Leptin Upregulates the Expression of ß3-Integrin, MMP9, HB-EGF, and IL-1ß in Primary Porcine Endometrium Epithelial Cells In Vitro.

Obesity has become a global health problem. Research suggests that leptin, a hormone that responds to fat deposition, may be involved in mammalian reproduction; however, its precise role in embryo implantation is poorly understood. Here, primary porcine endometrium epithelium cells (PEECs) were cultured in vitro and used to evaluate the regulatory role of different leptin levels on ß3-integrin, MMP9, HB-EGF, and IL-1ß, which are, respectively, involved in four critical steps of embryo implantation. Results showed that only 0.01 nM leptin significantly improved ß3-integrin mRNA expression (p < 0.05). MMP9 and HB-EGF mRNA expressions were upregulated by 0.10-10.00 nM leptin (p < 0.05). The IL-1ß expression level was only increased by 10.00 nM leptin (p < 0.05). ß3-integrin, MMP9, HB-EGF, and IL-1ß mRNA and protein have a similar fluctuant response to increased leptin. Leptin's influence on ß3-integrin, MMP9, HB-EGF, and IL-1ß disappeared when the JAK2, PI(3)K, or MAPK signaling pathways were blocked, respectively. In conclusion, leptin affected porcine implantation by regulating the expression of ß3-integrin, MMP9, HB-EGF, and IL-1ß in a dose-dependent manner. The signaling pathways of JAK2, PI(3)K, and MAPK may participate in this regulatory process. These findings will contribute to further understanding the mechanisms of reproductive disorders in obesity.

Genome-wide DNA methylation and transcriptome analyses reveal genes involved in immune responses of pig peripheral blood mononuclear cells to poly I:C.

DNA methylation changes play essential roles in regulating the activities of genes involved in immune responses. Understanding of variable DNA methylation linked to immune responses may contribute to identifying biologically promising epigenetic markers for pathogenesis of diseases. Here, we generated genome-wide DNA methylation and transcriptomic profiles of six pairs of polyinosinic-polycytidylic acid-treated pig peripheral blood mononuclear cell (PBMC) samples and corresponding controls using methylated DNA immunoprecipitation sequencing and RNA sequencing. Comparative methylome analyses identified 5,827 differentially methylated regions and 615 genes showing differential expression between the two groups. Integrative analyses revealed inverse associations between DNA methylation around transcriptional start site and gene expression levels. Furthermore, 70 differentially methylated and expressed genes were identified such as TNFRSF9, IDO1 and EBI3. Functional annotation revealed the enriched categories including positive regulation of immune system process and regulation of leukocyte activation. These findings demonstrated DNA methylation changes occurring in immune responses of PBMCs to poly I:C stimulation and a subset of genes potentially regulated by DNA methylation in the immune responses. The PBMC DNA methylome provides an epigenetic overview of this physiological system in response to viral infection, and we expect it to constitute a valuable resource for future epigenetic epidemiology studies in pigs.

Expression of obesity gene and obesity gene long form receptor in endometrium of Yorkshire sows during embryo implantation.

There is accumulating evidence that leptin may be directly involved in mammalian reproduction, however, the potential role of obesity gene/obesity gene long form receptor (ob/ob-Rb) system in porcine implantation is poorly understood. To further confirm this role, mRNA and protein expression of ob/ob-Rb in implantation site and inter-implantation sites of porcine uterus on pregnancy day 13, 18 and 24 were compared in this study. Ob mRNA level went up with the advance of pregnancy and was higher in implantation site than inter-implantation site (P < 0.05). But ob-Rb mRNA, which was negative-regulated by leptin, went down with the advance of pregnancy and lessened in implantation site compared with inter-implantation site (P < 0.05). During the three implantation phase, leptin protein peaked at day 18 pregnancy (P < 0.05) and leptin protein at implantation site were always higher than inter-implantation site (P < 0.05). The higher ob-Rb protein in implantation site compared with inter-implantation site (P < 0.05) only appeared at day 18 pregnancy. Localization of ob/ob-Rb protein in porcine uterus was assayed using immunohistochemistry and found that ob/ob-Rb protein mainly located in luminal epithelium and glandular epithelium in pregnant pigs, but distinct immune-staining of leptin also detected in stroma in non-pregnancy porcine uterus except for luminal epithelium and glandular epithelium. In conclusion, the peak of leptin and the peak of ob-Rb protein in implantation site specifically appeared on day 18 pregnancy of pig. Another funning discovery is ob-Rb mRNA in porcine endometrium was mainly negative-regulated by leptin. The space-time difference of gene and protein expression for ob/ob-Rb confirmed ob/ob-Rb system role as delicate regulator of porcine implantation process.

Effect of polymorphism in the peroxisome proliferator-activated receptor gamma gene on litter size of pigs.

