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Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova
Theriogenology40:1281-1291,1993
STRUCTURAL AND VIRAL ASSOCIATION COMPARISONS OF BOVINE ZONAE PELLUCIDAE FROM FOLLICULAR OOCYTES, DAY-7 EMBRYOS AND DAY-7 DEGENERATED OVA K.P. Riddell,' D.A. Stringfellow,' B.W. Gray,2 M.G. Riddell, J.C. Wright' and P.K. Galikl 'Department of Pathobiology 2Department of Anatomy and Histology 3Department of Large Animal Surgery and Medicine College of Veterinary Medicine Auburn University, Alabama 36849-5519, USA Received
for publication: Accepted:
November 12, 2991 October 7, 1983
ABSTRACT Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined. Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7pm/12.3pm, The mean total 161.3pm/12.6pm, and 158.9pm/12.8pm, respectively. protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 pg, 0.349 pg, and 0.254 pg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.O;P
Copyright
embryo, oocyte, IBRV, zona pellucida
0 1993 Butterworth-Heinemann
Theriogenology
1282
INTRODUCTION The mammalian zona pellucida (ZP) is a relatively thick, transparent, glycoproteinaceous shell to which are attributed the functions of species specificity of fertilization, block to polyspermy, and protection of the conceptus during early stages of development (28). With some variation between species, the ZP is composed of 2 to 4 distinct but microheterogeneous components when examined by 2 dimensional polyacrylamide gel electrophoresis (3,28). The glycoproteins that comprise the ZP are thought to be secreted exclusively by the oocyte prior to ovulation, with some components from oviductal epithelial cells being added after ovulation (2,4,14,18,28). Most investigations of the primary resistance to infectious agents that is provided by the bovine ZP have been done with Day7 embryos, and the value of the ZP of these transfer-stage embryos for disease control is well established (22,26). However, most of the biochemical, immunochemical and morphological information on this structure in cattle was derived through work with ZPs of ovarian origin (g-11,27). Essentially no comparable information on the Day-7 structure is available. It is known through work in other species that the ZP is an extracellular matrix that is subject to functional and biochemical changes after ovulation and fertilization and during the course of early embryonic development (14,17,21). Thus, it is possible that the ZP of the bovine follicular oocyte may function differently, as a barrier to infectious agents or in adherence of Refinement of pathogens, than the ZP of a Day-7 bovine embryo. systems for in vitro fertilization (IVF) in cattle have already led to proposed applications of the technology for commercial production of embryos (5), yet few studies of embryo-pathogen interactions around the time of fertilization have been conducted (6-8). Since most of the disease resistance studies of the bovine ZP were conducted at Day 7, and since most of the structural studies were conducted with ovarian oocytes, our initial objective was to make structural comparisons between these stages An additional objective of to determine if differences existed. the current study was to determine if there were differences in the amount of infectious IBRV associated with ZP-intact (I) follicular oocytes, Day-7 embryos, and Day-7 degenerated ova after in vitro exposure of a limited number of each of these to the virus and to washing, using standards established by the International Embryo Transfer Society (1). MATERIALS
AND METHODS
Ovaries from heifers slaughtered at a local abattoir were The oocytes were aspirated the source of follicular oocytes. Cumulus cells from l- to 5-mm follicles through a 23-g needle. were removed by pipetting vigorously through a glass micropipette Day-7 embryos and degenerated ova were collected (150 pm i.d.).
Theriogenology
from 11 superovulated dairy and beef cows and were washed according to International Embryo Transfer Society guidelines prior to use (1). When the Day-7 embryos and degenerated ova were to be exposed to IBRV, sera collected from donor cows were examined for anti-IBRV antibody by virus neutralization assay (20) to determine the serological status of donors at the time of collection: however, as stated, the embryos were washed 10 times prior to exposure to the virus in order to dilute any specific antibody as well as contaminants that might have been present in the embryo collection fluid. Washing medium contained fetal bovine serum that failed to neutralize IBRV at dilutions of 1:2 and greater. The washing procedure, which conformed to the recommended standards of the International Embryo Transfer Society (l), is described briefly as follows. Only ZP-I embryos/ova (examined at x50 magnification) were washed 10 times in groups of 10 or fewer. The washing medium consisted of Dulbecco's phosphate-buffered saline with 2% fetal bovine serum and antibiotics (100 U penicillin, 100 pg streptomycin, and 0.25 pg amphoteracin B/ml of Separate sterile micropipettes was used to carry medium). The ratio of volume of medium embryos/ova between wash dishes. containing embryos/ova in the micropipette to volume of medium in each wash dish was at least 1:lOO. For scanning electron microscopy, the oocytes, ova, and embryos were fixed in 1% glutaraldehyde, washed 5 min in distilled water, dehydrated in graded ethanol, treated with hexamethyldisilizane for 5 to 10 min, mounted on double-sided tape and immediately coated with gold. The diameters and thicknesses of ZPs of ZP-intact (I) oocytes (after cumulus cells were removed), ova., and embryos were