| Through somatic cell nuclear transfer technology,oocyte reprogramming factors can reprogram highly differentiated somatic cells to form reconstituted cloned embryos and develop into intact animal individuals.The quality and reprogramming ability of oocytes are one of the most critical limiting factors of somatic cell cloning.The oocyte is in a hypoxic environment during normal development in the body,and the follicular fluid contains a free radical scavenger and an antioxidant enzyme to protect the oocyte from oxidative damage.Compare with mature oocytes in vivo,in vitro maturation（IVM）oocytes are in a relatively hypoxic environment and lack the protection of free radical scavengers and antioxidant enzymes,thus having higher levels of reactive oxygen species.This results in lower quality and developmental potential.Excessive production of reactive oxygen species causes oxidative stress,destroys cell membranes and induces apoptosis.As an effective free radical scavenger and a broad-spectrum antioxidant,melatonin directly scavenges reactive oxygen species and reduces cellular oxidative damage.Recent studies have shown that melatonin is present in follicular fluid,and that melatonin supplementation during in vitro culture can increase the fertilization and developmental capacity of oocytes.Supplementation of melatonin during early embryonic development can significantly improve SCNT embryos developmental capacity.However,further research is needed on how melatonin can improve the potential mechanisms of oocyte quality and developmental potential.In addition,the effect of adding melatonin during IVM on subsequent cloned embryo development remains unclear.Therefore,in this study,we investigated the mechanism of effect on the quality of bovine oocytes in vitro and the ability of subsequent cloned embryos by supplementing melatonin in IVM medium.The following are the main test contents:1.GV oocytes were cultured in vitro using conventional in vitro maturation medium and in vitro maturation medium supplemented with different concentrations of melatonin.It was found that adding 10-9 M or 10-7 M melatonin can effectively increase eggs.The maternal cell maturation rate;the obtained MII oocytes were separately subjected to IVF and SCNT,and the subsequent cleavage and blastocyst development rate of the 10-9 M melatonin-added group and the 10-7 M melatonin-added group oocytes were found to be significant improve.2.Take 10-9 M melatonin added group and control oocytes for a series of experiments:through reactive oxygen species and early apoptosis test,it was found that melatonin can effectively remove reactive oxygen species and reduce the early apoptosis of oocytes;-tubulin antibody binding assay found that melatonin effectively improved spindle morphological malformation and chromosome alignment disorder;melanin was found to increase and evenly distribute mitochondrial activity by using mitochondrial fluorescent probes;melatonin increased oocytes by immunofluorescence staining The level of whole-genome H3K9ac in cells decreased the level of H3K9me3 and had a weak effect on the levels of DNA 5mC and 5hmC.3.Take 10-9 M melatonin added group and control oocytes for IVF and SCNT respectively,and the obtained blastocysts were subjected to CDX2 differential staining test and TUNEL apoptosis test.It was found that 10-9 M melatonin was found.The quality of blastocysts derived from the addition of oocytes was higher than that of the control group.Immunofluorescence staining showed that melatonin-treated oocytes had a reprogramming potential close to that of the IVF control group in SCNTs.4.Whole-genome expression analysis of melatonin-treated and untreated oocytes using RNA high-throughput sequencing revealed that 26 genes were differentially expressed between the treatment and control groups,such as glutathione S-transferase gene GSTP1,mitochondrial DNA polymerase gene POLG,mitochondrial ATP synthase gene ATP5F1E,centromeric enrichment gene CEP295,spindle assembly-related gene TCTP,cytoprotective and anti-apoptosis related gene HSP27,and gene UBB involved in protein degradation and DNA damage repair,showing that these genes are likely to play a key role in the mechanism by which melatonin affects oocyte development.Our results indicate that melatonin can effectively alleviate oxidative stress in oocytes,significantly reduce its early apoptosis,protect its spindle and mitochondrial integrity,improve its apparent modification,and thus improve oocyte quality and development.Potential and significantly promote the development of its subsequent cloned embryos. |
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