluorescence (625-nm line) and yellow-green fluorescence (567-nm line) of the microspheres. Extended focus images were obtained with Bio-Rad COSMOS and Lasersharp software (Bio-Rad House). Some caution with interpretation of the results is desirable since the possibility exists that the pressure of the deposition may cause some beads to progress further into the ZP than a nonmotile virus might.
In an additional experiment, ZP-intact cumulus-free embryos were incubated for 24 h in medium containing a mixture of the two types of microspheres. The localization of the microspheres in the ZP was visually demonstrated as described above.
Experimental Design
Experiment 1 The ZP of 10 IVM oocytes, 10 zygotes, 10 8-cell stage embryos, and 10 morulae were studied. All these embryonic stages were collected in a single sequence of in vitro maturation, fertilization, and culture. The outer pores present within five surface areas (arbitrarily selected) of 100 µm2 each per embryo were examined by SEM. The actual percentage of ZP surface area surveyed was about 2.5% of the total ZP surface. For each pore, the smallest diameter (nm) was measured by an image analysis system (NIH Image 1.60). The percentage of pores that were large enough to allow the entry of BHV-1 or BVDV within the examined surface of the ZP was calculated. The ultrastructure of the ZP surface areas was evaluated and compared within and between the stages of development.
Experiment 2 The ZP of 10 IVM oocytes, 10 zygotes, 10 8-cell stage embryos, and 10 morulae were studied by CLSM as described above. All these embryonic stages were collected in the same sequence of IVM/IVF/IVC as in Experiment 1. The passage and the localization of fluorescent microspheres of different size, respectively through and in the ZP, were evaluated and compared within and between the stages of development.
Statistical Analysis
The results of this completely hierarchical design were analyzed following a general linear procedure (SPSS for Windows, 7.0, Chicago, IL) with stage of embryo (S) as a fixed main factor, embryo per stage (E), surface area (A) per embryo, and pores (P) per surface area as consecutive random subfactors. The model was: Y = S + E/S + A/E/S + P/A/E/S, with Y the observed diameter of the pores, or the number of pores per area. In the latter case, the last term in the model is not considered. Equality of variances within groups was checked before proceeding with analysis. In the analysis of the observations of the diameter of pores, Type I Sums of Squares were chosen. This is recommended as the unequal number of pores per surface area, resulting in an unequal number of observations, and it is a reflection of the population size of pores in an embryo [23]. Variance components among groups were calculated for the random factors. Scheffé's procedure was followed for comparison of means of the fixed factor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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General observations The outer ZP-surface of the investigated embryos showed two principal patterns: 1) a network with a spongy appearance containing numerous pores, and 2) a more compact structure with fewer pores, which were mostly larger in comparison with the pores of the spongy surface. In all four stages of development, the deeper lying pores were smaller in comparison with the superficial pores (the diameters of the pores decreased the nearer the pores were situated to the inner ZP; Fig. 1). The pore shape was in general circular or elliptical, and the pores were arbitrarily distributed.
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Embryonic stage-dependent observations In observations of matured oocytes (Fig. 2A), SEM analysis showed that the ZP surface was mainly characterized by a spongy network with a rough surface. Remnants of presumably cellular material (> 1 µm in diameter) were regularly perceived. Globular as well as knobby processes (± 250–500 nm in diameter) were observed in all investigated surface areas and were seen to be touching the ZP.
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Examination of zygotes (Fig. 2B) revealed that the spongy network had become more compact and the pores were less numerous in comparison with the pores in the ZP of the oocytes. This compact pattern, which was found in all the embryos, displayed a smooth appearance. Sometimes the surface of the ZP had a melted look with only a few pores, which were mostly not as deep as the outer pores visible in oocytes. No remnants of cellular material were detected, and only a few processes as seen on the ZP of matured oocytes were observed. Some crevices with the print of a sperm head were seen. Occasionally, a sperm cell was detected near such a fissure, which was at least 1 µm deep and 1–2 µm x 4–6 µm in surface area. Penetrating and penetrated sperm cells were regularly noticed; of the latter, only the tails were visible. Completely melted ZP surfaces, without pores, were always observed in the immediate environment of the sperm cells.
In 8-cell-stage embryos (Fig. 2C), the two principal patterns were found to the same extent. The largest pores were found in surface areas that displayed a more compact appearance. In certain areas, granular depositions were observed on the ZP surface. There were no traces of penetrated or penetrating sperm cells. Sometimes an adhering sperm head or sperm cell (with tail) was seen. Stratified processes of the ZP were visible.
