| Melatonin(MLT),distributed wildely in animal body,is a multifunction endogenous hormone which plays cucial roles in the development of animal germ cells and embryos.However,exact effect of melatonin and its underlying mechanisms on development of pig oocytes and embryos remains poorly understood.Therefore,the present study aims to investigate influences of exogenous MLT on maturation of oocyte and development of embryons,in order to optimize in vitro culture media and provide basis for future uncovering mechanisms of MLT's function.The experiments and results are as follows:Experiment one: Exogenous melatonin at different concentrations(control group: 0nM and experimental groups: 1nM,10 nM,100nM)were supplementd in maturation medium,in order to explore the effect of melatonin on in vitro maturation of porcine oocytes.Nuclear and cytoplasmic maturaion were evaluated by polar body extrusion,staining of H3K27ac/H4K16 ac signal in nuclear matured oocytes,and subsequent preimplantational development after parthenogentic activation.It was found that melatonin-treated groups(1,10,100nM),compared with control group,could significantly increase nuclear maturation rate of oocytes(P<0.05),but the cleavage rate and total cell number per blastocyst are similar among groups(P>0.05).Blastocyst rate of parthenotes derived from oocytes treated with 1nM MLT was significantly improved(P<0.05),while no obvious changes was found from 10 nM and 100 nM groups(P> 0.05).Expression of H3K27ac/H4K16 ac was notably decreased in matured oocytes from 1nM MLT group(P <0.05).Experiment two: Based on the above experiment,1nM melatonin was added into maturaion medium in order to exmine the effects of treatment duration using on in vitro maturation of swine oocytes.The results showed that both 0-22 h and 0-42 h group could enhance polar body extrusion rate of oocytes and following blastocyst rate after activation(P<0.05),while the 22-42 h group displayed no obvious changes(P> 0.05).As for cleavage rate and total cell number per blastocyst,no significant differences were observed among groups(P> 0.05).Experiment three: Impact of 1nM melatonin supplementation in maturation medium on in vitro maturation of pig oocytes derived from medium follicles(diameter at 4-7mm)and small follicles(follicular diameter 1-3mm)was investigated.It was showed that 1nM treatment could increase significantly polar body extrusion rate of oocytes from small follicles as compared with the control(P <0.05),but no significant differences was found in rates of cleavage and blastocyst,and total number cell per blastocyst of parthernogenticembryos among groups(P>0.05).However,for oocytes frommedium follicules after 1nM treatment,the nuclear maturation rate,blastocyst rate and total cell number per blastocyst all except cleavage rate were significantly improved(P <0.05).Experiment four: The effects of 1nM melatonin treatment on in vitro maturation of oocytes from COCs of poor morphology(cumulus cells layer ?1)and artificial denuded oocytes were evaluated.It was found that 1nM melatonin treatment could greatly improve blastocyst rate after activation of oocytes either from poor morphology COCs or artificial denuded ones compared with the control group(P <0.05);while in terms of nuclear maturation rate,the cleavage and total cell number per blastocyst,no significant difference was observed among groups(P> 0.05).Experiment five: Melatonin of different concentrations were added into PZM-3(control group: 0nM and experimental groups: 1nM,10 nM,100nM)in order to investigate effects of MLT on in vitro development of pig parthenogenetic embryos.The results showed that MLT treatment could significantly enhance blastocyst rate(P<0.05),and 10 nM was the best among MLT-treated groups.Moreover,10 nM treatment could also significantly increase total cell number per blastocyst compared to the control(P <0.05),while 1nM and100 nM treatment showed no significant differences compared to the control(P> 0.05).Experiment six: Based on Experiment five,effects of different 10 nM MLT-treatment duration on in vitro preimplantational development of porcine parthenogenetic embryos were studied.The results revealed that blastocyst rate are significantly inceased in both the0-72 h and 0-168 h group supplemented with 10 nM MLT compared with the control group(P<0.05),but no obvious changes were seen in the 72-168 h group compared to the control group(P> 0.05);for cleavage rate and total cell number per blastocyst,no significant differences was observed among groups(P> 0.05).Experiment seven: Based on the findings of Experiment one and five,the effects of combination treatment using MLT,at concentrations most beneficial to oocytes and embryos,in both oocyte maturation and embryo culture on preimplantation development of embryos were explored.Groups(IVM+IVC)were: Control(0nM and 0nM),Treatment I(0nM+10nM),Treatment II(1nM+0nM),and Treatment III(1nM+10nM).It was showed that the blastocyst rate in all three combine treatment groups was significantly increased than the control group(P<0.05);for the total cell number per blastocyst,Treatment III was better than the control(P<0.05),and no statistically significance was observed among the other three groups(P> 0.05).Experiment eight: Effects of MLT treatment on expression of genes in COCs relatedwith apoptosis,autophagy and cumulus expansion were determined.After maturation,MII oocytes and cumulus cells in the control group and 1nM MLT treatment group were collected.Expression of genes EFNB2,HAS2,PTX3),AKT2,BMP15,GDF9,p34 was detected using quantitative real time PCR.It was found that 1nM melatonin-treatment significantly promoted expression of EFNB2,HAS2,PTX3 in cumulus cells(P<0.05),and expression of oocyte maturation-related genes such as AKT2,BMP15,GDF9,and p34 increased significantly in MII oocytes(P<0.05).In conclusion,the current study indicated that addition of 1nM and 10 nM MLT into culture medium of maturation or embryo culture are helpful to in vitro maturation of pig oocytes and subsequent parthenotes in vitro development,and the positive influences of MLT usually happened in earlier half duration of culture;1nM MLT treatment could enhance oocyte maturation and subsequent parthenote development,related closely with regulation of genes involved in histone modification,oxidative stress,cumulus expansion,and apoptosis in oocytes or cumulus.The finding of this study lay a solid foundation for revalling mechanism of MLT's role in porcine oocyte maturation and preimplantation embryo development. |
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