Penetration of Fluorescent Nanospheres through the Canine Zona Pellucida

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