The influence of the in vitro culture on the structure of the embryonic coats (zona
pellucida)-a scpellucida)-anningelectronic- pellucida)-and lightmicroscopic study
Significant lower pregnancy rates after transfer of in vitro produced embryos may, besides an influence of in vitro culture on the embryo proper, also indicate an influence on the oocyte and embryo surrounding coats, the zona pellucida (ZP) or embryonic coats (EC).
The ZP/EC has in addition to its fundamental significance for a successful fertilisation also important functions for early embryonic development. To determine the influence of the in vitro culture on the structure of the ZP/EC, a comparative investigation of immature, in vivo and in vitro matured oocytes and identically produced embryos (zygotes, 2- / 4 -, 8- / 16-cells, morula, blastocysts) was carried out. The reticular, spongy part of the ZP/EC and the thickness of the ZP/EC were determined via lightmicroscopic investigation. By scanning-electronic microscopy the surface structure, the number and the area of the pores of the samples were compared. The surface of the immature and in vivo matured oocytes showed well-developed, spherical granules and a very large number of pores, whereas the majority of the samples of in vitro matured oocytes had spherical and flattened granules and a poreless surface structure. The rest of the in vitro matured oocytes showed the same surface structure as immature and in vivo matured oocytes. In the embryonic stages, the reticular part of the EC was larger for all in vivo stages than for the equivalent in vitro stages, which indicates a condensation of the EC of the in vitro embryos. 100% of the in vivo stages showed a EC surface with spherical granules. 100% of the in vivo zygotes and the 2- / 4- cells had a very large number of pores, whereas a great part of the in vivo morulae (61,5%) and blastocysts (33,3%) showed a poreless structure and the remaining part showed a small number of pores. 100% of the in vitro embryos showed only flattened granules, which may be a result of a retarded development or a degeneration of the surface under in vitro conditions. The zygotes make an exception, the majority of the samples had flattened and spherical granules. 100% of the in vitro embryos had a porous
surface structure with a very large number of pores, whereas the major part (55,8%) of the in vitro morulae and blastocysts had a large number of pores. Only in vitro embryos showed signs of degradation. The thickness of the EC was significantly lower for the in vitro morulae and the blastocysts than for the equivalent in vivo stages. The thickness of the EC increased for the in vivo morulae in comparison to the 8- / 16- cells for about 30%.
This thickening might be caused by maternal factors and takes place without a change of the reticular structure. The area of the pores variated extremely (between 6,37 and 2508,19nm2) on the surface structure of the samples, however there was no correlation between the stage of development or in vivo/vitro production. According to that an important influence of the in vitro maturation and culture on the structure of the ZP/EC takes place. These structural changes may be a sign of a retarded development or a degeneration under in vitro conditions, and may, according to the functions of the ZP/EC mentioned above, have negative effects on a successful fertilisation of the oocyte and the early embryonic development. No change of the structure of the ZP/EC after a successful fertilisation could be observed. The assessment of VANROOSE et al. (2000), that BHV-1 and BVDV cannot be transmitted by the embryo transfer of in vitro produced embryos, cannot necessarily be transferred to the transfer of in vivo embryos, because of the important differences in the ZP/EC structure of in vivo and in vitro produced embryos and should, because of its important practical relevance, be verified with in vivo embryos.
Furthermore it could be determined by this investigation, that in cattle, as described for many other species, a physiological modification of the EC structure during the early embryonic development takes place. These structural developments might be influenced or affected by maternal factors, because they could not be assessed in in vitro produced embryos. Whether these structural dynamics may be due to oviductal or uterine secretions or if also maternal mediators may cause these structural modifications in embryos need to be investigated in further studies.
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