In our previous study  we determined the location of ovine prnt along the developing stages of ram germinal cells and specifically at the sperm head apical ridge subdomain of ejaculated and capacitated ovine spermatozoa. Therefore, the aim of our present work was to determine if Prt, like Doppel [5, 51], could also play a role in the fertilization process. In order to elucidate the molecular foundations of fertilization, we employed an antibody based approach by which we blocked Prt function via an anti-Prt antibody, following previous similar approaches by other authors . Herein we showed that adding APPA, an antibody that binds and subsequently blocks Prt function, to a culture medium where the spermatozoa are actively attaching, penetrating and fertilizing the oocytes, effectively and significantly (P = 0.006) decreases the fertilization rate, with the overall fertilizing ability of ram spermatozoa being lower than in control (fertilization rates: 46 ± 6.8 vs. 78.5 ± 7.5%, P = 0.001, Table 1). This seems to indicate a key role for Prt during fertilization. The cleavage rate of APPA group, which is as an indicator of the progress of fertilization was also lower than in control (19.7 ± 4.2 vs. 44.1 ± 4.2%, P < 0.0001; Table 2), although not significantly different from C Serum group. Interestingly, no differences were identified for D6-7 embryo rates among groups, implying that no repercussions of APPA blockage on the development from 2–4 cells embryos to blastocyst were identified. Taken together, these observations suggest that Prt could exert its main function in the fertilization process, potentially in the initial steps due to its location in the acrosome of ovine spermatozoa.
Ram Prt is readily detected in germ cells (but not in testis somatic cells) throughout spermatogenesis and spermiogenesis (the final stage of spermatogenesis, that consists of the complex differentiation of spermatids into spermatozoa), on fresh sperm and along the capacitation process .
In the case of the other prion family proteins, Dpl was detected both in germ and somatic Leydig, Sertoli and epididymal epithelial cells [6, 9, 53–56], and PrPC was detected in germ and somatic epididymal epithelial cells [3, 56–60]. Finally, the protein Shadoo (Sho), a paralogue of prion protein , also reveals testis expression but only in specific somatic cell types of the testicle (namely interstitial Leydig cells) .
From the outlined data, it is possible to observe that the prion family genes (prnp, prnd, sprn and prnt) exhibit different expression patterns in mammalian testis, so it seems conceivable that they might also present gentle deviations in terms of their function in reproduction, as has been previously suggested by Makrinou et al. . Thus, it appears reasonable to theorize that Prt could be involved in the early steps of fertilization, along the sperm-zona binding process. A hypothesis-driven tridimensional computational prediction was therefore developed to help understand if the study of Prt/ZP binding could represent an interesting path that might be followed in future research. For that purpose, the predicted 3D structure (I-TASSER; [24, 25]) of ovine Prt was refined with the Amber software  and compared with data obtained from CD spectroscopy (Figure 1) which confirmed the α-helical structure. Prediction of bovine ZPs (ZP2, ZP3 and ZP4) three-dimensional structures was also undertaken with the I-TASSER software.
In the present report we predicted a preferential binding ability between Prt and ZP domains (Figures 3 and 4). Computational modelling predicts a preferential docking for Prt within the ZP2 domain (residues 366–632), predominantly in its C-terminal region, downstream of the interdomain linker IHP (Internal Hydrophobic Patch; residues 485–491) and without any residues located inside the homologous ZP2 N-terminus domain (corresponding to bovine residues 36–150) described by Baibakov et al.  at the humanized zona pellucida. With respect to Prt/ZP3 binding, all predicted polar contacts are also located in the ZP domain (residues 44–306), with >85% of polar contacts inside the ZP-N subdomain, upstream of the predicted bovine IHP (residues 166–172). Also, predicted polar contacts for Prt/ZP4 binding seem to locate predominantly inside the N-terminal region of the ZP domain.
Preliminary findings and predicted analysis emphasize the role of ‘ZP domain’ modules, along with Prt, at the early steps of mammalian fertilization. ZP domains are structural elements, found in hundreds of extracellular proteins having diverse functions [64, 65], and from a wide variety of organisms, from nematodes to mammals, consisting of ∼ 260 aminoacids not amino acids positioned close to the C-terminus of the polypeptide . This structural element characterized by a set of eight highly conserved cysteines, is predicted to exist as a bipartite structure corresponding to ZP-N and ZP-C regions tethered by a linker . Recent in vivo analyses indicate that the functional organization of ZP-N is conserved throughout species, suggesting also that alteration of ZP-N structure affects polymerization of ZP-domain proteins . Also, the conserved duplicated motif that include the IHP linker located between ZP-N and ZP-C domains, and the external hydrophobic patch (EHP; an integral part of the ZP-C domain according to Lin et al., assume an important role in the assembly of mouse ZP proteins . Moreover, preliminary findings suggest that ZP domain (aa residues 273–551) of human ZP1 is sufficient to induce an acrosome reaction .
Data presented in this manuscript further highlight the importance of Zp domains and prion-like proteins in reproduction, requesting further studies so as to unearth the motifs and molecular mechanisms involved in the fertilization process, hoping that with such background information, results may then be interpreted in a more reliable manner.