Effects Of Methylene Blue On In Vitro Maturation And Parthenogenetic Embryo Development Of Porcine Oocytes

Methylene blue(MB)is a phenothiazine salts widely used in biological and medical fields.It is used in neurological diseases and metabolic diseases.Current study has shown that MB could act as electron carrier in mitochondria to enhanced the function of respiratory chain and help cells to alleviate mitochondrial damage,improve oxidative stress and ATP production in mitochondria.However,there are no studies reporte about the protective effect of methylene blue on germ cells.Therefore,the purpose of this study is to provide a theoretical basis for further optimization of porcine oocytes In Vitro Maturation system by inquiring into the effects of methylene blue on the development of In Vitro Maturation(IVM)and parthenogenetic embryos in vitro.First,the effects of adding different concentrations of MB(control group: 0nM;test group: 10 nM,50nM,100 nM,200nM)44h-46 h on IVM of porcine oocytes were discussed.According to count cumulus cell expansion and oocyte first polar body excretion rate,early embryo cleavage rate and blastocyst rate after parthenogenetic activation of MII oocyte,the results showed that cumulus cell expansion score(2.588±0.018)and oocyte first polar body excretion rate(69.73±3.56)of MB group were significantly higher than those of 0 nmol/L control group(2.372±0.029 ?60.13±3.31)(P < 0.05);subsequently,the cleavage rate(84.24±1.7)and blastocyst rate(34.54±3.56)of 10 nmol/L treated group were observed after parthenogenetic activation of early embryos was significantly higher than 0 nmol/L(75.71±3.24 ?25.69±3.85)in the control group(P < 0.05),but there was no significant difference incleavage rate and blastocyst rate between the other groups and the control group(P >0.05).Second,according to the treatment effect of methylene blue with different concentrations,10nmol/L MB was selected to treat porcine oocytes 44h-46 h,and further explored its possible mechanism.The results showed that compared with 0nmol/L control group,10 nmol/L MB treatment significantly decreased the fluorescence intensity of ROS in oocyte(12.8+3.28 VS 7.5+3.19,P<0.05);10 nmol/L MB treatment significantly increased the total glutathione content in oocyte(13.95+2.78 VS 11.86+1.28,P<0.05)and mitochondrial membrane potential(13.42+3,98 VS 20.7+2.67,P<0.05).It was also found that the expression of CyclinB1,SHMT2 and PGC1 alpha in oocyte could be significantly increased by adding 10 nmol/L MB treatment(P < 0.05),while the expression of C-MOS,CDC2 and ELASTIN had no significant effect(P > 0.05).Results showed that:(1)adding 10nmol/L methylene blue to promote the maturation of oocytes and the development of subsequent parthenogenetic embryos during the maturation of porcine oocytes and the development of parthenogenetic embryos;(2)methylene blue may play an important role in promoting oocyte maturation by promoting the proliferation and expansion of cumulus cells,reducing cell apoptosis and maintaining normal physiological activities;Scavenging free radicals in oocyte,improving the antioxidant ability of oxidative system in oocyte;improving the activity of mitochondria in oocyte,making more biosynthesis of mitochondria,in order to provide materials needed in oocyte maturation process and improve its quality.