190 INFLUENCE OF IN VITRO CULTURE OF BOVINE EMBRYOS ON THE STRUCTURE OF THE ZONA PELLUCIDA

Abstract

The zona pellucida is an extracellular structure at the direct interface between the maternal and embryonic sides, through which all signals of the embryo–maternal dialogue as well as nutritional factors have to pass. Up to now there has been no investigation as to whether in vitro culture influences the structure of the zona pellucida compared to that of in vivo embryos. Therefore, in vitro (oocyte, zygote, 2-, 4-, 8-, 16-cell, morula, and Day 7 blastocyst, using the protocol published by Nganvongpanit et al. 2006 Reproduction 131, 861–874) and in vivo (zygote, 4-cell, morula, and blastocyst) embryos were prepared for microscopical investigation. In total, 191 in vitro and 99 in vivo embryos of quality 1 and 2 from at least 2 replicates were used. Araldit-embedded embryos were semi-thin-sectioned and stained with hematoxilin. A morphometrical evaluation was performed to determine the percentage of the more reticular outer part compared to the total thickness of the zona. Furthermore, the total thickness of the zona pellucida was compared between in vitro and in vivo embryos. In parallel, embryos were analyzed by scanning electron microscopy. Up to the 16-cell stage, the zona of in vivo and in vitro embryos is similar, but in vivo morulae and blastocysts show significantly thicker zonae than the in vitro stages. The reticular part of the zona is thicker in in vivo embryos than in in vitro embryos (30.2 ± 2.1% vs. 12.4 ± 1.8%, respectively). Investigating the pores of the zona pellucida, in vivo morulae/blastocysts show smaller-sized pores than in those derived in vitro. Most of the in vivo morulae/blastocysts are totally covered by secreted granules, and therefore the pores could not be investigated. Furthermore, 30-50% of the in vitro embryos show partly degenerated outer layers of the zona pellucida. This investigation demonstrates that in vitro and in vivo zonae pellucidae are significantly different, reflecting a negative influence of the IVM and IVC.