R.M. Pereira

Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition.

The reproductive performance of dairy cows may be improved by feeding conjugated linoleic acid (CLA) supplements during early lactation. The mechanism of action of t10,c12 CLA is not clearly known. Our objective was to investigate the effect of t10,c12 CLA on oocyte maturation and lipid composition of cumulus oocyte complexes (COC). The developmental potential of oocytes incubated in in vitro maturation (IVM) medium supplemented with t10,c12 CLA to the blastocyst stage and embryo quality were also assessed. In experiment 1, abattoir-derived oocytes were matured in TCM199 + 10% serum supplemented with 100 μM t10,c12 CLA (t10,c12 CLA n = 672) or without it (control n = 672). Mature oocytes were either stained for chromatin configuration or inseminated and cultured for embryo development assessment. In experiment 2, COC and IVM culture media were subjected to fatty acid (FA) analysis prior and after maturation with t10,c12 CLA or without it (control). Total lipids and FA profiles in oocytes, cumulus cells and culture media were determined by gas chromatography. t10,c12 CLA supplementation to IVM medium improved (p = 0.05) embryo quality evaluated morphologically. This effect was associated with t10,c12 CLA presence (3.1 ± 0.7%, p = 0.04) and lower levels of arachidonic acid in FA profile of t10,c12 CLA mature oocytes (immature oocytes = 4.4 ± 1.9%, t10,c12 CLA mature oocytes = 1.0 ± 0.7%, p = 0.05). Differences in myristic and eicotrienoic acids, saturated and unsaturated FA concentrations between oocytes and cumulus cells were detected (p ≤ 0.05). In conclusion, the presence of t10,c12 CLA during maturation interfered on lipid metabolism improving bovine oocyte competence to develop into higher quality embryos.

A Role of Lipid Metabolism during Cumulus-Oocyte Complex Maturation: Impact of Lipid Modulators to Improve Embryo Production

Oocyte intracellular lipids are mainly stored in lipid droplets (LD) providing energy for proper growth and development. Lipids are also important signalling molecules involved in the regulatory mechanisms of maturation and hence in oocyte competence acquisition. Recent studies show that LD are highly dynamic organelles. They change their shape, volume, and location within the ooplasm as well as their interaction with other organelles during the maturation process. The droplets high lipid content has been correlated with impaired oocyte developmental competence and low cryosurvival. Yet the underlying mechanisms are not fully understood. In particular, the lipid-rich pig oocyte might be an excellent model to understand the role of lipids and fatty acid metabolism during the mammalian oocyte maturation and their implications on subsequent monospermic fertilization and preimplantation embryo development. The possibility of using chemical molecules to modulate the lipid content of oocytes and embryos to improve cryopreservation as well as its biological effects during development is here described. Furthermore, these principles of lipid content modulation may be applied not only to germ cells and embryo cryopreservation in livestock production but also to biomedical fundamental research.

Doppel gene polymorphisms in Portuguese sheep breeds: Insights on ram fertility

Transgenic knockout of the gene encoding the prion-like protein Doppel leads to male infertility in mice. The precise role of Doppel in male fertility is still unclear, but sperm from Doppel-deficient mice appear to be unable to undergo the normal acrosome reaction necessary to penetrate the zona pellucida of the oocyte. The objective of this study was to characterize Doppel (Prnd) gene polymorphisms in eight Portuguese sheep breeds and to determine a possible relationship between these polymorphisms and ram fertility. Ovine genomic DNA of 364 animals of different breeds (Bordaleira entre Douro e Minho, Churra Badana, Churra Galega Mirandesa, Churra Mondegueira, Merino da Beira Baixa, Merino Branco, Saloia and Serra da Estrela) were analysed by multiple restriction fragment-single-strand conformation polymorphism (MRF-SSCP). This analysis revealed a synonymous substitution G-->A in codon 26 of Prnd gene. Churra Galega Mirandesa and Saloia breeds were more polymorphic (P=0.005 and P=0.04, respectively) than the overall population, while Serra da Estrela and Merino Branco animals were less polymorphic (P=0.007 and P=0.04). No polymorphism was found in Churra Mondegueira breed. Semen from 11 rams of Churra Galega Mirandesa breed (7 homozygous wildtype GG and 4 heterozygous GA) routinely used in the Portuguese Animal Germoplasm Bank was collected and frozen for fertility tests. A classification function was estimated, using data from post-swim-up semen motility and concentration and Day 6 embryo production rate, allowing the identification of the Doppel homozygous GG genotype with 86.7% of accuracy. This preliminary study detected the presence of only one polymorphism in codon 26 of Prnd gene in the Portuguese sheep breeds. In the polymorphic Churra Galega Mirandesa breed, GG genotype could be characterized through a model using three fertility traits, suggesting a relationship with male reproduction. Any future research should investigate not only AA genotype and its influence on ram fertility but also the possible consequences of the European Community selection program to eradicate Scrapie on the Prnd genotypes and indirectly on sheep breeds viability and preservation.

