Structural aspects of the zona pellucida of in vitro-produced bovine embryos: A scanning electron and confocal laser scanning microscopic study

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (I VM) oocytes and in vitro-produced (IVP) embryos were studied in two experim ents to find a tentative explanation for the zona's barrier function agains t viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. Th e diameter (nm) and the number of the outer pores within an area of 5000 mu m(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morula e were evaluated by scanning electron microscopy. In oocytes and morulae, t he 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 mu m(2)) and the mean diameter of the outer por es 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 i n morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards th e embryonic cells was examined by confocal laser scanning microscopy. There fore, the passage through and the location in the ZP of fluorescent microsp heres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-l (BHV-1; 180-200 nm), were evaluated. For all s tages, the smallest beads were detected halfway through the thickness of th e ZP, whereas the beads with a size of 200 nm were found only within the ou ter-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 BH V-1 should not be able to traverse the ZP and reach the embryonic cells. Ho wever, the risk exists that viral particles can be trapped in the outer lay ers of the ZP.