Effects Of Regulating Sirt1 Activity On Lipid Metabolism Of Mature Porcine Oocytes In Vitro

Lipid metabolism can provide energy for cell proliferation,oocyte maturation,fertilization and subsequent embryo development.SIRT1 has ADP-ribotransferase and substrate specific deacetylase activities,and is widely expressed in somatic cells and reproductive cells,and plays an important role in cell proliferation,differentiation,apoptosis and glycolipid metabolism.At present,it has not been reported whether SIRT1 is involved in regulating oocyte lipid metabolism,so in this study,porcine oocytes were used as materials and SIRT1 activator was added to understand the relationship of SIRT1 gene expression and lipid metabolism by immunohistochemistry,q RT-PCR and other methods.The mechanism of SIRT1/AMPK on oocyte lipid metabolism was preliminarily studied.These results provide theoretical basis for further elucidating the role of SIRT1 in in vitro maturation of porcine oocytes and promoting lipid metabolism of porcine oocytes by regulating its expression,thus improving the maturation rate of porcine oocytes and the potential of early embryo development.The results are as follows.1.Expression analysis of SIRT1 in porcine oocytes matured in vitro and early IVF embryos.q RT-PCR results showed that compared with GV stage,the expression of SIRT1 gene was significantly increased in MI,MII and 4-cell stage(P<0.05),significantly decreased in 2-cell stage(P<0.05),and significantly decreased in 8-cell,16-cell and blastocyst stages(P<0.01).Immunofluorescence results showed that compared with GV stage,the expression of SIRT1 protein in MI and 4-cell stage was significantly increased(P<0.01),and that in the stage of MII,2-cell,8-cell and blastocyst was significantly increased(P<0.05),but there was no significant difference in the expression of SIRT1 protein in 16-cell stage(P>0.05).2.Effects of SIRT1 activator on lipid metabolism of porcine oocytes in vitro maturation.The results showed that adding 3 μM SRT 2104 in in vitro maturation medium could significantly increase the first polar body excretion rate and blastocyst rate(P<0.05).SRT 2104 supplementation significantly increased SIRT1 protein expression,number of mitochondria and lipid droplets,and fatty acid content in porcine oocytes compared with untreated group(P<0.05).q RT-PCR results showed that compared with the untreated group,the expression of mitochondria generation-related genes PGC-1α,TFAM,and lipid generation-related genes ACACA and FADS1 were significantly up-regulated in SRT 2104 treated porcine oocytes(P<0.05),and the expression of FASN was extremely significantly up-regulated(P<0.01).SREBE and PPARγexpressions were significantly down-regulated(P< 0.05).The expression of lipidolytic related genes ATGL,CGI-58 and HSL was significantly increased(P<0.05),the expression of MGL was extremely significantly increased(P<0.01),and there was no significant difference in PLIN2expression(P>0.05).The expression of fatty acid β-oxidation-related genes CPT1 a and ACADS was significantly increased(P<0.05),but there was no significant difference in the expression of CPT1 b and CPT2(P>0.05).AMPK related gene expression was significantly increased(P<0.05).Compared with untreated group,SRT 2104 treatment significantly increased the ATP content of oocytes(P< 0.05).3.Study on the mechanism of SIRT1 regulating lipid metabolism in porcine oocytes.There was no significant difference in the first polar body excretion rate and blastocyst rate of porcine oocytes treated with different AMPK inhibitor compound C compared with the untreated group(P>0.05).10 and 20 μM compound C significantly reduced the first polar body excretion rate and blastocyst rate(P<0.05),and the higher the concentration used,the effect was more obvious.Compared with the untreated group,compound C treatment significantly decreased mitochondrial content(P<0.05),increased the number and volume of lipid droplets,and significantly increased fatty acid content(P<0.05).q RT-PCR results showed that compared with the untreated group,compound C treatment significantly decreased the expression of PGC-1α and TFAM(P<0.05).The expression of lipid synthesis related genes FADS1 and FASN was significantly down-regulated(P<0.05),while the expression of ACACA was not significantly different(P>0.05),and the expression of SREBE and PPARγ was significantly up-regulated(P<0.01).The expressions of lipidolytic related genes ATGL,CGI-58,HSL and PLIN2 were significantly down-regulated(P<0.01),while MGL expression was not significantly affected(P>0.05).The expressions of fatty acid β-oxidation-related genes CPT1 a,CPT1b,CPT2 and ACADS were significantly decreased(P<0.05).The AMPKα1 expression was significantly decreased(P<0.05),but AMPKα2 expression was not significantly affected(P>0.05).Compared with the untreated group,compound C treatment significantly reduced the ATP content of oocytes(P<0.05).The above results suggest that activation of SIRT1 can improve mitochondrial activity and promote lipid metabolism of porcine oocytes in vitro maturation.It may improve in vitro maturation of porcine oocytes and early embryo development potential by regulating AMPK related gene expression.