measured by an ocular micrometer at x200 magnification using brightfield microscopy. Total proteins were determined using the bicinchoninic acid (BCA) method, with bovine serum albumin as a standard (Pierce The ZPs for protein Micro-BCA, Rockfield, IL, USA 61105). The ZPs were ruptured and the analysis were prepared as follows: contents were removed by vigorous pipetting through a glass micropipette (80 pm i.d.). The empty ZPs were washed a minimum of 3 times in 2 ml of a tris-buffered solution (pH 8.0) containing 1OmM MgC12, 24mM KCl, 1OmM tris base, and about 0.05% (v/v) Tween 80. The ZPs were heat solubilized (SO'C for 35 min) in groups of 7 to 12 in 1 ml of phosphate buffered saline that was then used for protein determination. Virus exposures and plaque assays were performed according to general procedures described previously (25). Briefly, ZP-I oocytes (without cumulus cells), ova, and embryos were exposed to IBRV-infected (Colorado strain) Madin Darby Bovine Kidney cells (MDBK) for 24 h. After exposure they were washed 10 times
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according to standardized procedures of the International Embryo Transfer Society (1); they were then sonicated individually in 1 ml of minimum essential medium, after which the sonicate fluids were placed onto la cm2 of confluent MDBK cells for 1 h prior to overlay with agarose. The monolayers were examined and the plaques were counted at the peak of development on Days 3, 4 or 5. The titer of IBRV to which the oocytes, ova, and embryos were exposed was determined by plaque assay at the end of each exposure period. As a final confirmation that the plaques were due to the cytopathic effects of IBRV, virus from plaques in each assay was further propagated in MDBK cells, and the identity of the expanded virus was confirmed by neutralization with anti-IBRV antibody. For each plaque assay, noninfected MDBK cells were overlaid with agarose to serve as the negative controls, while a simultaneous titration of the stock virus served as the positive control. Differences among group means of plaque-forming units (PFU), diameters, thicknesses and total proteins were analyzed using the General Linear Model for analysis of variance and Scheffe's test for multiple comparisons (19). RESULTS Scanning electron microscopy of the external surfaces of the ZPs of follicular oocytes revealed a very irregular exterior with uneven distribution of numerous pores, crevices and projections (Figure 1A). In contrast, the surfaces of the ZPs of Day-7 embryos were slightly smoother, with fewer obvious pores and less distinctive ridges and protrusions (Figure 1B). The smoothness and scarcity of distinct protrusions were even more evident on the surfaces of ZPs of degenerated Day-7 ova (Figure 1C). A summary of dimensional measurements of ZPs from follicular oocytes and from Day-7 embryos and degenerated ova is presented in Table 1. There was substantial variation between individual ZPs in measurements of the thicknesses and overall diameters: however, there was no significant difference among the mean The mean diameter thicknesses of the 3 groups of ZPs (P=O.6543). of ZPs of Day-7 embryos was significantly greater (P
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Although IBRV was associated with embryos/ova from all 3 sources after exposure and washing, there was substantial variation in the number of PFU detected within groups (Table 3). The mean PFU from the follicular oocytes was significantly greater than the mean PFU from Day-7 embryos (PcO.05) and degenerated Day-7 ova (PcO.01). There was no significant difference in the mean PFU from Day-7 embryos and the mean PFU from degenerated Day-7 ova. Moreover, there were no apparent differences between quantities of virus associated with Day-7 embryos/ova collected from 4 donor cows with anti-IBRV antibody in the blood serum on the day of collection when compared with that of embryos/ova collected from 7 donor cows without anti-IBRV antibody in the blood.
Figure 1. Scanning electron micrographs (x5,000) of the surface of the bovine zona pellucida of A) a follicular oocyte, B) a Day-7 embryo, and C) a Day-7 degenerated ovum. Table 1.
Mean dimensions in microns of a bovine zona pellucida (ZP) from follicular oocytes and Day-7 embryos and from degenerated ova
Source of ZPs
Diameter
(range)
Follicular (n=30)
156.7
(142.5-167.5)b
12.3
(7.5-17.5)a
Day-7 embryos (n=27)
161.3
(147.5-175.0)a
12.6
(10.0-15.0)a
Day-7 ova (n=28)
158.9
(130.0-172.5)a>b
12.8 (8.8-15.0)a
Thickness
(range)
oocytes
DMeans that do not share common superscripts a, different at PcO.05.
were significantly
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Table 2.
Mean value for total protein per zona pellucida from follicular oocytes and Day-7 embryos and degenerated ova.
(ZP)
Source of ZPs
Protein/ZP
Follicular oocytes (n=5 groups)
0.331 + 0.032a'b
(0.285 - 0.375)
Day-7 embryos (n=4 groups)
0.349 f 0.083a
(0.260 - 0.448)
Day-7 ova (n=6 groups)
0.254 + 0.027b
(0.230 - 0.290)
(pg)
ayDMeans that do not share common superscripts different at PcO.05. Table 3.
(range)
are significantly
Mean number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) adhering to the zona pellucida (ZP) of follicular oocytes and Day-7 embryos and degenerated ova after in vitro exposure
Source of ZP
Mean PFU (range)
Exposure level (PFU/ml)
Follicular (n=29)
68.1 (0 - 222)a
2 to 4x107
oocytes
Day-7 embryos (n=36)C
43.0
(1 - 118)b (P
2 to 6X107
Day-7 ova (n=31)d
31.9 (2 - 121)b (P
2 to 6X107
a$%eans that do not share different at the P values 'From 11 cows. dFrom 6 cows (blood serum cows ranged from negative
common superscripts shown.
