Characterisation of intracellular molecular mechanisms modulated by carnosine in porcine myoblasts under basal and oxidative stress conditions.

Carnosine is a naturally occurring histidine-containing dipeptide present at high concentration in mammalian skeletal muscles. Carnosine was shown to affect muscle contraction, prevent the accumulation of oxidative metabolism by-products and act as an intracellular proton buffer maintaining the muscle acid-base balance. The present study was undertaken to gain additional knowledge about the intracellular mechanisms activated by carnosine in porcine myoblast cells under basal and oxidative stress conditions. Satellite cells were isolated from the skeletal muscles of 3 to 4 day-old piglets to study the effect of 0, 10, 25 and 50 mM carnosine pre-treatments in cells that were exposed (0.3 mM H2O2) or not to an H2O2-induced oxidative stress. Study results demonstrated that carnosine acts differently in myoblasts under oxidative stress and in basal conditions, the only exception being with the reduction of reactive oxygen species and protein carbonyls observed in both experimental conditions with carnosine pre-treatment. In oxidative stress conditions, carnosine pre-treatment increased the mRNA abundance of the nuclear factor, erythroid 2 like 2 (NEF2L2) transcription factor and several of its downstream genes known to reduce H2O2. Carnosine prevented the H2O2-mediated activation of p38 MAPK in oxidative stress conditions, whereas it triggered the activation of mTOR under basal conditions. Current results support the protective effect of carnosine against oxidative damage in porcine myoblast cells, an effect that would be mediated through the p38 MAPK intracellular signaling pathway. The activation of the mTOR signaling pathway under basal condition also suggest a role for carnosine in myoblasts proliferation, growth and survival.

Uterine signaling at the emergence of the embryo from obligate diapause.

Embryonic diapause is the reversible arrest of embryo development prior to implantation under a regime of uterine control that is not well understood. Our objective was to explore uterine modifications associated with the emergence of embryonic diapause in the mink, a species in which embryonic diapause characterizes every gestation. We investigated the uterine transcriptome at reactivation using the suppressive subtractive hybridization technique. A library of 123 differentially expressed genes between uteri with blastocysts in diapause and reactivated blastocysts was generated. Among those genes, 41.5% encode for potential secreted products that are implicated in regulation of cell proliferation (14%), homeostasis (14%), protein folding (11%), electron transport chain (8%), and innate immune response (8%), therefore suggesting that these biological processes are implicated in blastocyst reactivation. Two genes, the high-mobility group nucleosome binding domain 1 (HMGN1), a chromatin remodeling factor, and the secreted protein acidic and cystein-rich (SPARC), which is implicated in extracellular cell-cell interactions, were submitted to more detailed analysis of expression patterns in the mink uterus at blastocyst reactivation. Expression of both HMGN1 and SPARC was increased significantly in the uterus at embryo reactivation compared with diapause, principally in the endometrial epithelium and subepithelial stroma. These results provide new insight into uterine signaling at the emergence of the blastocyst from diapause and highlight the factors HMGN1 and SPARC as potential inductors of uterine environment modifications underlying uterine signaling during emergence of the embryo from embryonic diapause.

Molecular characterization and expression analysis of the porcine paraoxonase 3 (PON3) gene.

The paraoxonase (PON) gene family has 3 members, PON1, PON2 and PON3, which are known to be involved in oxidative stress-associated processes such as dyslipidemia, diabetes and coronary heart disease. Although PON3 is the least studied paraoxonase, recent findings have shown that it can significantly reduce atherosclerotic lesion formation and obesity in PON3 transgenic mice. Here, we describe the isolation and molecular characterization of the cDNA encoding the porcine PON3 gene. We also report the cloning of three porcine PON3 transcript variants resulting from alternate splicing of exons. Our results show that PON3 mRNA and protein are ubiquitously expressed in pig tissues. Moreover, the relative abundance of PON3 mRNA, measured in perirenal and subcutaneous fat tissues, is higher in obese Upton Meishan gilts compared with the leaner Large White and Ham Line gilts. PON3 mRNA levels measured in fat tissues positively correlate with subcutaneous, visceral and total body fat weights. Four single nucleotide polymorphisms (SNP) were identified in the PON3 coding sequence, and among these, an association was found between the c.449G>A polymorphism and the longissimus dorsi depth estimated breeding value (EBV) trait. Knowledge of the structure, distribution and expression profile of the porcine PON3 gene provides insights into its physiological function. Our results provide further support for involvement of PON3 in obesity-related disorders.

Visfatin expression is not associated with adipose tissue abundance in the porcine model.

