Abstract
BackgroundThe 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.MethodsMature 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.ResultsThe 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.ConclusionsOur 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.
Highlights
The impact of prion proteins in the rules that dictate biological reproduction is still poorly understood
A novel possibility for the function of prion proteins has emerged from the identification of the paralogue prnd, that lies downstream of prnp and encodes the Doppel (Dpl) protein [1]. prnd expression focuses on testis tissue at the adult, and takes an important role in maintaining sperm integrity, normal fertility and motion ability [1,4] and eventually in the sperm-oocyte interaction [5], which might be linked to its physiological role in acrosome biogenesis [6]
Fertilization and embryo rates A total of 516 ovine mature oocytes were used to assess the fertilizing ability of ovine spermatozoa incubated in the presence and absence of anti-protein testis specific (Prt) serum and pre-immune serum
Summary
The impact of prion proteins in the rules that dictate biological reproduction is still poorly understood. The role of prnt gene, encoding the prion-like protein testis specific (Prt), in ram reproductive physiology remains largely unknown. The expression of prnp is found in the gonads (testis and ovary) [2,3], indicating a role in germ cell differentiation during mammalian spermatogenesis [3]. A third member of the prion gene family, prnt (that encodes the Prt protein), was later described as being closer to prnd than prnp in the human genomic sequence [7,8]. Expression in the nuclei of spermatogonia and spermatocytes, and subsequently in the elongated spermatides and in the spz acrosome, unrelated to spz capacitation, indicate that ovine Prt may play an important role in ram spermatogenesis, throughout spermatogenic cell proliferation and sperm maturation, as well as in fertilization
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