A Preliminary Study On Effects Of Gallic Acid In Vitro Maturation Of Bovine Oocytes And Its Mechanism

In vitro maturation of oocytes is an important step in assisted reproductive technology.However,the oxygen environment in vitro,does not favor the development of oocytes of in vitro maturation.Gallic acid is a natural organic acids of existing widely in plants,research has shown that it has strong antioxidant properties,which can help cells to scavenge free radicals and protect the cells.However,no studies have been reported on the role of gallic acid in the reproductive cells,and the mechanism of gallic acid is rarely reported.Therefore,the purpose of this study was to investigate the effect of gallic acid in vitro maturation of bovine oocytes and subsequent early embryo development.Meanwhile,the mechanism of action is clarified for optimizing the IVM culture system with a theoretical basis.Firstly,the effects of gallic acid on the maturation of bovine oocyte in vitro and embryogenesis were discussed.COCs of bovine were matured in vitro on culture medium with different concentrations(0,10,30,50,100 ?mol/L)of gallic acid for 22-24 h.Subsequently,the first polar rate of oocyte and the expansion of cumulus cell were calculated;The matured oocytes were fertilized,and then eggs' cleavage rates,the blastocyst formation rates,the number of blastomere and the apoptosis rate of blastomere were evaluated.The results reveal that the first polar rate and the sore of expansion of cumulus cell of oocytes in the mature medium with 30 ?mol/L GA is higher than the control group of 0 ?mol/L significantly(P<0.05).Observing embryonic development found that the treatment group's embryo cleavagerate and blastocyst rate was higher than the control group of 0 ?mol/L,and 30 ?mol/L group's cleavage rate,blastocyst rate and the number of blastocyst cells were significantly higher than 0 ?mol/L control group(P<0.05),but the apoptosis rate of blastula cells doesn't significantly differ(P>0.05).Secondly,According to the treatment effect of gallic acid in different concentrations,the optimal concentration(30 ?mol/L)is selected and the mechanism of action is preliminarily discussed.These results showed that the levels of reactive oxygen species(ROS)were significantly decreased(16.65±4.03 VS 10.80±3.36;P<0.05)and the content of total glutathione and catalase increased significantly for 30 ?mol/L(0.00498±0.00091 VS 0.00777±0.0010 pmol/oocyte,0.517 ±0.845 VS 1.169±0.845U/mg protein;P<0.05)compareding to the 0 ?mol/L control group;The nf-?B signaling pathway analysis found that which ROS levels decline caused by gallic acid in cattle oocytes will weaken to the stimulation of the nf-KB signaling pathways,meanwhile nf-KB mRNA and protein level were significantly lower than 0 p,mol/L group(P<0.05).And combineding with fluorescence quantitative PCR found that the nf-KB signaling pathways was suppressed may firther promote downstream antioxidant gene Superoxide Dismutasel(SOD1)and Glutathione Peroxidase 4(GPX4)mRNA expression(P<0.05).In addition,the effects of gallic acid on the cumulus cell during in vitro maturation of COCs were discussed.The results showed that compared with the control group,the levels of ROS in the cumulus cell of the 30 ?mol/L group were significantly decreased,and the total glutathione and catalase content increased significantly(P<0.05).Fluorescence quantitative PCR results show that the cumulus cell apoptosis gene expression(Bax,Bad)is restrained,and the extension gene(PTGS2/PTX3/HAS2F/TSG62),antiapoptotic gene(Bcl2),prolifer-ation(spyl/spy2/spy3)and antioxidant gene expression(GPX4 and SOD1)significantly increased for the treatment group(30 ?mol/L)(P<0.05).Finally,combined with fluorescence quantitative PCR and cellular immunofluorescence,the effect of gallic acid on the mitochondrial content in the oocyte was analyzed,the results show that during the oocyte maturation,the processing of gallic acid(30 ?mol/L)can significantly improve mitochondrial transcription factor PGC-1?,TFAM,UCP2 mRNA expression.At the same time,immunofluorescence results showed that gallic acid(30 mol/L)could also promote the translation of mitochondrial transcription factor TFAM protein(P<0.05).The above results show that:(1)The addition of a suitable amount of gallic acid in vitro maturation of the bovine oocyte can promote the mature quality.of the oocyte and the development of embryo;(2)the gallic acid promotes the oocyte maturation may be through the following ways:a?The anti-oxidation system of oocytes was regulated by NF-?B signaling pathway,and the oocyte was protected from the damage of reactive oxygen;b?To promote the physiological function of cumulus cell and indirectly contribute to the maturation process of oocytes;c?Gallic acid can promote the biosynthesis of mitochondria in oocyte and then improve the mature quality of oocyte.