Developmental expression of the cellular prion protein (PrP(C) ) in bovine embryos

Abstract

The mammalian cellular prion protein (PrP(C) ) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc) ), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrP(Sc) conversion and its role in TSEs, the normal function of PrP(C) has not been elucidated. In adult mammals, PrP(C) is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrP(C) is expressed during neurogenesis throughout development, and it has recently been proposed that PrP(C) participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell-specific expression of PrP(C) during mammalian development, we examined PrP(C) expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre-attachment embryos. PrP(C) was detected in the developing central and peripheral nervous systems in Day-27, 32-, and -39 embryos. PrP(C) was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrP(C) cell-specific pattern of expression was detected in non-nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrP(C) in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis.

Figure 1

Relative Prnp mRNA levels in bovine cumulus cells, gametes, and embryos during early development. Prnp mRNA levels were detected using quantitative PCR, normalized to 18s rRNA, and compared to oocyte expression. Cumulus cells (CC) showed significantly higher Prnp mRNA levels compared to oocytes. During development, the highest (P < 0.05) Prnp mRNA levels were detected in Day-4 and Day-18 embryos. (*) Indicates significant difference (P < 0.05). Abbreviations: Sp, spermatozoa; Oc, oocytes.

Figure 2

Expression of PrP^C in bovine oocytes and pre-attachment embryos. PrP^C was immunodetected in whole-mounted oocytes and in Day-4 and -8 embryos. Saggital histological sections (oriented as dashed line) were obtained from Day-14 and Day-18 embryos (M–Q). PrP^C was detected using SAF-32 antibody followed by Alexa Fluor 594 secondary antibody, and counterstained with DAPI. Specific PrP^C immunofluorescence was observed in the oocyte cytoplasm (B red and C merged), blastomeres in Day-4 embryos (F red and G merged), trophoblast (tf) and inner cell mass (icm) in Day-8 embryos (J red and K merged), and trophoblastic cells in Day-18 embryos (white arrow, R red and S merged). A weak PrP^C immunostaining was observed in Day-14 embryos (N red and O merged). No labeling was observed in the negative controls incubated with non-immune serum instead of SAF-32 antibody (D,H,L,P,T). Phase contrast (PC) (A, E, I, M and Q). Abreviation: ed, embryonic disc. Scale bars: 25 μm (B,C,D,F,G,H); 50 μm (J,K,L).

Figure 3

Expression of PrP^C protein in Day-27 bovine embryos. PrP^C was immunodetected using SAF-32 antibody followed by peroxidase-conjugated goat anti-mouse IgG, and counterstained with hematoxylin. Low magnification show PrP^C specific immunostaining associated to the developing brain (arrow), spinal cord (arrowhead), and mesonephros (*). Stereoscope image (A). Negative control incubated with non-immune horse serum instead of SAF-32 and counterstained with hematoxilyn (B). Higher magnification showed PrP^C-associated peroxidase in the marginal regions (mr) of the brain (D), spinal cord (E) (arrow), and mesonephros (F) (arrowhead). Cell-specific staining was detected in the liver (G–I). Abbreviations: drg, dorsal root ganglion; he, heart; liv, liver; me, mesonephros, mes, mesencephalon; met, metencephalon; mye, myelencephalon; sc, spinal cord; sto, stomach; tel, telencephalon.

Figure 4

Expression of PrP^C protein in Day-32 bovine embryos. PrP^C-specific immunostaining was detected in the dorsal root ganglion, spinal cord (C, arrow), and mesonephros (arrowhead). Stereoscope image (A). Negative control incubated with with non-immune horse serum instead of SAF-32 and counterstained with hematoxilyn (B). PrP^C specific immunostaining was detected in the marginal region (mr) of the brain (D), spinal cord (E), and dorsal root ganglion (F). PrP^C cellular-specific immunostaining was observed in epithelial cells and glomeruli of the mesonephros (G) and scattered multi-nucleated cells in the liver (H–I). Abbreviations: drg, dorsal root ganglion; he, heart; liv, liver; me, mesonephros, mes, mesencephalon; mye, myelencephalon; tel, telencephalon.

Figure 5

Expression of PrP^C protein in Day-39 bovine embryos. At low magnification, PrP^C-specific immunostaining was detected in the CNS (arrow) and mesonephros (arrow-head) (C). Stereoscope image (A). Negative control incubated with with non-immune horse serum instead of SAF-32 and counterstained with hematoxilyn (B). At higher magnification, PrP^C labeling was observed in the marginal region (mr) of the brain (D) and the spinal cord (E), but not in the periventricular region (pv). Staining was also detected in the mantle layer (F), dorsal root ganglions (G), sympathetic trunks (H–J), and peripheral nerves associated to the intestine (K) and lungs (L). In peripheral organs, PrP^C expression was evident in the mesonephros (M) and in multi-nucleated cells in the liver (N–O). Abbreviations: cc, cord canal; drg, dorsal root ganglion; he, heart; li, liver; lu, lung; me, mesonephros; met, metencephalon; telencephalon; sc, spinal cord; st, sympathetic trunks.

Figure 6

Western blot analysis of PrP^C expression in bovine embryos. PrP^C was detected by Western blot using SAF-32 anti-PrP antibody in Day-27, -32 and -39 embryos. PrP^C displayed two predominant migratory bands at 31 and 29 kDa, representative of different isoforms of the protein (A). Obex tissue was used as a positive control. Relative levels of 29-kDa PrP^C isoform were higher (P<0.05) in Day-39 compared to Day-27 embryos (B). Levels of 31- and 35-kDa PrP^C isoforms and total PrP^C were not different between days of development. (*) Indicates significant difference (P < 0.05).