Visfatin was recently identified as a novel adipokine highly enriched in visceral adipose tissue and suggested to play a role in the pathophysiology of obesity and type 2 diabetes mellitus. However, the biological role of visfatin remains elusive since subsequent studies failed to repeat some of the original findings. We report here the cloning of six porcine visfatin transcript variants, resulting from alternate polyadenylation or alternate splicing of exons. It is further demonstrated that the porcine visfatin gene and protein expression measured in fat tissues correlate negatively with subcutaneous (s.c.), visceral and total body fat tissue weights. Moreover, there was no correlation between visfatin mRNA or protein levels and fasting glucose or insulin. No correlation could be found between circulating visfatin and any of the carcass and metabolic parameters. Our results also demonstrate that the tumor necrosis factor (TNF)alpha increases porcine visfatin gene expression in stromal-vascular (SV) cell cultures, thus suggesting an intermediary role for TNFalpha in visfatin response. In conclusion, our results demonstrate that the porcine visfatin gene cannot be considered as a marker of fat accumulation since the highest visfatin expression levels were associated with the leaner animals.

Modulation of peroxisome proliferator-activated receptor delta and gamma transcripts in swine endometrial tissue during early gestation.

Recent evidence points to a role for peroxisome proliferator-activated receptors (PPARs) delta and gamma in embryo implantation and survival. In this study, we report the porcine PPARdelta complete coding sequence and mRNA abundance of PPARdelta, PPARgamma1 and gamma2, angiopoietin-like protein 4 (ANGPTL4) and adipocyte determination and differentiation-dependent factor 1 (ADD1) genes in the pregnant sow endometrium. Real-time PCR analysis was used to study the effect of parity (Yorkshire-Landrace multiparous (YL) and nulliparous (YLn)), site of endometrial tissue sampling (between and at embryo attachment sites) in crossbred DurocxYorkshire-Landrace (DYL) sows and stages of pregnancy (non-pregnant, day 15 and day 25 after mating) in Meishan-Landrace (ML) on mRNA levels. Parity effects were observed for PPARdelta, ANGPTL4, and ADD1, with higher mRNA levels in YL than YLn sows. In DYL sows, lower mRNA levels were present at attachment sites compared to between attachment sites for PPARdelta, PPARgamma1, and ANGPTL4. Finally, day 15 pregnant ML sows had lower PPARdelta mRNA levels compared to day 15 cycling ML sows. A significant increase of PPARgamma1 mRNA levels was found on day 25 pregnant ML and DYL sows relative to day 15 ML or DYL pregnant sows. PPARdelta and gamma immunostaining was detected in endometrial tissue of day 15 cycling sows, day 15 and 25 pregnant sows and epithelial cells of day 25 embryos. Collectively, our results suggest a role for PPARdelta, PPARgamma1, and ANGPTL4, but not PPARgamma2, during the peri-implantation period in pregnant sows.

Isolation of differentially expressed genes in conceptuses and endometrial tissue of sows in early gestation.

The implantation period is a critical time for embryonic survival in pigs. During this period, numerous growth factors are secreted by the conceptuses and the uterine endometrium in order to establish pregnancy and to provide a proper environment for embryonic development. It is well known that the Chinese Meishan sows have a larger litter size when compared with occidental sows mainly because of a superior embryonic survival rate. As a further step toward understanding the mechanisms involved in embryonic survival, we used a suppression subtractive hybridization technique to identify genes that were differentially expressed in Meishan-Landrace conceptuses and endometrial tissue at Day 15 of gestation when compared with conventional Landrace sows. Of the 1000 subtractive clones isolated from each library, 137 endometrial and 166 conceptus-enriched cDNAs were single-pass sequenced and examined by BLAST analysis for identification. Sixty-two percent of the clones found in the endometrial library and 78% of the clones found in the conceptus library showed homology with known genes. Among these genes, the 20 most relevant to embryonic survival based on the available literature were validated through real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Our results show that suppression subtractive hybridization is a powerful method applicable in identifying putative candidate genes that might be used for selection of high litter-size breeds.

Effects of breed, parity, and folic Acid supplement on the expression of folate metabolism genes in endometrial and embryonic tissues from sows in early pregnancy.

Folic acid and glycine are factors of great importance in early gestation. In sows, folic acid supplement can increase litter size through a decrease in embryonic mortality, while glycine, the most abundant amino acid in the sow oviduct, uterine, and allantoic fluids, is reported to act as an organic osmoregulator. In this study, we report the characterization of cytoplasmic serine hydroxymethyltransferase (cSHMT), T-protein, and vT-protein (variant T-protein) mRNA expression levels in endometrial and embryonic tissues in gestating sows on Day 25 of gestation according to the breed, parity, and folic acid + glycine supplementation. Expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. We also report, for the first time, an alternative splicing event in the porcine T-protein gene. Results showed that a T-protein splice variant, vT-protein, is present in all the tested sow populations. Further characterizations revealed that this T-protein splice variant contains a coding intron that can adopt a secondary structure. Results demonstrated that cSHMT mRNA expression levels were significantly higher in sows receiving the folic acid + glycine supplementation, independently of the breed or parity and in both endometrial and embryonic tissues. Upon receiving the same treatment, the vT-protein and T-protein mRNA expression levels were significantly reduced in the endometrial tissue of Yorkshire-Landrace sows only. These results indicate that modulation of specific gene expression levels in endometrial and embryonic tissues of sows in early gestation could be one of the mechanism involved with the role of folic acid on improving swine reproduction traits.