The Effect Of Melatonin On The In Vitro Maturation And Embryo Development Of Old Mice Oocytes

With the increase of age,the reproductive ability of female mammals decreases significantly,which is mainly manifested in the relative decrease of hormone secretion in vivo,the gradual decline of ovarian function,and the decrease of the quality and quantity of oocytes,thus affecting the efficiency of embryo production in vitro.Melatonin,as an effective antioxidant and anti-aging substance,is secreted by the pineal gland and exists in the follicular fluid,which can protect oocytes from oxidative stress during ovulation.However,the role of melatonin in in vitro maturation and embryonic development of aged mice oocytes were rarely reported,so it is of great significance to study the mechanism of melatonin in in vitro embryo production of aged mice oocytes in vitro.In this study,the oocytes obtained from 10-12 months old ICR female mice were injected with PMSG 44 h(superovulation).Melatonin was added to the in vitro maturation medium(the final concentration reached 0,10-4,10-6,10-8 mol/L).The effects of melatonin on in vitro maturation of aged mice oocytes were studied by means of in vitro maturation,in vitro fertilization and in vitro early embryo development.The results showed that 10-6 mol/L melatonin could significantly increase the first polar body excretion rate of oocytes compared with the control group after 16-18 h maturation in vitro.And 10-4 mol/L,10-6 mol/L and 10-8 mol/L melatonin significantly increased the cleavage rate of oocytes after in vitro fertilization.Further early embryo development culture in vitro showed that 10-6 mol/L and 10-8 mol/L groups could effectively improve the blastocyst rate of aged mice oocytes.Through comprehensive comparison,it was concluded that 10-6 mol/L was the best concentration.Then,a series of experiments were carried out with 10-6 mol/L melatonin group and control group:through the detection of reactive oxygen species,glutathione level,mitochondrial distribution,ATP content,early apoptosis,immunofluorescence and real-time fluorescence quantitative PCR detection,it was found that 10-6 mol/L melatonin could effectively eliminate reactive oxygen species,increase GSH content,reduce oxidative stress,and improve mitochondrial distribution.Maintain the normal energy supply of the cell(ATP),to ensure the normal shape of the spindle,make its meiosis accurately completed,and reduce the early apoptosis rate.This study also confirmed that melatonin can improve oocytes quality and promote oocyte maturation by increasing the expression of antioxidant and anti-aging genes(GPX4,SOD1,SOD2,SIRT1,SIRT3)in oocytes of aged mice.To sum up,adding 10-6 mol/L melatonin to the maturation medium in vitro can reduce the oxidative stress level of oocytes of aged mice,improve the function of mitochondria,ensure the normal meiosis of oocytes,and then improve the fertilization rate and blastocyst rate after in vitro fertilization,which can provide reference for the related research of artificial assisted reproductive technology and the fertility of elderly pregnant women in human medicine.