Fatty Acids Promote Bovine MDSCs Differentiation Through ELOVL3

In addition to genetic factors,the process of muscle growth and development is regulated by a series of environmental factors,such as fatty acids,vitamins,hormones,amino acids,and so on.Muscle-drived satellite cells(MDSCs)are muscle stem cells.During the process of muscle injury,some MDSCs are able to self-renew through mitosis,some can differentiate into myotubes and fuse with each other into new muscle fibers,and others can fuse with existing muscle fibers during differentiation.This potential muscle enhancement characteristic of MDSCs plays a significant role in the production of livestocks.Discovering the novel factors and understanding their roles in the processes of proliferation and differentiation in MDSCs enable us to develop new breeding strategies to improve the muscle production of livestocks.Recently,there are some researches have shown that certain fatty acids play a regulatory role in the proliferation and differentiation of myogenic cells in mice,rats and chickens.However,the corresponding researches on whether fatty acids can regulate the proliferation or differentiation of bovine MDSCs have not been reported.Before this research,there are some researches have shown that the mRNA expression of ELOVL3 was significantly increased in the differentiation process of bovine MDSCs.Moreover,after treating bovine MDSCs with exogenous fatty acids,the mRNA expression of ELOVL3 was significantly increased.Therefore,we speculate that ELOVL3 and fatty acids may play important roles in the differentiation of bovine MDSCs.ELOVL3 which is also called very long-chain fatty acid elongase 3,is a member of the fatty acid elongase family(Elovls)and is expressed mainly in the liver and brown adipose tissue of animals.ELOVL3 can be involved in the initiation and rate control of the elongation process of the carbon chains of the saturated or unsaturated very long chain fatty acids(VLCFAs)whose carbon chains contain less than 24 carbon atoms.A large number of studies have shown that,in addition to the liver and brown adipose tissue,ELOVL3 also expressed a lot in white adipose tissue and skin.However,few reports have shown the expression and function of ELOVL3 in animal muscle cells.Therefore,the main purposes of this study are as follows: investigating the effect of FAs on the proliferation and differentiation processes in bovine MDSCs;investigating the expression pattern of ELOVL3 during bovine MDSCs differentiation;investigating the effect of ELOVL3 on the differentiation process in bovine MDSCs;investigating the relationship between the FAs-drived MDSCs differentiation and ELOVL3.In order to achieve the above purposes,we conducted the following studies:1.Three kinds of exogenously FAs(PA,OA and DHA)were added to the differentiation culture medium of bovine MDSCs in vitro,immunofluorescence technique and western blot technique were used to detect the effects of FAs on the differentiation process of bovine MDSCs.The results showed that the myotube fusion indexs and the myotube lengths in each FA-treated group increased significantly and the protein expression levels of muscle differentiation markers MYOG and MYH3 also increased significantly;2.Three kinds of exogenously FAs(PA,OA and DHA)were added to the proliferation culture medium of bovine MDSCs in vitro,flow cytometry and EdU incorporation assay were used to detect the effects of FAs on the cell cycle and proliferation process of bovine MDSCs.The results showed that the cell cycles and the proliferation rates in each group have no significant difference;3.Inducing the differentiation of bovine MDSCs,immunofluorescence and western blot were used to detect the expression pattern of ELOVL3 protein during the differentiation of bovine MDSCs in vitro.The results showed that the expression of ELOVL3 protein increased with the differentiation of bovine MDSCs;4.CRISPR-activation technology and siRNA interference technology were used to activate or interference the expression of ELOVL3 in bovine MDSCs,immunofluorescence technique and western blot technique were used to detect the effects of up-regulating or down-regulating the expression of ELOVL3 on the differentiation process of bovine MDSCs.The results are as follows: when activating the expression of ELOVL3,the myotube fusion indexs and the myotube lengths in bovine MDSCs increased significantly and the protein expression levels of muscle differentiation markers MYOG and MYH3 also increased significantly;when interference the expression of ELOVL3,the myotube fusion indexs and the myotube lengths in bovine MDSCs decreased significantly and the protein expression levels of muscle differentiation markers MYOG and MYH3 also decreased significantly;5.Three kinds of exogenously FAs(PA,OA and DHA)were added to the differentiation culture medium of bovine MDSCs,meanwhile,interference the expression of ELOVL3,immunofluorescence and western blot techniques were used to detect the relationship between the functions of FAs and the expression levels of ELOVL3.The results showed that the myotube fusion indexs and the myotube lengths in each FA-treated group decreased significantly and the protein expression levels of muscle differentiation markers MYOG and MYH3 also decreased significantly;6.In order to detect the relationships between ELOVL3 and lipid droplets,fluorescence staining and confocal microscopy imaging techniques were used to detect the subcellular localization of ELOVL3 and lipid droplets in bovine MDSCs during proliferation and differentiation.The results showed that the subcellular localizations of ELOVL3 were consistent with the subcellular localizations of neutral lipids and lipid droplets in bovine MDSCs.The results discussed above showed that: PA,OA and DHA can promote the differentiation process of bovine MDSCs but have no effect on bovine MDSCs proliferation;ELOVL3 can promote the differentiation of bovine MDSCs;the FAs-derived bovine MDSCs differentiation relies on the high expression levels of ELOVL3.The knowledge gained from these studies enables us to develop the new breeding strategies and nutritional approaches to improve the muscle production of livestocks.