In morulae (Fig. 2D), a more rough surface of the ZP was demonstrated. An occasional single sperm cell was detected on the surface of some morulae. There were many small pores, which were accompanied by deposits of layers of biological material. Granular depositions were noticed, which were sometimes closely associated with the ZP. In 8-cell-stage embryos and morulae, narrow bridges of biological material were present between 2 pores, as if these bridges were splitting up one large pore (Fig 3).
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Statistical Analysis
The observed number of outer pores in 5 surfaces of 10 oocytes or embryos (a total area of 5000 µm2) and the mean diameter of these pores are summarized in Table 1. In matured oocytes, 1511 pores/5000 µm2 were detected. After fertilization, the number of pores remained more or less stable at first; but from the third day onwards, a dramatic increase was seen, with the largest number of pores being present in morulae (3259 pores/5000 µm2) (P < 0.001). The mean diameter of the outer pores was significantly different between the 4 stages of development (F = 14.14, df = 3 and 36, P < 0.001), with the largest diameter in zygotes (223 nm). The diameter decreased significantly in more developed stages (P < 0.001).
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A significantly negative correlation was observed between the number of pores per area and the mean diameter of these pores (r = -0.56 ; P < 0.001). This correlation was clearly demonstrated when the percentage of pores with a diameter of 
200 nm were studied. In morulae (the stage with the highest number of pores per area) only 19% of these pores was large enough to allow entry of BHV-1, whereas in the other 3 stages these percentage were more than 34%. At least 97% of the pores (in all stages) were large enough to allow the entry of BVDV. The distributions of the pores in relation to their smallest diameter are shown in Figure 4.
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In order to establish the source of the observed differences (number and diameter of pores), the variation in the observations was partitioned for each possible source of the variation: fixed (stage) or random (embryo, surface area, pores) (Table 2). The percent variation in the number of pores between stages (55%) far exceeds the percent random variation between embryos (25%) and between different areas (20%). As for the variation in diameter, the main contribution (64%) comes from differences between pores per observed area; the remaining variation is due mainly to differences of the mean diameter between the stages (25%).
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Experiment 2: CLSM Study
After CLSM analysis, a homogeneous dispersion of the microspheres was detected in the outer layers of the ZP. The smallest microspheres (40 nm) were shown to be deposited obviously deeper in the ZP in comparison with the beads of a size of 200 nm (Fig. 5). Microspheres were not detected in the inner layers of the ZP or close to the oocyte membrane.
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After incubation of the embryos in a suspension of the microspheres for 24 h, the smallest beads (40 nm) were detected halfway through the thickness of the zona. The beads with a size of 200 nm were found only within the first one-fourth of the thickness of the ZP (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results clearly demonstrated that the surface of oocytes and embryos show a variety of morphological structures. We have distinguished two principal patterns: 1) a network with a rough and spongy appearance containing numerous pores, and 2) a more compact and melted structure with fewer pores, which are mostly larger in comparison with the pores of the spongy surface. The same patterns have also been demonstrated in IVM human oocytes [24]. In our study, all oocytes and embryos were found to display the two patterns. However, the proportion of these two patterns was dependent upon the stage of embryonic development. In matured oocytes, the surface was rougher, probably due to remnants of cumulus cells and corona radiata cells. Suzuki et al. [12–14] reported similar observations in immature, maturing, and matured bovine oocytes. They inferred that the rough surface was composed of residuals of the corona radiata cells. The latter cells form cytoplasmic processes that pass through the zona and terminate with gap junctions on the vitelline membrane of the oocyte [25]. It is known also that after maturation, most of these cell processes retract or disintegrate and that the matrix of the ZP closes the channels left by the cytoplasmic processes [26]. The pores that we detected on the outer surface of mature oocytes probably marked the entries of cell processes. Whereas in pig and rat the diameter of these processes is about 50–100 nm [26], we found in bovine oocytes a mean diameter of 182 nm. The surface of the zygotes was found to have a smooth, melted appearance with only a few pores. On some of the surface areas investigated, we detected fewer than 5 pores. The melted look of the ZP, without pores, in the direct proximity of the penetrating sperm cells is probably caused by the lytic action of acrosomal enzymes such as acrosin and hyaluronidase. This acrosome reaction is necessary for spermatozoa to traverse the ZP in order to fuse with the oolemma. The lytic action of the enzymes probably explains the general melted view of the ZP shortly after fertilization. The same observation was made by Suzuki and coworkers [13], who described a dramatic change of the ZP network after fertilization. This structural change, accompanied by modification of ZP glycoproteins and by changed permeability of macromolecules (dextrans and lectins), is known as "hardening", which plays a critical role in preventing polyspermy [27,28].
