Open Access Research

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

Jorge Pimenta12, João Sardinha3, Carla C Marques1, Ana Domingos4, Maria C Baptista1, João P Barbas1, Ivo C Martins5, Patrícia Mesquita1, Pedro Pessa6, Rui Soares3, Aldino Viegas3, Eurico Cabrita3, EM António Horta1, Carlos A Fontes2, AM José Prates2 and Rosa MLN Pereira17*

Author Affiliations

1 Unidade de Biotecnologia e Recursos Genéticos, Instituto Nacional de Investigação Agrária e Veterinária Santarém, Quinta da Fonte Boa, Vale de Santarém, 2005-048, Portugal

2 CIISA, Faculdade de Medicina Veterinária (FMV), Universidade Técnica de Lisboa, Lisbon, Portugal

3 REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal

4 IHMT-CMDT – Instituto de Higiene e Medicina Tropical, Centro de Malária e Doenças Tropicais, Lisbon, Portugal

5 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon, 1649-028, Portugal

6 Hospital Universitário de Coimbra, Coimbra, Portugal

7 Escola Universitária Vasco da Gama, Coimbra, Portugal

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Reproductive Biology and Endocrinology 2013, 11:25  doi:10.1186/1477-7827-11-25

Published: 26 March 2013

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.

Keywords:
Prion proteins; Prt; Zona pellucida; Circular dichroism; Docking; Ovine; Reproduction