PRION EXPRESSION IN MALE AND FEMALE RUMINANT REPRODUCTIVE SYSTEMS

The cellular prion protein (PrPC) is a 29- to 35-kDa, host-encoded, GPIanchored membrane glycoprotein. The function of PrPC is unknown; however, it has been implicated as an antioxidant, anti-apoptotic factor and cell adhesion molecule. The pathogenic isoform of the prion protein (PrPSc) is believed to be the infectious agent in transmissible spongiform encephalopathies (TSE). Tissues expressing PrPC are potential sites for conversion of PrPC to PrPSc during TSE pathogenesis. A PrPC tissue expression map, together with relative expression levels, should provide insight into the elusive function of PrPC. The aim of this study was to examine relative PrPC expression in bovine and ovine reproductive tissues. Angus cattle (n=4) and Merino sheep (n=3) were slaughtered and tissue samples were collected from testis, epididymis, vas deferens, prostate, seminal vesicles, penis, glans, prepuce, ovary, oviduct, uterus and vagina. Samples were frozen at −70°C and analyzed for PrPC expression by Western blot. Total protein (20 micrograms) extracted from each tissue was separated by SDS-PAGE in a 12% gel and blotted onto a PVDF membrane. PrPC was detected using a primary anti-PrP mouse monoclonal antibody (SAF 32 (1:400) or 12F10 (1:200; both from Cayman Chemicals). A polyclonal rabbit anti-GAPDH antibody (Santa Cruz Biotechnology) was used to detect GAPDH. Rabbit anti-mouse IgG or goat anti-rabbit IgG secondary antibodies, each labeled with a different fluorescent tag, allowing simultaneous detection of both targets using an Odyssey Infrared Imaging System (LI-COR). Protein was quantitated in each band as integrated fluorescent pixel intensity (I.I.) with Odessey software. PrPC expression within each sample was normalized to corresponding GAPDH expression (PrPC I.I./GAPDH I.I.). Normalized PrPC levels were then compared to GAPDH-normalized PrPC expression in bovine or ovine brain (obex) tissue (i.e., GAPDH-normalized sample tissue PrPC I.I./normalized obex PrPC. PrPC expression was detected in all tissues analyzed. Testis and epididymis showed the highest relative PrPC expression in both cattle (0.071 and 0.80 realtive to obex) and sheep (0.92 and 0.82 relative to obex). In female tissues, the highest relative PrPC expression was found in the ovine ovary (0.35 relative to obex). PrPC bands of differing molecular weights were observed in different tissues, presumably corresponding to the di-, mono- and un-glycosylated states of PrPC. Bovine testis, epididymis and vas deferens expressed three MW species (∼42.6, 35.4 and 28.4 kDa) whereas only two bands were detected in ovine ovary and oviduct (∼31.3 and 23.7 kDa). The relatively high PrPC expression in the testis and epididymis suggests a functional role for PrPC in spermatogenesis or sperm maturation and marks these tissues as potential sites for pathogenic prion conversion. (platform)