262 TISSUE-SPECIFIC ANALYSIS OF PRION EXPRESSION IN EARLY BOVINE FETUSES

Abstract

The prion protein (PrP) is best known for its mis-folded, pathogenic isoform, which is widely regarded as the infectious agent in transmissable spongiform encephalopathies. However, the role of normal, cellular PrP, a host-encoded 29-kD glycoprotein tethered to the cell membrane by a phosphatidyl-inositol glycane (GPI) anchor, is poorly understood. PrP binds copper with high affinity, has antioxidant activity and may play a role in cell adhesion and/or signaling. PrP is expressed in mouse embryos on 6.5 days post-coitum in extra-embryonic tissue and at 13.5 days in the central and peripheral nervous system, intestine, and dental lamina. Our previous data revealed PrP gene expression in bovine embryos throughout pre-implantation embryo development. As part of a larger effort to map the ontogeny of cellular PrP expression in cattle, we sought here to analyze in early bovine fetuses (1) total PrP gene expression by real-time quantitative PCR (QPCR), and (2) tissue-specific PrP expression by immunohistochemistry. Fetuses were obtained from donor cattle bred by artificial insemination (AI; Day 0) and subjected to mid-ventral laparotomy on Days 32 (n = 2) and 39 (n = 2). Immediately upon recovery, one fetus from each stage was placed in RNAlater for RNA isolation and the other fixed in 10% formalin for immunohistochemistry. RNA was isolated using an RNeasy^® mini kit (Qiagen, Valencia, CA, USA). cDNA was generated by reverse transcription with random hexamer priming and used the ΔΔcT method for estimation of PrP expression by QPCR. Tissue-specific expression was determined by immunohistochemistry. Formalin-fixed fetuses were embedded in paraffin, sagittally sectioned, dehydrated, and subjected to an unmasking protocol that employed Vectorlab unmasking solution and autoclaving. Tissues were then probed with a primary anti-PrP monoclonal antibody (SAF 32; Cayman Chemical Company, Ann Arbor, MI, USA). Bound primary antibody was detected with a biotinylated horse anti-mouse secondary antibody complexed to horseradish peroxidase using the ABC kit (Vector Laboratories, Burlingame, CA, USA). Probed sections were then counterstained with hematoxolin and eosin. Neighboring sections, processed identically but to which no primary antibody was added, served as controls. PrP gene expression was detected by QPCR at both stages examined and tended to be higher in Day 39 compared to Day 32 fetuses. PrP immunoreactivity was found throughout the central and peripheral nervous systems, ganglia, nerve trunks, and neural cell populations of sensory organs in both Day 32 and Day 39 fetuses. PrP immunolabeling was also observed in the mesonephric kidney, liver, and heart in the Day 39 fetus. At both stages, immunoreactivity was most intense in the nervous system. Thus, PrP is expressed in a tissue-specific pattern in early bovine fetuses. Tissue distribution of fetal PrP expression appears similar to that of adult PrP. Moreover, PrP appears to be expressed in a developmentally regulated fashion in some tissues.