Is prnt a Pseudogene? Identification of Ram Prt in Testis and Ejaculated Spermatozoa Jorge Pimenta 1,2 , Ana Domingos 3 , Pedro Santos 4 , Carla C. Marques 1 , Ca ´ tia Cantante 5 , Ana Santos 5 , Joa ˜ o P. Barbas 1 , Maria C. Baptista 1 , Anto ´ nio E. M. Horta 1 , Aldino Viegas 6 , Patrı´cia Mesquita 1 , Joa ˜o Gonc ¸alves 5 , Carlos A. Fontes 2 , Jose ´ A. M. Prates 2 , Rosa M. L. N. Pereira 1,7 * 1 Unidade de Recursos Gene ´ticos, Reproduc ¸a ˜o e Melhoramento Animal, Instituto Nacional dos Recursos Biolo ´ gicos (INRB) L-INIA Santare ´m, Quinta da Fonte Boa, Vale de Santare ´m, Portugal, 2 CIISA, Faculdade de Medicina Veterina ´ria (FMV), Universidade Te ´cnica de Lisboa, Lisboa, Portugal, 3 IHMT-CMDT – Instituto de Higiene e Medicina Tropical, Centro de Mala ´ria e Doenc ¸as Tropicais, Lisboa, Portugal, 4 Hospital Universita ´rio de Coimbra, Coimbra, Portugal, 5 Unidade de Retrovı ´rus e Infecc ¸o ˜ es Associadas (URIA), ADEIM-Centro de Patoge ´ nese Molecular/Instituto de Medicina Molecular (IMM), Faculdade de Farma ´cia, Universidade de Lisboa, Lisboa, Portugal, 6 REQUIMTE/ CQFB Departamento de Quı ´mica, Faculdade de Cie ˆncias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal, 7 Escola Universita ´ria Vasco da Gama, Coimbra, Portugal Abstract A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP C ), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP Sc ) 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. Citation: Pimenta J, Domingos A, Santos P, Marques CC, Cantante C, et al. (2012) Is prnt a Pseudogene? Identification of Ram Prt in Testis and Ejaculated Spermatozoa. PLoS ONE 7(8): e42957. doi:10.1371/journal.pone.0042957 Editor: Jean-Pierre Rouault, Ecole Normale Superieure de Lyon, France Received January 9, 2012; Accepted July 16, 2012; Published August 24, 2012 Copyright: ß 2012 Pimenta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by the Portuguese Foundation for Science and Technology (FCT), Portuguese Ministry of Science and Technology, under the project PTDC/CVT/98607/2008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rosalnp@gmail.com Introduction 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]. Mammalian prnp, that encodes the cellular prion protein (PrP C ), 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 (or Dpl) is located 16–52 kb downstream from prnp, depending on the species [7–10]. 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’’. Although the normal physiological function of the cellular prion protein, PrP C , remains largely unclear, an aberrant conformation with amyloid-like features of PrP C is thought to be the essential component of the infectious particle, involved in transmissible spongiform encephalopaties (TSE) in mammals [11]. The normal prion protein exists in the a- helix-rich form and is harmless while the pathogenic form exists as rod-like particles with 10–12 nm in diameter. Besides TSE, these rod-like particles known as amyloids, are associated to a large number of diseases such as Alzheimer’s disease, diabetes mellitus type 2, Parkinson’s disease, among many others [12,13]. prnp is a single-copy gene, but other prnp-related proteins, may also contribute to the pathogenesis of TSEs [7] demanding further investigation. Several studies reported that prnd as well as prnt are highly expressed in the testicular tissue [7,9,14], which allied to the PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42957