Imaging the Zona Pellucida of Canine and Feline Oocytes Using Scanning Electron Microscopy

Abstract

Ultrastructure of the zona pellucida (ZP) of canine and feline oocytes has not been fully investigated. The objective of the study was to evaluate the potential use of the low vacuum scanning electron microscope (LVSEM) with oocytes. This required development of a method to prepare canine and feline cumulus-oocyte complexes (COCs) for LVSEM to provide ultrastructural information on the ZP. COCs were collected from ovaries, and cumulus cells were either partially or completely removed to reveal the ZP. COCs and zona-intact oocytes were fixed at 4 degrees C for 1 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and subsequently viewed wet or further processed by critical point drying, and viewed uncoated or sputter coated with gold. Although the spongy surface of the ZP was visible at low vacuum in uncoated oocytes, coated oocytes had more details at high vacuum. The ZP surface of canine and feline oocytes contained numerous various-sized, spherical or elliptical pores that narrowed centripetally splitting into several smaller, deep pores. The round to oblong cumulus cells tightly surrounded the ZP. Each corona radiata cumulus cell tapered into a thin projection that entered the ZP. Our detailed techniques will enable future studies connecting ultrastructural and molecular aspects of oocyte maturation and development in mammals.

The Zona Pellucida Porosity: Three-Dimensional Reconstruction of Four Types of Mouse Oocyte Zona Pellucida Using a Dual Beam Microscope

“…5) showed that whereas in the two types of ZP with lowest porosities (types B and D) holes are uniformly distributed across the thickness of the ZP, in the two types of ZP with the highest porosities (types A and C) a considerable variation was found. This variation in the number of holes across the ZP suggests that pores differ in length and that most of them do not extend across all the ZP, at least in types A and C. These results agree with the observations of Lunn and Wright (2009) in canine oocytes, who reported that the ZP presents a multilayered aspect, where pores narrow and bifurcate across the ZP thickness. It is important to note that in type A ZP, despite the fact that most of the pores seem to be truncated, the absolute number of pores extending across the ZP is still higher than in the other types of ZP.…”

“…The porosity of the ZP has been previously analyzed in bovine oocytes and embryos at different development stages (Vanroose et al, 2000; Santos et al, 2008), in porcine oocytes and embryos (Michelmann et al, 2007), and in canine and feline oocytes (Lunn & Wright, 2009), among others, showing that ZPs from different mammalian species present pores at their surface. The porosity together with the network aspect of the ZP are the characteristics that have been used to establish different types of ZP depending on the species analyzed, the degree of oocyte maturity, or the embryo stage of development.…”

The canine oocyte: uncommon features of in vivo and in vitro maturation

“…8). The surface shows various-sized (up to 5 mm in diameter) spherical or elliptical pores that narrow centripetally, splitting into several smaller, deep pores (Lunn and Wright 2009). The thickness of the zona pellucida was measured to be between 9.5 and 15.0 mm (Barber et al 2001;Viaris de Lesegno et al 2008a;Lunn and Wright 2009), in the same range as in other mammals.…”

“…The surface shows various-sized (up to 5 mm in diameter) spherical or elliptical pores that narrow centripetally, splitting into several smaller, deep pores (Lunn and Wright 2009). The thickness of the zona pellucida was measured to be between 9.5 and 15.0 mm (Barber et al 2001;Viaris de Lesegno et al 2008a;Lunn and Wright 2009), in the same range as in other mammals. The zona pellucida, as well as the cytoplasmic organisation of the oocytes, is modified during IVM: the zona pellucida becomes thinner with enlarged pores (Viaris de Lesegno et al 2008a;De Los Reyes et al 2009) and oocyte diameter (zona pellucida excluded) increases by 10% (Viaris de Lesegno et al 2008a).…”

“…7;De Lesegno et al 2008). The invaginations in the oocyte membrane may even penetrate to the vicinity of the nucleus (Lunn and Wright 2009). Upon gross observation, these cells remain firmly attached to the zona pellucida for 2-3 days after ovulation and, at the ultrastructural level, degeneration of transzonal projections starts 2 days after ovulation and is completed 3 days after ovulation Figs 5, 6a).…”