were significantly
titers of anti-IBRV antibody of donor at 1:lO to positive at 1:32). DISCUSSION
The fenestrated lattice-like or sponge-like appearance of the ZP has been observed by scanning electron microscopy in a The source of the ZPs for these variety of species (3,11,22). examinations generally was ovarian oocytes or recently ovulated ova. The appearance of follicular oocytes observed by scanning electron microscopy in this study was consistent with previously reported observations: however, the surfaces of Day-7 embryos and degenerated ova were markedly different from traditional descriptions of the bovine ZP. The smoother surfaces
Theriogenology
and relative scarcity of observable pores on the ZPs of Day-7 embryos and degenerated ova compared with those of follicular oocytes may be due to exposure to proteolytic enzymes and/or the That these types of addition of glycoproteins after ovulation. changes have an effect on the biochemical makeup of the ZP after ovulation has been demonstrated in other species (14,21). Further, it has been reported that changes associated with either in vivo or in vitro ageing of murine oocytes give rise to surface changes similar to those that we observed (17). The progressively smoother surface and apparent reduction in porosity associated with the ageing of the bovine ZP in vivo might or might not result in differences in zona-pathogen interactions. Previously, it was reported that the mean values for diameter/thickness of ZPs of normal, degenerating and degenerated Day-7 embryos were 160@m/12~m, 159~m/12~m and 159fim/12@m, respectively (15). Our values for both follicular oocytes and However, in Day-7 embryos or degenerated ova were very similar. a report by Gwatkin et al. (Q), the average diameter of bovine ovarian oocytes was stated to be 146.2 ? 5.7 pm. The smaller mean diameter reported by these investigators may have been due to the use of methods for retrieval of the ZPs that yielded a large proportion of relatively immature oocytes. Gwatkin et al. (9) also reported an estimated mean total protein per ZP (36.0 * 2.9 ng/ZP) that was considerably lower than the amount that we found for ZPs of follicular oocytes or Day-7 embryos. The difference in values could have been due to differences in sources of ZPs, methods of handling ZPs, methods for protein determination, or a combination of these factors. Gwatkin et al. (9) used the method of Lowry (16) while we used the bicinchoninic acid method. It is not likely that differences in methodology accounted for all of the difference in values, since these 2 methods were compared previously for determination of total protein in porcine ZPs with only a 5% variation reported (12). It is more likely that differences in the source of ZPs and possibly, to a lesser degree, variations in methods for handling of raw material resulted in the discrepancy. The ZPs used by Gwatkin et al.(Q) were recovered from the ovaries of 4to 5-mo-old calves by grinding and enzymatic dispersion with collagenase. Our ZPs were aspirated from visible ovarian follicles of slaughter-age, cyclic heifers that were more than 1 yr of age. We removed cumulus cells and contents mechanically without the use of proteolytic enzymes, That the use of collagenase for preparation of ZPs results in some biochemical alteration in the structure has been demonstrated with porcine ZPS (13). The use of this enzyme could have reduced the total amount of protein somewhat, but a more likely reason for the large differences in total protein is the average stage of development of the oocytes used. Developing oocytes are continuously secreting zonal proteins while increasing in diameter at a tremendous rate (up to 300 times increase in volume), resulting in a structure that is larger, thicker and denser internally than on the surface (28). Gwatkin et al.(Q)
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did not report the average thickness of the ZPs that they analyzed, but as stated above, the average diameter of the oocyte was approximately 146 pm compared to an average of 157 pm for the more mature oocytes used in our analyses. The larger size and presumed greater density of ZPs from more mature oocytes could account for most of the difference in total proteins found. Perhaps more important than the comparison to previous findings of absolute values for total protein are the comparative values between follicular oocytes, Day-7 embryos, and Day-7 degenerated ova analyzed in this study. Zonae pellucidae from follicular oocytes and Day-7 embryos had approximately the same amount of protein, while ZPs from degenerated ova at Day-7 had significantly less (P~0.05) protein than the ZPs of the Day-7 embryos. This finding is consistent with the more eroded appearance of degenerated Day-7 ova seen with scanning electron microscopy. It is important to emphasize that neither the structural differences detected by scanning electron microscopy and protein determinations nor the differences in diameter detected by measurement were discernable when observed at x200 magnification. It is generally accepted that pathogens that are shown to adhere to the ZPs of Day-7 transferable bovine embryos are likely to adhere equally well to Day-7 ZP-I nontransferable or degenerated bovine ova. It was cited as unpublished information that IBRV adheres to the ZPs of both Day-7 transferable bovine embryos and Day-7 ZP-I nontransferable or degenerated ova (19), but quantitative comparisons or examinations of the consistency with which IBRV is associated with each have not been reported. Since one protocol for health certification of embryos for international movement suggests the testing of nonfertile/ degenerated ova recovered from a superovulated cow as an indication of the infective status of transferable embryos from the same collection (l), and in light of obvious structural differences, we decided to compare the quantities of IBRV that were associated with ZP-I Day-7 embryos and Day-7 degenerated ova. We chose to work with IBRV because it is one of the few viral pathogens known to adhere to the bovine ZP, and in our experience, it adheres in relatively large quantities. We also chose to compare simultaneously the quantities of IBRV that were associated with ZP-I bovine follicular oocytes because recent moves toward the commercial production of in vitro-derived embryos (5) have raised health concerns, and a previous report of association of IBRV with in vitro matured and fertilized oocytes did not provide comparative quantitative data (8). Despite a considerable variation in the quantity of the virus, IBRV was isolated from all Day-7 embryos and degenerated ova after washing, thus providing additional support for the suggested practice of testing degenerated embryos/ova in a collection as an indicator of the IBRV-infective status of transferable embryos (1). Obviously, the lower total protein of ZPs from degenerated ova was not accompanied by structural
