Study Of The Effect Of Melatonin And CNP On Oocyte In Vitro Maturation And Overexpression Of AANAT In Ovine

In vitro maturation(IVM)of oocyte is the important basic link of embryo engineering research,but the quality of the in vitro maturation oocytes is still poorer than the in vivo maturation oocytes.Therefore,in this study melatonin(MT)and c-type natriuretic(CNP)were used to study the in vitro maturation of sheep oocytes,in order to further improve the in vitro culture system and further study the regulation mechanism on in vitro maturation of oocytes.Experiment I In this study the potential beneficial effects of melatonin on the in vitro maturation of ovine oocytes and in vitro development of embryos was analyzed.First,immunostaining and western blot methods showed that MT1 and MT2 were both detectable in ovine GV cumulus,M? cumulus,GV oocyte,M? oocyte and granule cells,moreover the expression of MT1 was notable higher than the other three groups,and the expression of MT2 in M? oocytes was significantly higher than the other four groups.The rate of cumulus expansion and polar body extruding were remarkably increased at 10"9?10-7 mol/L MT group,moreover 10-7 mol/L MT improved the cleavage rate and blastocyst rate.Quantitative real-time polymerase chain reaction results indicated that MT had no effect on the expression of GDF9,BMP15 and DNMT1 of oocyte,but enhanced the expression of PTX3,HAS2 and EGFR of cumulus cells.The beneficial effects of melatonin on ovine oocyte maturation may be mediated by the MT receptor,this is based on the facts that the polar body extruding rate,the cleavage rate,the blastocyst rate and the hatched blastocyst rate of luzindole group were inferior to the MT group,while had no difference with the control group.Furthermore,the cAMP level of MT group oocytes was significantly lower than the control group,luzindole group and MT+luzindole group,but the cGMP level was higher than the three groups.Experiment II Quantitative real-time polymerase chain reaction(qRT-PCR)results showed that CNP and NPR2 mRNA were both expressed in ovine mural granulosa cells,cumulus cells and oocytes,and the expression of CNP mRNA is predominantly expressed in ovine mural granulosa cells,whereas cumulus cells surrounding oocytes mostly express mRNA of the receptor NPR2.200 nM CNP treatment for different time(4 h,8 h,12 h,16 h,20 h,24 h)showed that CNP inhibited ovine oocytes meiosis within 6 h,and after 6 h the inhibition rapidly reduced,and after 16 h the cumulus cells already appear complete expansion which illustrated that the optimum time of CNP for inhibiting the ovine oocytes meiosis was 6 h,and had no effect on the cumulus expansion.The expression of related genes were tested of the CNP processing 4 h,8 h and 12 h oocytes and cumulus cells of ovine and the results showed that the expression of CNP,NPR2 and MT1 of oocytes rising from GV to 4 h,and amount the highest at 8 h,and declined at 12 h;the expression of CNP and FSHR in cumulus cells was highest at 12 h,and NPR2 showed the highest expression at 4 h while the expression of EGFR decreased at first and then increased.When co-cultured the GC and COCs,CNP treatment for 12 h had the highest expression of CNP,NPR2,MT1 and LHR,while MT2 had the highest expression at 8h;CNP,MT1,MT2 and LHR had the highest expression at 12 h in COCs,while NPR2 showed the highest expression at 4 h,and peak expression amount were significantly higher than other time.Research showed that within the first 4 h of in vitro meiosis mature of sheep COCs CNP played the best inhibition effect,which mainly through affect the NPR2 receptor of cumulus cells,and also could affect the expression of the mature related genes.Experiment ? studied the influence of the CNP and MT treatment on oocyte in vitro maturation and embryo in vitro development of ovine.The experimental results showed that pre-treatment of CNP for 4h and then maturation for 24 h and 28 h significantly promoted the cleavage rate when compared to the control group,and maturation for 24 h group had higher blastocyst rate,while maturation for 28 h group had no difference with the control group of blastocyst rate.The results manifested that regardless of CNP and MT treated separately or combined,the cleavage rate,the blastocyst rate and the total cell number of blastocyst were all higher than that of the control group;Quantitative real-time polymerase chain reaction results indicated that when compared to the control group,the expression of CNP,NPR2,FSHR and EGFR of CNP group,MT group and CNP + MT group were all remarkable increased,and the rate of mitochondria distribution even and cortical granule full migration were all improved when compared to the control group.Experiment IV studied the generation of the AANAT transgenic offspring via pronuclear embryos microinjection and frozen-thawed procedure in ovine.The results showed that there were no significant differences among transgenic positive,farrowing and lamb survival rates between the frozen and non-frozen AANAT microinjected pronuclear embryos.It appears that the OPS vitrification and thawing process has limited influence on the embryo quality.The main reason may be that the AANAT gene transferred to the pronuclear embryo overexpressed and resulted in elevated melatonin levels in the embryos and consequently advanced the efficiency of embryo transfer and positive rate and reproduction efficiency of the offspring.In conclusion,MT could promote in vitro maturation of oocyte and the in vitro development of parthenogenetic embryos of ovine;within 4 h CNP can inhibit oocyte meiotic division recover;CNP and MT treated separately or combined both in favour of in vitro maturation of ovine oocyte and enhance the expression of CNP,NPR2,FSHR,LHR,EGFR,MT1 and MT2,and moreover improve the rate of mitochondria distribution even and cortical granule full migration.The overexpression of AANAT gene can advance the efficiency of embryo transfer and positive rate and reproduction efficiency of the offspring.