Structural Aspects of the Zona Pellucida of In Vitro-Produced Bovine Embryos: A Scanning Electron and Confocal Laser Scanning Microscopic Study1

Abstract

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. 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 microm(2) 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 microm(2)) 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.

In vitro interaction of bovine herpesvirus 1 with uterine tube epithelial cells and oocytes

“…ZP proteins have been implicated in specific sperm binding of species, the prevention of polyspermy, and embryo protection between fertilization and implantation (Dunbar et al, 1991;Lasiene et al, 2009;Gupta et al, 2012;Yonezawa, 2014). However, there is a risk that virus particles could be trapped in the layers of the ZP (Vanroose et al, 2000;Van Soom et al, 2010). Singh et al (1982) demonstrated that bovine embryos (from 16-cell to blastocyst stage) with intact ZP that had been exposed to BoHV-1 for 24 hours did not show embryonic infection; but the virus was recovered from most of these embryos even after extensive washing.…”

“…Dubuc (1994) demonstrated that oocytes recovered from BoHV-1 infected cows could be matured and in vitro fertilized, resulting in transferable embryos; however, the ratio of morphologically normal transferable blastocysts was decreased. According to Vanroose et al (2000), the zone pellucida (ZP) can protect against viral infection. Nonetheless, there is a risk that viral particles may be trapped on the outer layers of the ZP.…”

Texture of the zona pellucida of the mature pig oocyte. The mammalian egg envelope revisited

“…The ZP is perforated by radial canaliculi containing the cytoplasmic processes of the corona cells and contacting the surface of the immature oocyte [23]. It should be stressed that these corona cell expansions are not at any time actively penetrating the ZP; the latter is built around these processes as well as around the short oocyte microvilli [24].…”

“…Effectively, although the ZP is permeable to a large spectrum of components [23,44], differential staining of the ZP layers was obtained by treatment en bloc of the oocytes with antibodies or lectins [19,45,46]. The diffusion of (immuno)cytochemical reactions must be easy in the spongy outer layer; it may be progressively hampered while the ZP is becoming a plugged filter or when reagents precipitate in the periphery [47,48].…”

Risks of transmissible diseases in relation to embryo transfer

“…Vanroose et al [62] concluded that the intact bovine ZP of IVM oocytes and IVF embryos is constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, it appears that few small viruses like BVDV can be caught in the outer layers of the ZP and contaminate the embryo at hatching.…”