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changes that prevented interaction for significantly larger quantities follicular oocytes could have been
with the virus. of virus to be due to entrapment
The tendency isolated from
in the more numerous pores and grooves on the rougher surface, or it may have been due to adherence to remnants of follicular cell membranes, although this cannot be stated definitively. Conversely, the association of IBRV with the relatively smooth Day-7 embryos and ova seems to indicate that the mechanism of interaction is at least. not entirely by entrapment, but could be by some specific affinity for the structure that is maintained despite changes that occur in the ZP during the first 7 d of development. In summary, although differences cannot be detected by light micro8scopy in the thickness or appearance of bovine ZPs of follicular oocytes, Day-7 embryos or Day-7 degenerated ova, it is clear from scanning electron microscope examination and total This is protein analysis that the structures are not equivalent. consistent with the results of biochemical and electron microscope examinations in other species (14,17). Further, IBRV was associated with the ZPs from all sources examined, and there were quantitative differences in the limited numbers examined. It is important to place our results in proper perspective. The value of the intact ZP of Day 7 bovine embryos as a barrier to pathogens was established through the cumulative results of numerous studies conducted over a lo-year period (22-24,26). It may ultimately be shown that subtle differences in the structure of thfe ZP of follicular oocytes will not affect its capability to Further studies are serve as an equivalent barrier to pathogens. needed for a proper evaluation. REFERENCES 1. Anonymous. Recommendation for the sanitary handling of embryos. In: Stringfellow DA, Seidel SM (eds), Manual of the International Embryo Transfer Society. IETS, Champaign, IL, 1990;41-45. 2. Boice ML, McCarthy TJ, Maurogianis PA, Fazleabas AT, Verhage HG. Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (w anubis) . Bio Reprod 1990;43:340-346. 3. Dunbar BS. Morphological, biochemical, and immunochemical characterization of the mammalian zona pellucida. In: Hartmann JF Mechanism and Control of Animal Fertilization. Academic Press, (ed), Mew York, 1983;139-175. 4. Gandolfi F, Brevini TAL, Richardson L, Brown CR, Moor RM. Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 1989;106:303-312. 5. Gordon I, Lu KH. Production of embryos in vitro and its impact on livestock production. Theriogenology 1990;33:77-87. 6. Guerin B, Le Guienne B, Chaffaux S, Harlay T, Allietta M, Thibier M. Contamination des ovocytes et des embryons fecondes in vitro apres infection experimentale, de vaches donneuses par le virus herpes bovin de type 1 (BHVl). Ret Med Vet 1989;165:827-833.
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7. Guerin B, Le Guienne B, Thibier M. Absence de contamination microbiologigue des embryons bovins fecondes in vitro. Bull Acad Vet de France 1988:61:513-520. 8. Guerin B, Marguant-Le Guienne B, Allietta M, Harlay T, Thibier M. Effets de la contamination par le BHVl sur la maturation et la fecondation in vitro des ovocytes de bovins. Ret Med Vet 1990;166:911917. 9. Gwatkin RBL, Andersen OF, Williams DT. Large scale isolation of bovine and pig zonae pellucidae: chemical, immunological, and receptor properties. Gam Res 1980;3:217-231. 10. Gwatkin RBL, Williams DT. Immunization of female rabbits with heatsolubilized bovine zonae: production of anti-zona antibody and inhibition of fertility. Gam Res 1978;1:19-26. 11. Gwatkin RBL,' Williams DT, Meyenhofer M. Isolation of bovine zonae pellucidae from ovaries with collagenase: antigenic and sperm receptor properties. Gam Res 1979;2:187-192. 12. Hedrick JL, Wardrip NJ. Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Analy Biochen 1986;157:63-70. 13. Hedrick JL, Wardrip NJ. Proteolysis by collagenase preparations alters the macromolecular composition of the porcine zona pellucida. Biol Reprod 1986;35:677-682. 14. Hedrick JL, Wardrip NJ, Berger T. Differences in the macromolecular composition of the zona pellucida isolated from pig oocytes, eggs and zygotes. J Exp Zoo1 1987;241:257-262. 15. Linares T, King WA. Morphological study of the blastocyst with phase contrast microscopy. Theriogenology 1980;14:121-133. 16. Lowry OH, Roseberg MJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 17. Nogoies C, Ponsa M, Vidal F, Boada M, Egozcue J. Effects of aging on the zona pellucida of mouse oocytes. J IVF Emb Trans 1988;5:225-229. 18. Oikawa T, Sendai Y, Kurata S, Yanagimachi A glycoprotein of R. oviductal origin alters biochemical properties of the zona pellucida of hamster egg. Gam Res 1988;19:113-122. 19. SAS User's Guide: Statistics. SAS Institute, Inc, Cary, NC, 1982;139200. 20. Schmidt NJ. Tissue culture methods and procedures for diagnostic Procedures for virology. In: Lennette EH, Schmidt NJ (eds), Diagnostic Viral and Rickettsial Diseases. American Public Health Association, New York, 1964;120-121. 21. Shabanowitz Characterization of the human zona RB, O*Rand MG. pellucida from fertilized and unfertilized eggs. J Reprod Fertil 1988;82:151-161. 22. Shisong C, Wrathall The importance of the zona pellucida for AE. disease control in livestock by embryo transfer. Br Vet J 1989;145:129-140. 23. Singh EL. Rev sci Disease control: procedures for handling embryos. tech off int Epiz 1985;4:867-872. 24. Singh EL. The disease control potential of embryos. Theriogenology 1987;27:9-20. 25. Stringfellow DA, Lauerman LH, Nasti KB, Galik PK. Trypsin treatment of bovine embryos after in vitro exposure to infectious bovine rhinotracheitis virus qr bovine herpesvirus-4. Theriogenology 1990:34:427-434.
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26. Stringfellow DA, Riddell KP, Zurovac 0. The potential of embryo transfer for infectious disease control in livestock. NZ Vet J 1991;39:8-17. Inhibition of fertilization in cattle 27. Tsunoda Y, Soma T, Sugie T. by passive immunization with anti-zona pellucida serum. Gam Res 1981;4:133-138. Ann Rev Biochem 28. Wassarman PM. Zona pellucida glycoproteins. 1988:57:415-442.
