Is prnt a pseudogene? identification of ram prt in testis and ejaculated spermatozoa

A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrPC), expressed by the prion gene (prnp), into an abnormally folded isoform (PrPSc) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific - Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.publishersversionpublishe

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

NEW TRENDS IN ON-LINE RHEOMETRY TO STUDY THE RHEOLOGICAL PROPERTIES OF NANOCOMPOSITES

ABSTRACT On-line rheometers are usually employed for quality control purposes, but they can also be used to detect changes in structure, or composition of a given material system, thus assisting materials research and processing optimization. A new on-line rotational rheometer was developed with the intention of studying the morphological changes of complex materials during melt compounding. The scope of this work is to address on-line rheometry as an emergent characterization technique to study the evolution of exfoliation/intercalation of layered silicates during compounding and to validate its scale-up from the lab to the industrial scene

Sequence alignment of the amino acid sequences of the ovine, caprine, human and monkey Prt (prion protein testis-specific).

<p>Top lane: complete ovine Prt sequence GenBank: ABO86196.1. Second lane: complete caprine Prt sequence, GenBank: CAL85353.1. Third lane: truncated (42–94 a.a.) human Prt sequence, UniProtKB/Swiss-Prot: Q86SH4.1. Forth lane: truncated (42–94 a.a.) Rhesus monkey (<i>Macaca mulatta</i>) Prt sequence, NCBI Reference Sequence: XP_001112273.2. Blue letters with arrows on top: different amino acids between sheep and goat Prt; Asterisk (*) denotes identical residue; colon (:), conservative change; period (.), related substitution. Dark red indicates residues aligned in a similar fashion among all the individual MSAs; Dark yellow, orange and red residues can be considered to be reliably aligned. Alignment using the M-Coffee program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042957#pone.0042957-Moretti1" target="_blank">[28]</a>.</p

Immunofluorescence images of prion protein testis specific (Prt) location in spermatozoa, using mouse anti-ovine Prt polyclonal antibody (APPA).

<p>Ovine Prt (Churra breed) is present on the ram spermatozoon head apical ridge subdomain, corresponding to the membranes over the acrosome, after ejaculation (A and B) and <i>in vitro</i> capacitation (C). Same results were obtained for the Merino breed animals (data not shown). No immunofluorescence staining was detected with APPA on goat (Serrana breed) spermatozoa (F). Negative controls (D, E, G and H) were carried out with samples processed exactly the same way but using either pre-immune serum from the same BALB/c mouse (E and H), or TBS (D and G) as the primary antibody. Anti-mouse FITC antibody (green), nucleus (DAPI/blue), Scale bar: 10 µm.</p

Ovine prion protein testis specific (Prt) distribution in ram testis using immunohistochemistry, and produced anti-ovine Prt polyclonal antibody (APPA).

<p>APPA labeling of ram germinal cells in seminiferous tubules (E). Positive cells were detected (arrows) in spermatogonia (C), spermatocytes (D), spermatides (A), and spermatozoa (B). Negative control using mouse control serum (pre-immune), as the primary antibody (F). Same results were obtained when incubating sections with the secondary antibody alone (data not shown). Vimentin (G) and Ki-67 antigen (H), immunodetection of Sertoli cells cytoskeletal and spermatides, respectively. Spermatogonia (Sg), spermatocytes (Sc), spermatides (Sp), spermatozoa (Spz), Sertoli cell (S). Magnifications (A, B, C and D) ×1000, (E and F) ×100.</p

Western blot analysis.

<p>Western blot analysis of crude preparations of adult ovine spermatozoa (sperm extracts) with the anti-ovine prion protein testis specific (Prt) polyclonal antiserum (A). No hybridization was detected when the primary antibody was preabsorbed with the immunizing peptide (B). Molecular masses are indicated.</p

Amyloid propensity prediction for ovine and caprine prion protein testis specific (Prt).

<p>Top lane: ovine Prt sequence ABO86196.1. Second lane: caprine Prt sequence CAL85353.1. Consensus predictions for amyloid propensity [ovine (8–13 and 24–34 residues) and caprine (6–13 and 24–34 residues)] with different methods (as described in <a href="http://biophysics.biol.uoa.gr/AMYLPRED" target="_blank">http://biophysics.biol.uoa.gr/AMYLPRED</a>) are labeled with a number sign symbol (#). Note the key role of phenylalanine residue (caprine aa position 6) in increased amyloid formation propensity.</p