Three days after IVF, the two principal patterns were found to the same extent on the ZP of 8-cell-stage embryos. The first signs of granular deposits on the surface of the ZP were noted. At 6 days postinsemination, the rough surface was the dominant pattern on the ZP surface of morulae. It is known that oviductal cells secrete proteins that attach to the ZP [29,30]. Therefore, we speculate that the rough aspect of post fertilization embryonic stages (8-cell stage and morula) could be a result of the depositions of biological material secreted by the oviductal cells used in our culture system. The outer pores were the smallest in morulae, and bridges were found in the middle of some large pores, features that may also have been caused by these depositions in and around the pores. All the aforementioned observations clearly demonstrate that the ZP is a dynamic structure that can be changed by its environment [29] and that is able to repair damage [24].
Concerning the risk of infection of embryonic cells with BHV-1 or BVDV, we studied the outer pores, which could be ports of entry for viruses. We have shown that pores at the surface of the ZP are large enough to allow entry of BHV-1 (180–200 nm) and BVDV (45–55 nm). In all embryonic stages investigated, more than 96% of the outer pores were large enough for the entry of BVDV. The percentage of pores large enough for the entry of BHV-1 was dependent on the embryonic stage, with a minimum of 19% in morulae and a maximum of 51% in zygotes.
By means of SEM, it was observed that the diameter of the channels in the ZP decreased centripetally, which is in agreement with reports in humans [15] and in other animals [8]. Our observation was confirmed by means of CLSM images. From these data, it may be assumed that intact ZP of IVP bovine oocytes and zygotes are constructed in such a way that BHV-1 and BVDV may enter the channels in the outer layers of the ZP but do not pass through inner layers of the ZP towards the embryonic cells. This centripetal narrowing architecture of the meshes of the ZP has been demonstrated in the hamster [9,10]. At present, only one virus has been demonstrated to be able to penetrate through the ZP of farm animals. This is the porcine parvovirus, which is a very small virus of only 20 nm diameter. It has been detected by means of a transmission electron microscope in embryonic cells of ZP-intact in-vivo-derived porcine embryos after incubation with the virus [31]. In mice, Mengo encephalitis virus (27–28 nm in diameter) has also been shown to pass through the ZP of 2-cell mouse embryos and mouse morulae [32,33]. It has been suggested that viral penetration presumably occurs along channels left when the follicle cell processes are withdrawn.
Although we have found no evidence in previous reports that viruses are able to cross the ZP [20,21], one particular observation in this study deserves further attention. In some fertilized zygotes, excavation or fissures with the exact dimensions of a sperm head (which was probably caused by enzymatic digestion) were observed. These holes could provide a point of entry for the virus at fertilization, besides serving as a potential hiding place for the virus. Virus resident in such a cavity might be protected against eventual sanitary measures, e.g. washings and trypsin treatment, and pose a danger for infecting susceptible cows after transference of such treated embryos. Possible virus entry in the ZP during fertilization has been previously reported for porcine parvovirus, pseudorabies virus, and porcine enteroviruses, which were found in sperm tracks in pores in the ZP [34]. However, these tracks tend to close up quickly after fertilization, thus it would be difficult for viruses, which are nonmotile, to traverse these channels in the ZP up to the embryonic cells [26]. Porcine parvovirus, pseudorabies virus, and porcine enteroviruses have also been associated with porcine sperm cells at the outer surface of the ZP of porcine embryos.
In conclusion, this study provides evidence that the intact ZP of IVM bovine oocytes and IVP bovine embryos are constructed in such a way that BHV-1 and BVDV should not be able to reach the embryonic cells. However, even though the structure of the ZP serves as a barrier, the risk exists that viral particles could be embedded in the outer layers of the ZP. Therefore, additional research is needed to determine if viral particles remain in the ZP after washing and/or after trypsin treatment, and to establish effective embryo treatments which can ensure that virus-free IVP embryos are produced.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grant no. 941437 from IWT Brussels. 
2 Correspondence and reprint requests: Geert Vanroose, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, B-9000 Gent, Belgium. Fax: 32 9 264 77 97; geert.vanroose{at}rug.ac.be
Accepted: September 24, 1999.
Received: June 25, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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