STRUCTURAL AND VIRAL ASSOCIATION COMPARISONS OF BOVINE ZONAE PELLUCIDAE FROM FOLLICULAR OOCYTES, DAY-7 EMBRYOS AND DAY-7 DEGENERATED OVA K.P. Riddell,' D.A. Stringfellow,' B.W. Gray,2 M.G. Riddell, J.C. Wright' and P.K. Galikl 'Department of Pathobiology 2Department of Anatomy and Histology 3Department of Large Animal Surgery and Medicine College of Veterinary Medicine Auburn University, Alabama 36849-5519, USA Received
for publication: Accepted:
November 12, 2991 October 7, 1983
ABSTRACT Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined. Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7pm/12.3pm, The mean total 161.3pm/12.6pm, and 158.9pm/12.8pm, respectively. protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 pg, 0.349 pg, and 0.254 pg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.O;P
Copyright
embryo, oocyte, IBRV, zona pellucida
0 1993 Butterworth-Heinemann
Theriogenology
1282
INTRODUCTION The mammalian zona pellucida (ZP) is a relatively thick, transparent, glycoproteinaceous shell to which are attributed the functions of species specificity of fertilization, block to polyspermy, and protection of the conceptus during early stages of development (28). With some variation between species, the ZP is composed of 2 to 4 distinct but microheterogeneous components when examined by 2 dimensional polyacrylamide gel electrophoresis (3,28). The glycoproteins that comprise the ZP are thought to be secreted exclusively by the oocyte prior to ovulation, with some components from oviductal epithelial cells being added after ovulation (2,4,14,18,28). Most investigations of the primary resistance to infectious agents that is provided by the bovine ZP have been done with Day7 embryos, and the value of the ZP of these transfer-stage embryos for disease control is well established (22,26). However, most of the biochemical, immunochemical and morphological information on this structure in cattle was derived through work with ZPs of ovarian origin (g-11,27). Essentially no comparable information on the Day-7 structure is available. It is known through work in other species that the ZP is an extracellular matrix that is subject to functional and biochemical changes after ovulation and fertilization and during the course of early embryonic development (14,17,21). Thus, it is possible that the ZP of the bovine follicular oocyte may function differently, as a barrier to infectious agents or in adherence of Refinement of pathogens, than the ZP of a Day-7 bovine embryo. systems for in vitro fertilization (IVF) in cattle have already led to proposed applications of the technology for commercial production of embryos (5), yet few studies of embryo-pathogen interactions around the time of fertilization have been conducted (6-8). Since most of the disease resistance studies of the bovine ZP were conducted at Day 7, and since most of the structural studies were conducted with ovarian oocytes, our initial objective was to make structural comparisons between these stages An additional objective of to determine if differences existed. the current study was to determine if there were differences in the amount of infectious IBRV associated with ZP-intact (I) follicular oocytes, Day-7 embryos, and Day-7 degenerated ova after in vitro exposure of a limited number of each of these to the virus and to washing, using standards established by the International Embryo Transfer Society (1). MATERIALS
AND METHODS
Ovaries from heifers slaughtered at a local abattoir were The oocytes were aspirated the source of follicular oocytes. Cumulus cells from l- to 5-mm follicles through a 23-g needle. were removed by pipetting vigorously through a glass micropipette Day-7 embryos and degenerated ova were collected (150 pm i.d.).
Theriogenology
from 11 superovulated dairy and beef cows and were washed according to International Embryo Transfer Society guidelines prior to use (1). When the Day-7 embryos and degenerated ova were to be exposed to IBRV, sera collected from donor cows were examined for anti-IBRV antibody by virus neutralization assay (20) to determine the serological status of donors at the time of collection: however, as stated, the embryos were washed 10 times prior to exposure to the virus in order to dilute any specific antibody as well as contaminants that might have been present in the embryo collection fluid. Washing medium contained fetal bovine serum that failed to neutralize IBRV at dilutions of 1:2 and greater. The washing procedure, which conformed to the recommended standards of the International Embryo Transfer Society (l), is described briefly as follows. Only ZP-I embryos/ova (examined at x50 magnification) were washed 10 times in groups of 10 or fewer. The washing medium consisted of Dulbecco's phosphate-buffered saline with 2% fetal bovine serum and antibiotics (100 U penicillin, 100 pg streptomycin, and 0.25 pg amphoteracin B/ml of Separate sterile micropipettes was used to carry medium). The ratio of volume of medium embryos/ova between wash dishes. containing embryos/ova in the micropipette to volume of medium in each wash dish was at least 1:lOO. For scanning electron microscopy, the oocytes, ova, and embryos were fixed in 1% glutaraldehyde, washed 5 min in distilled water, dehydrated in graded ethanol, treated with hexamethyldisilizane for 5 to 10 min, mounted on double-sided tape and immediately coated with gold. The diameters and thicknesses of ZPs of ZP-intact (I) oocytes (after cumulus cells were removed), ova., and embryos were measured by an ocular micrometer at x200 magnification using