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Insights into the role of “prion-like” genes and proteins on scrapie susceptibility and ram fertility
Publication . Pimenta, Jorge Manuel Botelho Garcia Andrade; Pereira, Rosa Maria Lino Neto; Prates, José António Mestre
The established association between polymorphisms of prnp prion gene and susceptibility to scrapie disease in sheep prompted the development of breeding programmes aimed at increasing the natural resistance to scrapie in the European Union. In order to study the possible undesirable consequences from the widespread selection for the prnp genotype on ovine genetic diversity and reproduction, we primarily focused our investigation in the ovine prnd gene which encodes a prion-like protein designated as Doppel that maps to the same chromosomal region as prnp, but is mainly expressed in testis. When prnd is overexpressed in the nervous tissue, Doppel is neurotoxic and causes neurodegenerative disease. We genotyped 460 animals (207 female and 253 male) from 8 Portuguese sheep breeds (Bordaleira entre Douro e Minho, Churra Badana, C. Galega Mirandesa, C. Mondegueira, Merino da Beira-Baixa, M. Branco, Saloia e Serra da Estrela), for prnp and prnd and established a parallel between a polymorphism in the prnd gene (78G>A) and scrapie susceptibility. We also identified an association between prnd polymorphisms and fertility traits. Upholding the emphasis on ovine reproduction, recombinant Doppel (rDpl) was supplemented in different concentrations to ram spermatozoa during the capacitation process. Regardless of dosage, rDpl improved sperm individual motility and vigour, and enhanced in vitro spermatozoa fertilizing ability. In addition, we determined by Nuclear Magnetic Resonance (NMR) spectroscopy the three-dimensional structure of the N-terminal (1-30) ovine Doppel (OvDpl) peptide, encompassing the 78G>A polymorphism and the entire signal peptide sequence. The new solved three-dimensional structure was subsequently used to construct molecular models with the M-domain of the signal recognition particle subunit (SRP54M). Simultaneously, we were able to obtain soluble mature Dpl protein through the co-expression with the SUMO chaperone. Finally, we developed a new polyclonal antibody (APPA) demonstrating for the first time that the recently discovered ovine prnt gene is a translated protein-coding gene and not a pseudogene, and identified through immunofluorescence and immunohistochemistry the location pattern of the encoded protein Prt in ram testis along spermatogenesis and in ejaculated spermatozoa. These results guided us to our last work, where data obtained through Prt blockage pointed to a physiological role for Prt in ovine fertilization (possibly through an interaction with zona pellucida proteins), reinforcing the importance of prion-like genes and proteins in the ovine reproductive physiology.
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Fundação para a Ciência e a Tecnologia
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3599-PPCDT
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PTDC/CVT/098607/2008