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.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

No abstract

Mammalian oocytes are surrounded by an extracellular glycoprotein matrix called the zona pellucida (ZP). Sperm bind and penetrate the ZP to fertilize the oocyte. ZP structure and composition varies depending on the species. Surprisingly little is known about the ultrastructure of the ZP in companion animals. Our previous studies have shown the canine ZP is porous. The objective of this study was to determine whether the canine ZP prevents nanospheres the size of canine viruses from penetrating the ZP. Oocytes (n = 184) were isolated from ovaries from 21 dogs aged 6 months to 9 years. Oocytes were exposed to fluorescent nanospheres (24, 100, 210 nm), and confocal microscopy was used to determine how far the nanospheres penetrated the ZP. The 210 nm nanospheres penetrated the entire width of the ZP in only 24.6% of the oocytes (n = 57), whereas the 100 nm and the 24 nm nanospheres penetrated the ZP more often, 41.5% (n = 53 oocytes) and 86.3% (n = 74 oocytes), respectively. In some oocytes, most of the ZP was resistant to penetration, however, the nanospheres penetrated the ZP in discrete patches reminiscent of the path of transzonal processes of cumulus cells. Since the ZP inconsistently blocks nanospheres, in vitro fertilization is not a means to consistently produce viable, virus‐free offspring from infected dogs and endangered canids.Grant Funding SourceAKC Canine Health Foundation

No abstract