In vitro and in vivo fertility of ram semen cryopreserved in different extenders.

Seminal traits of frozen-thawed (FT) ram semen and in vitro and field fertility in native Portuguese breeds were evaluated in 4 experiments. In exp. 1 and 2 the cryopreservation capacity of 2 extenders, E1 (15% egg yolk-EY) and E2 (4.5% EY and trehalose) was compared through morphological evaluation and in vitro fertilizability of FT ram semen. Exp. 3 aimed to determine the usefulness of in vitro homologous/heterologous fertilization tests as tools for predicting ram fertility. Exp. 4 was conducted to verify if the identified differences between the 2 extenders could be confirmed by field fertility. E1 showed a better cryoprotective action expressed by higher in vitro and field fertility results. In conclusion, EY is difficult to be replaced in ram semen extenders. Heterologous fertilization seems to be a useful tool for predicting fertility of FT ram semen.

Prion‐like Doppel gene polymorphisms and scrapie susceptibility in portuguese sheep breeds

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion-like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.

Embryos and culture cells: a model for studying the effect of progesterone.

A positive association between P4 concentration and initial bovine embryo survival has been reported. The objective of this study was to establish two coculture systems as a model to study the influence of progesterone on the initial bovine embryo development. Granulosa cells (GC) or bovine oviduct epithelial cells (BOECs) were used at the base of embryo culture medium microdroplets (TCM199 and 10% of superovulated oestrus cow serum, (SOCS)) supplemented or not with progesterone (P4, 33.4 ng mL(-1)) and/or a progesterone receptor antagonist (onapristone, OP, 2.2x10(-5)M). Presumptive zygotes were transferred to monolayers after in vitro maturation and fertilization of bovine oocytes with thawed swim-up selected sperm. Embryo development was carried out according to the following groups: experiment 1, BOEC (n=378) and BOEC plus OP (n=325); experiment 2, GC (n=514); GC plus OP (n=509); BOEC (n=490); BOEC plus P4 (n=500); BOEC plus P4 and OP (n=502). Embryos were checked for cleavage at day 2 and for stage development between days 8 and 12 of culture. In experiment 1, no differences (P>0.05) were identified between BOEC and BOECOP groups for embryo rates of development, quality or developmental stages. Also in experiment 2, no differences were found in embryo rates of development, quality or developmental stages between embryos cultured under the two coculture systems when no supplementation was added. Embryo development rates were not affected by OP presence in GCOP group. However, P4 negatively affected Day 8 (D8) embryo development rates in BOEC system (BOECP4=16.8+/-2.6% vs. BOEC=23.7+/-1.7%, P=0.02). This negative effect was abolished when P4 antagonist (OP) was added to the culture medium. BOEC supplementation with P4 also induced a delay on embryo development at D8 as confirmed by a lower development score (BOECP4=3.0+/-1.4 vs. GC=3.4+/-0.1, GCOP=3.5+/-0.1, BOEC=3.4+/-0.1 and BOECP4OP=3.5+/-0.1; P<0.05). These results demonstrate that OP supplementation had no harmful effect on embryo development either in granulosa, where P4 is naturally synthesised, or in BOEC coculture systems. Also we can not confirm a direct association between high P4 concentrations and embryo survival during early stages, although P4 may influence early embryo development through different mechanisms mediated by the type of cells present.

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.

The Prion-like Protein Doppel Enhances Ovine Spermatozoa Fertilizing Ability

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

NMR solution structure and SRP54M predicted interaction of the N-terminal sequence (1-30) of the ovine Doppel protein

Prion protein (PrP(C)) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrP(C) proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N-C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.

The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep

The objective of this study was to search for polymorphisms in the ovine prion-related protein (testis-specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene-coding region was analyzed by single-strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine-X-X-serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C-112G-129T-144A,17CT-112GC-129CT-144AG and 17T-112C-129C-144G), and the only three animals found homozygous at c.78A had the 17C-112G-129C-144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP).