Studies On Fat Deposition And MARK4 Regulatory Mechanism In Placental Of Sows With High Backfat Thickness

Reproductive performance is critical to the pig industry.High backfat thickness can lead to decreased reproductive performance and placental dysfunction.Therefore,the objectives of this study was to investigate the effect of high backfat thickness of pregnant sows on adipose deposition of placentas,and pig chorionic trophoblast cells were isolated and cultured to investigate the effect and mechanism of functional protein Mark4 on adipose deposition in pig chorionic trophoblast cells,the test includes three parts:1 Effects of high backfat thickness on fat deposition in placenta of pregnancy sowsThe aim of this study was to investigate the effects of high backfat thickness on fat deposition in placenta of pregnancy sows.In this study,46 sows were divided into two groups according to backfat thickness:24 sows' backtfat thickness is within 12-17 mm(LBF)and 22 sows' backtfat thickness is within 20-27 mm(HBF).Total litter size,live litter size,mummy foetus size,stillbirth size and litter birthweigt were recorded.Lipid metabolism factors of maternal blood and piglets umbilical cord blood were detected.The contents of triglyceride,cholesterol and free fatty acid in placental villous tissues after delivery were measured,and sections of placental villous tissues was made for HE staining and Oil red O staining.The mRNA expression of FASN1,ACCa,ACSL1,SCD,ATGL,HSL in placental villous tissues were detected,and the mRNA and protein levels of transcriptional regulatory elements PPAR?,PPAR?,SREBP-lc,C/EBP and Mark4,AMPK were detected.The results showed that total litter size,live litter size and litter birthweigt of the HBF group were significantly lower than the LBF group(P<0.05).The Oil red O staining results showed that the content of lipid droplets in the placenta of the HBF group was significantly higher than that in the LBF group,and the contents of triglyceride,cholesterol and free fatty acid in the placenta of the HBF group were significantly higher than in the LBF group(P<0.05).The mRNA expression level of FASNI,ACCa and ACSL1 genes in the placenta of the HBF group was significantly higher than in the LBF group(P<0.05),while the mRNA expression level of ATGL gene was significantly lower than that in the LBF group(P<0.05).The mRNA expression level of C/EBPa in placenta of the HBF group was significantly higher than that in the LBF group(P<0.05),while the mRNA expression level of PPARy was significantly lower than the LBF group(P<0.05).The protein level of SREBP-1 in the HBF group was significantly higher than that in the LBF group(P<0.01),while the protein level of PPARy was significantly lower than that in the LBF group(P<0.01).In addition,the mRNA and protein expression level of Mark4 in the placenta of the HBF group were significantly higher than those in the LBF group(P<0.05),while the mRNA and protein expression level of AMPK was significantly lower than that in the LBF group(P<0.05).Conclusions:High backfat thickness leads to a decrease in total litter size,litter size and primary litter weight,accompanied by increased of blood lipids,fat deposition level and expression of fatty acid synthesis factors in placenta of pregnancy sows.2 Primary culture of villous cytotrophoblasts isolated from pig full-term placentaThis research was conducted to establish a simple and effective method for isolation and purification of pig chorionic trophoblast cells.Single cell suspension was prepared from pig full-term placentas by the method of trypsin-DNase I digestion and two density(35%and 45%,v/v)Percoll gradient centrifugation.Morphology observation,growth curve measurement,immunofluorescence and transmission electron microscopy(TEM)were applied to cells identification.The results showed that the cells were polygon or quasi-circular in shape,which exhibited epithelioid and paving-like spreading growth.After 48 hour culture,some cells started developing into syncytiotrophoblasts.Immunofluorescence assay showed that the proportion of Cytokeratin 7 positive cells was more than 91%,indicated the high purity of pig chorionic trophoblast cells.The results of TEM revealed that the cells had typical structural features of trophoblasts.These results indicated that our research could be used to obtain high-purity pig chorionic trophoblast cells simply and effectively by the method of trypsin-DNase I digestion and two density(35%and 45%,v/v)Percoll gradient centrifugation.3 Effects and mechanism of mark4 on fat deposition in pig chorionic trophoblast cells under lipid toxicityThe aim of this research was to investigate the effect and mechanism of Mark4 on fat deposition in pig placental trophoblast cells under lipid toxicity.First,a mixture fatty acid of 0?M,50?M,100?M,200?M,400?M,and 500?M was added to the cell culture medium,and the optimum concentration of fatty acid inducing porcine trophoblast cell lipid toxicity(fat deposition)was detected by oil red O staining.At the same time,Mark4 gene interference vector was screened by Real-time PCR,and the overexpression efficiency and interference efficiency were detected.The content of triglyceride,transcriptional levels of?-catenin and adipogenic genes in trophoblast trophoblast cells were detected while overexpression or inhibition of Mark4 gene.Under the condition of overexpression or inhibition of Mark4 gene,JW74(Wnt inhibitor)or BML284(Wnt activator)were added,the content of triglyceride,transcriptional levels of ?-catenin and adipogenic genes ACCa,FASN1,ACSL1,PPAR? in trophoblast trophoblast cells were detected.The results showed that 400 ?M was the best concentration to induce lipid toxicity in pig placental trophoblast cells.The overexpression of Mark4 gene in pig placental trophoblast cells promoted the content of triglyceride(P<0.05)and transcriptional levels of ?-catenin and adipogenic genes ACCa,FASN1,ACSL1(P<0.01),and inhibition of Mark4 gene reduced the content of triglyceride(P<0.05)and transcriptional levels of P-catenin and adipogenic genes ACCa,FASN1,ACSL1(P<0.05).The addition of JW74 in the case of overexpression of Mark4 gene resulted the content of triglyceride and the expression of ?-catenin and adipogenic genes ACCa,FASN1,ACSL1 was decreased(P<0.05);while BML-284 was added in the case of inhibition of Mark4,resulting the content of triglyceride and the expression of?-catenin and adipogenic genes ACCa,FASN1,ACSL1 were increased(P<0.01).Thus,Mark4 can activate the Wnt/?-catenin signaling pathway to mediate lipid deposition in pig placental trophoblast cells under lipid toxicity.