title: Inhibition of ovine in vitro fertilization by anti-Prt antibody: hypothetical model for Prt/ZP interaction.
abstract: BACKGROUND
  The impact of prion proteins in the rules that dictate biological reproduction is still poorly understood. Likewise, the role of prnt gene, encoding the prion-like protein testis specific (Prt), in ram reproductive physiology remains largely unknown. In this study, we assessed the effect of Prt in ovine fertilization by using an anti-Prt antibody (APPA) in fertilization medium incubated with spermatozoa and oocytes. Moreover, a computational model was constructed to infer how the results obtained could be related to a hypothetical role for Prt in sperm-zona pellucida (ZP) binding.
  
  
  METHODS
  Mature ovine oocytes were transferred to fertilization medium alone (control) or supplemented with APPA, or pre-immune serum (CSerum). Oocytes were inseminated with ovine spermatozoa and after 18 h, presumptive zygotes (n=142) were fixed to evaluate fertilization rates or transferred (n=374) for embryo culture until D6-7. Predicted ovine Prt tertiary structure was compared with data obtained by circular dichroism spectroscopy (CD) and a protein-protein computational docking model was estimated for a hypothetical Prt/ZP interaction.
  
  
  RESULTS
  The fertilizing rate was lower (P=0.006) in APPA group (46.0+/-6.79%) when compared to control (78.5+/-7.47%) and CSerum (64.5+/-6.65%) groups. In addition, the cleavage rate was higher (P<0.0001) in control (44.1+/-4.15%) than in APPA group (19.7+/-4.22%). Prt CD spectroscopy showed a 22% alpha-helical structure in 30% (m/v) aqueous trifluoroethanol (TFE) and 17% alpha in 0.6% (m/v) TFE. The predominant alpha-helical secondary structure detected correlates with the predicted three dimensional structure for ovine Prt, which was subsequently used to test Prt/ZP docking. Computational analyses predicted a favorable Prt-binding activity towards ZP domains.
  
  
  CONCLUSIONS
  Our data indicates that the presence of APPA reduces the number of fertilized oocytes and of cleaved embryos. Moreover, the CD analysis data reinforces the predicted ovine Prt trend towards an alpha-helical structure. Predicted protein-protein docking suggests a possible interaction between Prt and ZP, thus supporting an important role for Prt in ovine fertilization.
authors.affiliation: Unidade de Biotecnologia e Recursos Genéticos, Instituto Nacional de Investigação Agrária e Veterinária Santarém, Quinta da Fonte Boa, Vale de Santarém, 2005-048, Portugal.
authors.forename: Jorge
authors.initials: J
authors.lastname: Pimenta
authors.forename: João
authors.initials: J
authors.lastname: Sardinha
authors.forename: Carla C
authors.initials: CC
authors.lastname: Marques
authors.forename: Ana
authors.initials: A
authors.lastname: Domingos
authors.forename: Maria C
authors.initials: MC
authors.lastname: Baptista
authors.forename: João P
authors.initials: JP
authors.lastname: Barbas
authors.forename: Ivo C
authors.initials: IC
authors.lastname: Martins
authors.forename: Patrícia
authors.initials: P
authors.lastname: Mesquita
authors.forename: Pedro
authors.initials: P
authors.lastname: Pessa
authors.forename: Rui
authors.initials: R
authors.lastname: Soares
authors.forename: Aldino
authors.initials: A
authors.lastname: Viegas
authors.forename: Eurico
authors.initials: E
authors.lastname: Cabrita
authors.forename: E M António
authors.initials: EM
authors.lastname: Horta
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Fontes
authors.forename: A M José
authors.initials: AM
authors.lastname: Prates
authors.forename: M L N  Rosa
authors.initials: ML
authors.lastname: Pereira
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D002942:Circular Dichroism; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro / Q000187:drug effects*; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013997:Time Factors; D014270:Trifluoroethanol / Q000737:chemistry / Q000494:pharmacology; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
chemical_list: D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011328:Prions; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; D014270:Trifluoroethanol