Inhibition of ovine in vitro fertilization by anti-Prt antibody: hypothetical model for Prt/ZP interaction

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.