brightfield microscopy. Total proteins were determined using the bicinchoninic acid (BCA) method, with bovine serum albumin as a standard (Pierce The ZPs for protein Micro-BCA, Rockfield, IL, USA 61105). The ZPs were ruptured and the analysis were prepared as follows: contents were removed by vigorous pipetting through a glass micropipette (80 pm i.d.). The empty ZPs were washed a minimum of 3 times in 2 ml of a tris-buffered solution (pH 8.0) containing 1OmM MgC12, 24mM KCl, 1OmM tris base, and about 0.05% (v/v) Tween 80. The ZPs were heat solubilized (SO'C for 35 min) in groups of 7 to 12 in 1 ml of phosphate buffered saline that was then used for protein determination. Virus exposures and plaque assays were performed according to general procedures described previously (25). Briefly, ZP-I oocytes (without cumulus cells), ova, and embryos were exposed to IBRV-infected (Colorado strain) Madin Darby Bovine Kidney cells (MDBK) for 24 h. After exposure they were washed 10 times
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according to standardized procedures of the International Embryo Transfer Society (1); they were then sonicated individually in 1 ml of minimum essential medium, after which the sonicate fluids were placed onto la cm2 of confluent MDBK cells for 1 h prior to overlay with agarose. The monolayers were examined and the plaques were counted at the peak of development on Days 3, 4 or 5. The titer of IBRV to which the oocytes, ova, and embryos were exposed was determined by plaque assay at the end of each exposure period. As a final confirmation that the plaques were due to the cytopathic effects of IBRV, virus from plaques in each assay was further propagated in MDBK cells, and the identity of the expanded virus was confirmed by neutralization with anti-IBRV antibody. For each plaque assay, noninfected MDBK cells were overlaid with agarose to serve as the negative controls, while a simultaneous titration of the stock virus served as the positive control. Differences among group means of plaque-forming units (PFU), diameters, thicknesses and total proteins were analyzed using the General Linear Model for analysis of variance and Scheffe's test for multiple comparisons (19). RESULTS Scanning electron microscopy of the external surfaces of the ZPs of follicular oocytes revealed a very irregular exterior with uneven distribution of numerous pores, crevices and projections (Figure 1A). In contrast, the surfaces of the ZPs of Day-7 embryos were slightly smoother, with fewer obvious pores and less distinctive ridges and protrusions (Figure 1B). The smoothness and scarcity of distinct protrusions were even more evident on the surfaces of ZPs of degenerated Day-7 ova (Figure 1C). A summary of dimensional measurements of ZPs from follicular oocytes and from Day-7 embryos and degenerated ova is presented in Table 1. There was substantial variation between individual ZPs in measurements of the thicknesses and overall diameters: however, there was no significant difference among the mean The mean diameter thicknesses of the 3 groups of ZPs (P=O.6543). of ZPs of Day-7 embryos was significantly greater (P
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Theriogenology
Although IBRV was associated with embryos/ova from all 3 sources after exposure and washing, there was substantial variation in the number of PFU detected within groups (Table 3). The mean PFU from the follicular oocytes was significantly greater than the mean PFU from Day-7 embryos (PcO.05) and degenerated Day-7 ova (PcO.01). There was no significant difference in the mean PFU from Day-7 embryos and the mean PFU from degenerated Day-7 ova. Moreover, there were no apparent differences between quantities of virus associated with Day-7 embryos/ova collected from 4 donor cows with anti-IBRV antibody in the blood serum on the day of collection when compared with that of embryos/ova collected from 7 donor cows without anti-IBRV antibody in the blood.
Figure 1. Scanning electron micrographs (x5,000) of the surface of the bovine zona pellucida of A) a follicular oocyte, B) a Day-7 embryo, and C) a Day-7 degenerated ovum. Table 1.
Mean dimensions in microns of a bovine zona pellucida (ZP) from follicular oocytes and Day-7 embryos and from degenerated ova
Source of ZPs
Diameter
(range)
Follicular (n=30)
156.7
(142.5-167.5)b
12.3
(7.5-17.5)a
Day-7 embryos (n=27)
161.3
(147.5-175.0)a
12.6
(10.0-15.0)a
Day-7 ova (n=28)
158.9
(130.0-172.5)a>b
12.8 (8.8-15.0)a
Thickness
(range)
oocytes
DMeans that do not share common superscripts a, different at PcO.05.
were significantly
Theriogenology
1286
Table 2.
Mean value for total protein per zona pellucida from follicular oocytes and Day-7 embryos and degenerated ova.
(ZP)
Source of ZPs
Protein/ZP
Follicular oocytes (n=5 groups)
0.331 + 0.032a'b
(0.285 - 0.375)
Day-7 embryos (n=4 groups)
0.349 f 0.083a
(0.260 - 0.448)
Day-7 ova (n=6 groups)
0.254 + 0.027b
(0.230 - 0.290)
(pg)
ayDMeans that do not share common superscripts different at PcO.05. Table 3.
(range)
are significantly
Mean number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) adhering to the zona pellucida (ZP) of follicular oocytes and Day-7 embryos and degenerated ova after in vitro exposure
Source of ZP
Mean PFU (range)
Exposure level (PFU/ml)
Follicular (n=29)
68.1 (0 - 222)a
2 to 4x107
oocytes
Day-7 embryos (n=36)C
43.0
(1 - 118)b (P
2 to 6X107
Day-7 ova (n=31)d
31.9 (2 - 121)b (P
2 to 6X107
a$%eans that do not share different at the P values 'From 11 cows. dFrom 6 cows (blood serum cows ranged from negative
common superscripts shown.
