Abstract
A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrPC), expressed by the prion gene (prnp), into an abnormally folded isoform (PrPSc) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific - Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the “prion gene complex”. Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.
Highlights
Prion gene family comprises four identified genes: the prion protein gene, prnp, the prion-like protein gene, prnd, the shadow of prion protein gene, sprn and the prion protein testis-specific gene prnt [1]. prnp, and its homologues, sprn, prnd and prnt, show similar gene organizations, which encompass two or three exons [2]
That encodes the cellular prion protein (PrPC), is a housekeeping gene, present in both eutherians and fish [3] that has been characterized in several species, like hamster [4], human, sheep, mouse [5] and bovine [6]. prnd, which encodes a prion-like protein designated as Doppel is located 16–52 kb downstream from prnp, depending on the species [7,8,9,10]. prnd contributes together with prnp, the recently discovered prnt, that encodes the prion protein testis specific – Prt [8], and the sprn shadow of prion protein gene, that encodes Shadoo [3], to the so called ‘‘prion gene complex’’
Several studies reported that prnd as well as prnt are highly expressed in the testicular tissue [7,9,14], which allied to the sterility of prnd 0/0 mice [15,16] and the enhanced fertilizing ability of ovine spermatozoa after Doppel supplementation [17], suggest an important physiological role on male fertility
Summary
Prion gene family comprises four identified genes: the prion protein gene, prnp, the prion-like protein gene, prnd, the shadow of prion protein gene, sprn and the prion protein testis-specific gene prnt [1]. prnp, and its homologues, sprn, prnd and prnt, show similar gene organizations, which encompass two or three exons [2]. Prion protein testis-specific gene, prnt, was described as being closer to prnd than prnp in the human genomic sequence. While the eutherian prnp and sprn promoters incorporate CGIs, the prnd and prnt promoters do not include CGIs [2,7] suggesting that these genes are expressed in a tissuespecific manner. Analysis of both adult and fetal human tissues confirmed the ubiquitous but variable expression profile of prnp, with the highest levels observed in the Central Nervous System and testis. Prt involvement in TSEs has not yet been confirmed, we tested for ovine and caprine Prt amyloid propensity as Prt is a prnprelated protein
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