| Melatonin,a small endogenous molecule,is characterized by its roles in the regulation of circadian rhythms and free radicals scavenging.In fact,the content of melatonin in follicles is much higer than that in circulation,and existence of melatonin receptors has also been identified in ovary cells.Then melation may take participate in the regulation of development of oocytes and embryos through its unique ways.In this research,we fisrtly investigated the relationship between melatonin and developmental competence of porcine SCNT?somatic cell nuclear transfer?embryos:i)The blastocyst rate was significantly improved when porcine zygote medium 3?PZM3?was supplemented with melatonin at concentrations of10-7M or 10-9M?22.6.7%vs 12.5%;21.2%vs 12.5%;18.4%vs 12.5%,p<0.05?,but there was no significant difference for the cleavage rate,compared to the control group?0M?;ii)The cleavage rate,4-cell rate and blastocyst rate were all improved when donor cells?porcine embryonic fibroblasts,PEFs?were treated with melatonin at a concentration of 10-7M?cleavage rate 79.8%vs 94.0%;4-cell rate 62.3%vs79.5%;blastocyst rate 13.2%vs 19.7%,p<0.05?;iii)The cleavage rate,4-cell rate and blastocyst rate were gradually improved with the elongation of treatment durations when PEFs were treated with a optimal concentration of 10-7M.There was no further improvement for those beneficial effects when the treatment time was beyond 48 hours.Next,we investigated the mechanisms underlying those beneficial effects of mealtonin on SCNT embryos.i)The results of cell proliferation test demonstrated that proliferation rate of PEFs was siginificantly improved with a concentration of10-9M.And cell cycle test revealed that the proportion of G0/G1 phase were improved with a treatment concentration of 10-7M.ii)Content of 5hmC and the expression of relevent genes?TET1/2/3,Ten to eleven translocation protein?were respectively detected with dot blotting and real-time RCR when PEFs were treated with different concentrations or duration time.The content of 5hmC were gradually elevated with the increase of treatment concentrations and treatment durations,which was consistent with expression of TET1 and TET3.However,the influence of melatonin treatment on expression of TET2 was much more particular.The expression of TET2was specially elevated with treatment concentration of 10-9M or concentration of10-7M for long time treatment?beyond 2 days?.iii)Content of 5mC and the expression of relevent genes?DNMT1/3A,DNA methyltransferase?were also detected.The expression level of DNMT1was gradually decreased with the increase of treatment concentrations and treatment durations.Short-period treatment with concentration of 10-7M increased the content of 5mC,which then was gradually decreased with the elongation of treatment duration time.Morever,the content of5mC was increasd with low concentrations treatment(10-7M-10-11M)and decreased with higer concentrations treatment(10-3M-10-5M).Variation of 5mC content might depended on the expression of DNMT3 for their similar fluctuation.All together,preimplantation development of porcine SCNT embryos can be appropriately improved by applicable treatment concentrations(10-7M and 10-9M)of melatonin.The mechanisms underlying those beneficial effects may concern with the regulation of cell cyle,variation of 5mC/5hmC and expression of the relevent genes?DNMT1/3A,TET1/2/3?. |
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