were significantly
titers of anti-IBRV antibody of donor at 1:lO to positive at 1:32). DISCUSSION
The fenestrated lattice-like or sponge-like appearance of the ZP has been observed by scanning electron microscopy in a The source of the ZPs for these variety of species (3,11,22). examinations generally was ovarian oocytes or recently ovulated ova. The appearance of follicular oocytes observed by scanning electron microscopy in this study was consistent with previously reported observations: however, the surfaces of Day-7 embryos and degenerated ova were markedly different from traditional descriptions of the bovine ZP. The smoother surfaces
Theriogenology
and relative scarcity of observable pores on the ZPs of Day-7 embryos and degenerated ova compared with those of follicular oocytes may be due to exposure to proteolytic enzymes and/or the That these types of addition of glycoproteins after ovulation. changes have an effect on the biochemical makeup of the ZP after ovulation has been demonstrated in other species (14,21). Further, it has been reported that changes associated with either in vivo or in vitro ageing of murine oocytes give rise to surface changes similar to those that we observed (17). The progressively smoother surface and apparent reduction in porosity associated with the ageing of the bovine ZP in vivo might or might not result in differences in zona-pathogen interactions. Previously, it was reported that the mean values for diameter/thickness of ZPs of normal, degenerating and degenerated Day-7 embryos were 160@m/12~m, 159~m/12~m and 159fim/12@m, respectively (15). Our values for both follicular oocytes and However, in Day-7 embryos or degenerated ova were very similar. a report by Gwatkin et al. (Q), the average diameter of bovine ovarian oocytes was stated to be 146.2 ? 5.7 pm. The smaller mean diameter reported by these investigators may have been due to the use of methods for retrieval of the ZPs that yielded a large proportion of relatively immature oocytes. Gwatkin et al. (9) also reported an estimated mean total protein per ZP (36.0 * 2.9 ng/ZP) that was considerably lower than the amount that we found for ZPs of follicular oocytes or Day-7 embryos. The difference in values could have been due to differences in sources of ZPs, methods of handling ZPs, methods for protein determination, or a combination of these factors. Gwatkin et al. (9) used the method of Lowry (16) while we used the bicinchoninic acid method. It is not likely that differences in methodology accounted for all of the difference in values, since these 2 methods were compared previously for determination of total protein in porcine ZPs with only a 5% variation reported (12). It is more likely that differences in the source of ZPs and possibly, to a lesser degree, variations in methods for handling of raw material resulted in the discrepancy. The ZPs used by Gwatkin et al.(Q) were recovered from the ovaries of 4to 5-mo-old calves by grinding and enzymatic dispersion with collagenase. Our ZPs were aspirated from visible ovarian follicles of slaughter-age, cyclic heifers that were more than 1 yr of age. We removed cumulus cells and contents mechanically without the use of proteolytic enzymes, That the use of collagenase for preparation of ZPs results in some biochemical alteration in the structure has been demonstrated with porcine ZPS (13). The use of this enzyme could have reduced the total amount of protein somewhat, but a more likely reason for the large differences in total protein is the average stage of development of the oocytes used. Developing oocytes are continuously secreting zonal proteins while increasing in diameter at a tremendous rate (up to 300 times increase in volume), resulting in a structure that is larger, thicker and denser internally than on the surface (28). Gwatkin et al.(Q)
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Theriogenology
did not report the average thickness of the ZPs that they analyzed, but as stated above, the average diameter of the oocyte was approximately 146 pm compared to an average of 157 pm for the more mature oocytes used in our analyses. The larger size and presumed greater density of ZPs from more mature oocytes could account for most of the difference in total proteins found. Perhaps more important than the comparison to previous findings of absolute values for total protein are the comparative values between follicular oocytes, Day-7 embryos, and Day-7 degenerated ova analyzed in this study. Zonae pellucidae from follicular oocytes and Day-7 embryos had approximately the same amount of protein, while ZPs from degenerated ova at Day-7 had significantly less (P~0.05) protein than the ZPs of the Day-7 embryos. This finding is consistent with the more eroded appearance of degenerated Day-7 ova seen with scanning electron microscopy. It is important to emphasize that neither the structural differences detected by scanning electron microscopy and protein determinations nor the differences in diameter detected by measurement were discernable when observed at x200 magnification. It is generally accepted that pathogens that are shown to adhere to the ZPs of Day-7 transferable bovine embryos are likely to adhere equally well to Day-7 ZP-I nontransferable or degenerated bovine ova. It was cited as unpublished information that IBRV adheres to the ZPs of both Day-7 transferable bovine embryos and Day-7 ZP-I nontransferable or degenerated ova (19), but quantitative comparisons or examinations of the consistency with which IBRV is associated with each have not been reported. Since one protocol for health certification of embryos for international movement suggests the testing of nonfertile/ degenerated ova recovered from a superovulated cow as an indication of the infective status of transferable embryos from the same collection (l), and in light of obvious structural differences, we decided to compare the quantities of IBRV that were associated with ZP-I Day-7 embryos and Day-7 degenerated ova. We chose to work with IBRV because it is one of the few viral pathogens known to adhere to the bovine ZP, and in our experience, it adheres in relatively large quantities. We also chose to compare simultaneously the quantities of IBRV that were associated with ZP-I bovine follicular oocytes because recent moves toward the commercial production of in vitro-derived embryos (5) have raised health concerns, and a previous report of association of IBRV with in vitro matured and fertilized oocytes did not provide comparative quantitative data (8). Despite a considerable variation in the quantity of the virus, IBRV was isolated from all Day-7 embryos and degenerated ova after washing, thus providing additional support for the suggested practice of testing degenerated embryos/ova in a collection as an indicator of the IBRV-infective status of transferable embryos (1). Obviously, the lower total protein of ZPs from degenerated ova was not accompanied by structural