The association of polymorphisms in peroxisome proliferator-activated receptor γ (PPARγ) gene with litter size was studied in Large White and Landrace pig. Three SNP loci (P1, P2 and P7) on PPARγ(2) gene were determined by PCR-SSCP and the results showed that there were A → G mutations at 220 and 324 bp in 5'-regulator region and at 147 bp in exon 6, respectively. Allele frequencies were analysed in two breeds. Information on 2341 litter records from 564 sows was used to analyse the trait total number born (TNB) and number born alive (NBA). In Large White, TNB and NBA of genotype BB for P2 locus were the lowest, and the TNB and NBA of third and following parities and all parities were 0.74 and 0.51 piglets per litter less (P < 0.001) than those of the highest genotype AB, respectively, but for P1 and P7 locus the beneficial genotype AA were more 0.4-0.8 piglets per litter (P < 0.05) than the inferior genotype AB. In landrace, TNB and NBA of the first parity of genotype BB for P1 locus were 2.0 piglets per litter higher than AA (P < 0.05), but for all parities the TNB and NBA of genotype BB were 0.66 and 0.97 piglets per litter (P < 0.05) higher than AA, respectively. At P2 locus, the TNB and NBA of the second parity of genotype AA were obviously higher than those of AB (P < 0.05). And at P7 locus, the TNB and NBA of each parity of genotype AA were both about 2 piglets per litter more than those of BB (P < 0.05). The results indicated that PPARγ gene was significantly associated with litter size in pigs.

Effect of simvastatin on culturing of kidney cells from pigs in vitro.

Statins is an inhibitor in the cholesterol biosynthesis pathway. Simvastatin (SIM) has been found to have other clinical benefits besides those resulted from its actions of reducing plasma level of low density lipoprotein (LDL) cholesterol. Both mevastatin (MEV) and parvastatin (PAR) can increase release of nitric oxide (NO) which is a potent inhibitor of platelet aggregation and endothelial cell conglutination. In this study, we found different concentrations of SIM had different effects on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) mRNA expression and NO and total cholesterol (TC) in normal cultural pig kidney cells. NO and TC were measured by using colorimetry in 550 nm and 546 nm, respectively. HMGR mRNA expression was tested by RT-PCR. Results showed that HMGR mRNA expression had a significant difference (P < 0.05) between different concentration of SIM treatment (0, 5, 10, or 25 micromol/l). HMGR mRNA expression and TC content decreased gradually with the elevation of SIM concentration. The content of NO increased with the elevation of SIM concentration, but the difference was not notable. SIM affects the expression of HMGR-CoA, TC and NO in normal cells, but the specific mechanism need to be further research.

Expression of alphaV and beta3 integrin subunits during implantation in pig.

Integrin are adhesion molecules involved in uterine-conceptus interactions during the perimplantation period. In this study, the expression of alphaV and beta3 integrin subunits in endometrium during implantation in pigs was investigated. The immunohistochemical location was performed on paraformaldehyde-fixed, paraffin-embedded tissue sections, and the mRNA expression of alphaV was detected in endometrium. In addition, serum levels of estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone were measured on Days 0, 12, 18, and 25 of pregnancy. The results indicate that endometrium expressed integrin alphaV and beta3 in all stages examined. The most intensive staining for integrin alphaV and beta3 was observed in endometrial stroma in porcine pregnancy on Day 18. The mRNA of alphaV integrin strongly expressed on Day 18, and moderately expressed on Days 12 and 25. The correlation between serum hormone level and the mRNA expression of alphaV integrin was not significant. The expression patterns of integrin alphaV and beta3 during implantation provide insights into the important physiological function of alphaVbeta3 integrin in pig, and the strong expression of integrin alphaV and beta3 in mid-implantation may indicate its crucial role in successful implantation and embryo survival.

[Effects of MTNR1A gene on litter size in a large white and a landrace herd].

Two pairs of primers were designed based on the known sequence in GenBank for amplification of MTNR1A gene in a Large White and a Landrace herd. Using PCR-SSCP (single strand conformation polymorphism), we found a single nucleotide polymorphism(SNP) within the product amplified from the first pair of primers. PCR products from randomly selected different genotypes were sequenced after were recovered and purified. Results revealed a synonymous single base mutation(G-->A) at +159bp(sequence numbering based on Genbank accession number U73326) for the BB genotype. When analyzed for association with litter size traits, this MTNR1A SNP was found to have no significant effect on litter size traits.

[Genetic relationships among seven sheep populations using four microsatellite markers].

