| Whereas the content of intramuscular fat(IMF)is crucial to the flavor,tenderness,juiciness,palatability and price of meat,accumulation of subcutaneous fat and visceral fat is a liability to livestock production,decreasing carcass classification,dramatically increasing production costs and reducing economic benefits of animal agriculture.Given that myogenic,adipogenic,and fibrogenic cells are all derived from a common pool of mesenchymal progenitor cells during embryonic development,by manipulating progenitor cell differentiation,promoting myogenic differentiation is beneficial to the growth of lean meat,strengthening intramuscular adipogenic differentiation increases IMF content,and decreasing intramuscular fibroblast differentiation improves overall tenderness of meat.Therefore,better understanding of the adipocyte biology in ruminants,especially the mechanism of intramuscular fat deposition,is of great significance in improving meat quality and maximizing animal production efficiency.The purposes of this study were to develop an effective method for inducing adipogenesis of primary cells in ruminant animals by establishing three dimensional(3D)microenvironment model to mimic adipose tissue development in vivo,and to investigate the regulation roles of vascular factors in adipogenesis of intramuscular adipose stromal vascular cells(ASVCs)by promoting or inhibiting the growth of vascular endothelium to elucidate the relationship between adipogenesis and angiogenesis in ruminant animals.Furthermore,we hypothesized that All-Trans Retinoic Acid(ATRA)plays a crucial role in the early commitment stage of adipogenesis of intramuscular ASVCs by promoting angiogenesis in ruminant animals.It is expected that the adipogenesis of mesenchymal progenitor cells and the hyperplasia of intramuscular fat cells will have enhanced by vitamin A administration of maternal and offspring in order to ultimately provide a feasible means of nutritional regulation for improving marbling.The main results were as follows:1.We developed a new protocol of 3D spheroid culture system to induce the adipogenic differentiation of ASVCs derived from bovine intramuscular fat.The ASVCs were resuspended in endothelial basal medium-2 at a concentration of 4×104 cells/ml.The hanging drops were maintained on the lid for 3 days,when cells formed spherical structures.After that,the spheroids were transferred one by one using a pipette with 200μl tips(cut the top to fit the spheroids).The spheroids were cultured in EBM-2 for 3 additional days to settle onto the cell culture plates(1.5 spheroids/cm~2).Endothelial basal medium-2 was replaced by DMEM supplemented with adipogenic induction medium(MDI):insulin(1μg/ml),dexamethasone(0.1μg/ml)and isobutylmethylxanthine(27.8μg/ml)for the following 3 days,and then just DMEM supplemented with insulin(1μg/ml)for the last 3 days of culture.The results of oil red O staining,fat content test and RT-PCR are all indicated that the adipogenic ablility of ruminant intramuscular ASVCs in 3D spheroid culture system is signifcantly higher than 2D traditional culture system.3D spheroid culture system likely provides physical supportive and interactive environment for adipogenesis of ruminant intramuscular ASVCs.In short,the spheroid culture system developed in this study is an efficient model for in vitro studies of adipogenesis concerning ruminant-derived ASVCs.In addition,this model can also be used for studying the role of angiogenesis in adipose tissue development in general.2.EBM-2 culture before adipogenic differentiation improves the adipogenesis of ruminants intramuscular ASVCs by promoting the angiogenesis.EBM-2 culture before adipogenic differentiation promotes angiogenesis,may provide a desirable environment for adipocyte formation,and further improves the adipogenesis of intramuscular ASVCs.The mRNA expression of C/EBPα、PPARγand FABP4 in EBM-2 group were significantly up-regulated,and the fat content is significantly higher than DMEM group(P<0.05)and EBM-2+SU5416 group(P<0.01),a large number of red lipid droplets were observed.RT-PCR and microscopic observation results further confirmed that the mRNA expression of VEGFA、VEGFR-2、EGF and FGF-2 in EBM-2 group were significantly up-regulated,and promoting the angiogenesis of runimants intramuscular ASVCs,A large number of vascular structures are formed around the spheroids.Immunofluorescence cytochemistry test showed that intramuscular ASVCs of ruminant animals can differentiate into endothelial cell phenotype in vitro and form blood vessels on matrigel.On the contrary,blocking the vascular endothelial growth factor receptor(VEGFR)by SU5416 affects the role of VEGF and inhibits angiogenesis,which result in decreasing adipogenesis of intramuscular ASVCs.The mRNA expression of ZFP423、C/EBPα、C/EBPβ、PPARγand FABP4 in EBM-2+SU5416 group were significantly down-regulated,and the fat content is significantly lower than DMEM group(P<0.01)and EBM-2 group(P<0.01),no obvious red lipid droplets were observed.RT-PCR and microscopic observation results further confirmed that the mRNA expression of VEGFA、VEGFR-1、VEGFR-2、EGF and FGF-2 in EBM-2+SU5416 group were significantly down-regulated,and angiogenesis was inhibited.Suggesting that the adipogenesis of intramuscular ASVCs in ruminant animals is tightly related to angiogenesis,the growth and development of adipose tissue depends on the angiogenesis,and blood vessels may serve as a reservoir of progenitor cells for adipocytes.In addition,we optimized the immunofluorescence staining method of matrigel stereoscopic culture of cells.3.ATRA treatment before adipogenic differentiation enhances the adipogenesis of intramuscular ASVCs in ruminant animals by promoting the expression of angiogenic factors.ATRA treatment before adipogenic differentiation dramatically enhances the adipogenesis of intramuscular ASVCs in ruminant animals.The mRNA expression of C/EBPα、PPARγand FABP4 in ATRA group were significantly up-regulated,and the fat content is significantly higher than Control group(P<0.05)and ATRA+SU5416group(P<0.01),a large number of red lipid droplets were observed.Blocking the VEGFR by SU5416 results in dramatically decreasing adipogenesis of intramuscular ASVCs in ruminant animals.The mRNA expression of ZFP423、C/EBPα、C/EBPβ、PPARγand FABP4 in ATRA+SU5416 group were significantly down-regulated,and the fat content is significantly lower than Control group(P<0.01)and ATRA group(P<0.01),no obvious red lipid droplets were observed.RT-PCR and microscopic observation results further confirmed that the mRNA expression of VEGFA and VEGFR-2 in ATRA group were significantly up-regulated,and promoting the angiogenesis of runimants intramusculat ASVCs,A large number of vascular structures are formed around the spheroids.The mRNA expression of VEGFA、VEGFR-1、VEGFR-2、EGF and FGF-2 in ATRA+SU5416 group were significantly down-regulated,and angiogenesis was inhibited.Therefore,ATRA plays a crucial role in the early commitment stage of adipogenesis of intramuscular ASVCs in ruminant animals by promoting the angiogenesis.In conclusion,this study provides a feasible means of nutritional regulation for improving marbling of ruminant animals through the vitamin A administration of maternal and offspring. |
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