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Theriogenology
changes that prevented interaction for significantly larger quantities follicular oocytes could have been
with the virus. of virus to be due to entrapment
The tendency isolated from
in the more numerous pores and grooves on the rougher surface, or it may have been due to adherence to remnants of follicular cell membranes, although this cannot be stated definitively. Conversely, the association of IBRV with the relatively smooth Day-7 embryos and ova seems to indicate that the mechanism of interaction is at least. not entirely by entrapment, but could be by some specific affinity for the structure that is maintained despite changes that occur in the ZP during the first 7 d of development. In summary, although differences cannot be detected by light micro8scopy in the thickness or appearance of bovine ZPs of follicular oocytes, Day-7 embryos or Day-7 degenerated ova, it is clear from scanning electron microscope examination and total This is protein analysis that the structures are not equivalent. consistent with the results of biochemical and electron microscope examinations in other species (14,17). Further, IBRV was associated with the ZPs from all sources examined, and there were quantitative differences in the limited numbers examined. It is important to place our results in proper perspective. The value of the intact ZP of Day 7 bovine embryos as a barrier to pathogens was established through the cumulative results of numerous studies conducted over a lo-year period (22-24,26). It may ultimately be shown that subtle differences in the structure of thfe ZP of follicular oocytes will not affect its capability to Further studies are serve as an equivalent barrier to pathogens. needed for a proper evaluation. REFERENCES 1. Anonymous. Recommendation for the sanitary handling of embryos. In: Stringfellow DA, Seidel SM (eds), Manual of the International Embryo Transfer Society. IETS, Champaign, IL, 1990;41-45. 2. Boice ML, McCarthy TJ, Maurogianis PA, Fazleabas AT, Verhage HG. Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (w anubis) . Bio Reprod 1990;43:340-346. 3. Dunbar BS. Morphological, biochemical, and immunochemical characterization of the mammalian zona pellucida. In: Hartmann JF Mechanism and Control of Animal Fertilization. Academic Press, (ed), Mew York, 1983;139-175. 4. Gandolfi F, Brevini TAL, Richardson L, Brown CR, Moor RM. Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 1989;106:303-312. 5. Gordon I, Lu KH. Production of embryos in vitro and its impact on livestock production. Theriogenology 1990;33:77-87. 6. Guerin B, Le Guienne B, Chaffaux S, Harlay T, Allietta M, Thibier M. Contamination des ovocytes et des embryons fecondes in vitro apres infection experimentale, de vaches donneuses par le virus herpes bovin de type 1 (BHVl). Ret Med Vet 1989;165:827-833.
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7. Guerin B, Le Guienne B, Thibier M. Absence de contamination microbiologigue des embryons bovins fecondes in vitro. Bull Acad Vet de France 1988:61:513-520. 8. Guerin B, Marguant-Le Guienne B, Allietta M, Harlay T, Thibier M. Effets de la contamination par le BHVl sur la maturation et la fecondation in vitro des ovocytes de bovins. Ret Med Vet 1990;166:911917. 9. Gwatkin RBL, Andersen OF, Williams DT. Large scale isolation of bovine and pig zonae pellucidae: chemical, immunological, and receptor properties. Gam Res 1980;3:217-231. 10. Gwatkin RBL, Williams DT. Immunization of female rabbits with heatsolubilized bovine zonae: production of anti-zona antibody and inhibition of fertility. Gam Res 1978;1:19-26. 11. Gwatkin RBL,' Williams DT, Meyenhofer M. Isolation of bovine zonae pellucidae from ovaries with collagenase: antigenic and sperm receptor properties. Gam Res 1979;2:187-192. 12. Hedrick JL, Wardrip NJ. Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Analy Biochen 1986;157:63-70. 13. Hedrick JL, Wardrip NJ. Proteolysis by collagenase preparations alters the macromolecular composition of the porcine zona pellucida. Biol Reprod 1986;35:677-682. 14. Hedrick JL, Wardrip NJ, Berger T. Differences in the macromolecular composition of the zona pellucida isolated from pig oocytes, eggs and zygotes. J Exp Zoo1 1987;241:257-262. 15. Linares T, King WA. Morphological study of the blastocyst with phase contrast microscopy. Theriogenology 1980;14:121-133. 16. Lowry OH, Roseberg MJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 17. Nogoies C, Ponsa M, Vidal F, Boada M, Egozcue J. Effects of aging on the zona pellucida of mouse oocytes. J IVF Emb Trans 1988;5:225-229. 18. Oikawa T, Sendai Y, Kurata S, Yanagimachi A glycoprotein of R. oviductal origin alters biochemical properties of the zona pellucida of hamster egg. Gam Res 1988;19:113-122. 19. SAS User's Guide: Statistics. SAS Institute, Inc, Cary, NC, 1982;139200. 20. Schmidt NJ. Tissue culture methods and procedures for diagnostic Procedures for virology. In: Lennette EH, Schmidt NJ (eds), Diagnostic Viral and Rickettsial Diseases. American Public Health Association, New York, 1964;120-121. 21. Shabanowitz Characterization of the human zona RB, O*Rand MG. pellucida from fertilized and unfertilized eggs. J Reprod Fertil 1988;82:151-161. 22. Shisong C, Wrathall The importance of the zona pellucida for AE. disease control in livestock by embryo transfer. Br Vet J 1989;145:129-140. 23. Singh EL. Rev sci Disease control: procedures for handling embryos. tech off int Epiz 1985;4:867-872. 24. Singh EL. The disease control potential of embryos. Theriogenology 1987;27:9-20. 25. Stringfellow DA, Lauerman LH, Nasti KB, Galik PK. Trypsin treatment of bovine embryos after in vitro exposure to infectious bovine rhinotracheitis virus qr bovine herpesvirus-4. Theriogenology 1990:34:427-434.
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26. Stringfellow DA, Riddell KP, Zurovac 0. The potential of embryo transfer for infectious disease control in livestock. NZ Vet J 1991;39:8-17. Inhibition of fertilization in cattle 27. Tsunoda Y, Soma T, Sugie T. by passive immunization with anti-zona pellucida serum. Gam Res 1981;4:133-138. Ann Rev Biochem 28. Wassarman PM. Zona pellucida glycoproteins. 1988:57:415-442.