The genetic polymorphisms of four microsatellite loci BM143, OarHH35, OarAE101, and BMS2508 were analyzed in 286 sheep of seven sheep populations (Small Tail Han sheep, Hu sheep, Ujumqin sheep, Suffolk sheep, Dorset sheep, Charolais sheep, F1 of Dorset male x Small Tail Han female sheep). The numbers of alleles for BM143, OarHH35, OarAE101, and BMS2508 are 9, 11, 14 and 9 in seven sheep populations, respectively. The polymorphism information content/number of effective alleles/ heterozygosity of BM143, OarHH35, OarAE101 and BMS2508 were 0.7073/3.7231/0.7314, 0.8267/6.4399/0.8447, 0.5743/2.5178/0.6028, 0.6172/3.0712/0.6744 in 286 sheep, respectively. The results revealed the greatest genetic variation at OarHH35 locus and the lowest at OarAE101, the greatest genetic variation in Small Tail Han sheep and the lowest in Hu sheep among seven sheep populations. In the unweighted pair group method with arithmetic mean (UPGMA) dendrograms based on Nei's D(A) distance and Nei's D(S) standard genetic distance, the Chinese native breeds (Small Tail Han sheep, Ujumqin sheep, Hu sheep) were grouped together, then with Charolais sheep. The F1 crossbred sheep, and the two British native sheep (Suffolk sheep, Dorset sheep) also clustered together. Microsatellite genotyping in sheep provided a useful tool for examining the genetic relationships among breeds (populations).

[Genetic polymorphisms of five microsatellite loci in Small Tail Han sheep].

Small Tail Han sheep is an excellent local sheep breed in China. Small Tail Han sheep had significant characteristics of high prolificacy. The lambing percentage averaged 260 percent in Small Tail Han sheep. The polymorphisms of 5 microsatellite loci OarAE101, BM1329, BMS2508, TGLA54 and TGLA68 which were closely linked to the fecundity genes FecB and FecXI in sheep were detected in 244 ewes of Small Tail Han sheep. The PCR amplified products of microsatellites were detected by non-denatured (natural) polyacry lamide gel electrophoresis. Allele frequency, polymorphism information content, gene homogeneity and heterozygosity for 5 microsatellite loci were calculated. The number of alleles for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 6, 9, 5, 2 and 6 in Small Tail Han sheep, respectively. The range of allele sizes for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 160 bp to 180 bp, 97 bp to 135 bp, 116 bp to 136 bp, 98 bp to 100 bp, and 93 bp to 115 bp, respectively. The alleles of the greatest frequency for BM1329, OarAE101, TGLA54 and BMS2508 were 164 bp, 97 bp, 134 bp and 99 bp, respectively. Polymorphism information content/gene homogeneity/heterozygosity for BM1329, OarAE101, TGLA54, TGLA68 and BMS2508 were 0.4481/0.4840/0.5160, 0.3516/0.6375/0.3625, 0.2528/0.7326/0.2674, 0.3733/0.5034/0.4966 and 0.5809/0.3581/0.6419 in Small Tail Han sheep, respectively. The results revealed the greatest genetic variation in BMS2508 and the lowest in TGLA54. These results could provide, basic molecular data for the research on the germplasm characteristics of Small Tail Han sheep.

[Cloning and sequencing of four microsatellite loci in Small Tail Han Sheep].

Small Tail Han sheep is an excellent local sheep breed in China. Small Tail Han sheep has significant characteristics of high prolificacy. The lambing percentage averaged 260 percent in Small Tail Han sheep. The genetic detection of 4 microsatellite loci OarAE101, OarHH35, BM143 and BMS2508 which were closely linked to the fecundity gene FecB in Booroola sheep was conducted in 159 sheep in Small Tail Han sheep, Dorset sheep, F1 hybzids(Dorset x Small Tail Han sheep). The results confirmed the genetic characteristics of codominance of microsatellite DNA. The PCR amplified products of microsatellites were detected by non-denatured (natural) polyacrylamide gel electrophoresis. The sequences of PCR amplification fragment of 6 clones of 4 microsatellite loci in Small Tail Han sheep were accepted by GenBank of National Center for Biotechnology Information, National Institutes of Health in USA, the GenBank accession numbers were AF394445, AF394446, AF394447, AF394448, AF394449, AF394450, respectively. The sequence homogeneity between OarAE101 in Small Tail Han sheep in this study and the ovine OarAE101 in GenBank was 98 percent. The sequence homogeneity between OarHH35 in Small Tail Han sheep in this study and the ovine OarHH35 in GenBank was 99 percent. The sequence homogeneity between BM143 in Small Tail Han sheep in this study and the bovine BM143 in GenBank was 95 percent. The sequence homogeneity between BMS2508 in Small Tail Han sheep in this study and the bovine BMS2508 in GenBank was 95 percent. OarAE101, OarHH35, BM143 and BMS2508 in Small Tail Han sheep were all perfect microsatellites. These results could provide molecular basic data for the research on the germplasm characteristics of Small Tail Han sheep.