TET-mediated Formation Of 5-hydroxymethylcytosine From 5-methylcytosine In Active DNA Demethylation In Vitro Fertilization And Somatic Cell Cloned Porcine Embryos

BackgroundDuring the formation of maternal and parental pronucleus after fertilization,both the maternal and paternal genomes undergo dramatic changes to prepare for reprogramming.The active demethylation of the paternal pronucleus is supposed as the most important process.In zygotes,the reprogramming occurred in parental pronucleus is substantially different from maternal pronucleus.After fertilization,the maternal pronucleus is almost protected against active demethylation by some DNA binding protein(Stella/Dppa3 ect.).The demethylation of the maternal pronucleus mainly take place in the cleavage stage of embryonic development.Compared with maternal pronucleus,the paternal genome rapidly demethylates,independent of genomic DNA replication,which is called active demethylation.Due to the fact that the process of active demethylation is independent of DNA replication,factors must exist in zygotes that possess the ability to actively demethylate.Over the past three decades,many enzymes have been implicated in the process of active DNA demethylation.In recent three years,Tet family has been discovered and it makes significant difference for demethylation research.In 2009,enzyme ten-eleven translocation 1(Tet 1)was reported as a demethylase which could catalyze the conversion of 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC)for the first time.During the early embryonic development in mice,the level of methylation and demethylation in maternal and parental pronucleus exists remarkable difference.It is detected that the 5hmC level is elevated while 5mC content comes down when the parental pronucleus develops,while 5hmC staining of maternal pronucleus exhibited no obvious differences.The signal of 5hmC in parental pronucleus would be found enhanced at the stage of pronucleus 3(PN3)and parental pronucleus becomes larger than maternal pronucleus.In parental pronucleus,the signal of 5mC becomes weaker at PN3 stage.Nonetheless,in maternal pronucleus the signal of 5hmC and 5mC makes no differences.Thepresent and the parental/maternal pronucleus asymmetry of 5hmC signal can also be found in other species'zygotes,such as cows and rabbits.This suggests that it is relatively conservative that 5hmC rapidly demethylates in parental pronucleus after fertilization.The qRT-PCR results shows that there was high level of Tet3 expression in fertilized oocytes,but not Tetl or Tet2.However Tet3 expression was decreased after the two-cell stage.A conditional knockdown of Tet3 in the mouse germ cells or RNA interference knockdown of Tet3 proteins in the zytoges will hinder the conversion of 5mC to 5hmC in parental genomes,and Tet3 protein will be specifically gathered in the parental pronucleus at the pronucleus stage of fertilized oocytes.These studies in mice have certified that Tet proteins play a biological and essential role in the early embryonic development and maintain the pluripotency and differentiation of stem cells.The demethylation in the parental pronucleus of mouse fertilized oocytes is the conversion of 5mC to 5hmC by Tet3 protein,and obviously asymmetrical demethylation is observed in parental pronucleus of mouse fertilized oocytes.Nevertheless,those processes in porcine early embryonic development remain several controversies and diverse conclusions were drawn by different researchers.Across the development of oocytes,zygotes,parthenogenesis embryos and cloned embryos in pig,multitudinous events remain uncovered at present.We still need to learn whether the conversion of 5mC into 5hmC will occur,what biological function will be in this process,whether Tet protein will play an important role in the process and what expression pattern will happen among Tet family members.ObjectivesThis study aimed to investigate the conversion from 5mC to 5hmC in zygotes,parthenogenetic embryos and somatic cell cloned embryos at pronucleus stage and the early cleavage stage,and to further verify whether this transformation were mediated by TET1-3.We hope we can explore the DNA demethylation mode during early embryonic development and clarify the demethylation mechanism.Our results will lay the foundation for improving the efficiency of nucleus reprogramming and cloned embryos.Methods1.Genomic methylation and hydroxymethylation were visualized with immunofluoresence(IF)staining of 5-methylcytosine and 5-hydroxylmethylcytosine for in vitro fertilization(IVF),Parthenogenetic and cloned(somatic cell nuclear transfer derived)embryos.2.The BSP were applied to detect the total genomic methylation level in SINE,POU5F1 and Satelite Region in zygotes from PN2 to PN5.3.The GluMS-PCR were applied to detect the ratio of 5hmC/5mC in SINE and POU5F1 in zygotes from PN2 to PN5.4.Tet3,Dnmt1 Dnmt3a were visualized with immunofluoresence(IF)staining of in IVF,Parthenogenetic and cloned embryos.5.The qRT-PCR were used to detect the expression of Tetl,Tet2 a,Tet3 and Dnmtl at the Pronuclear stage in IVF,parthenogenetic and cloned embryos.6.Genomic methylation and hydroxymethylation were visualized with immunofluoresence(IF)staining of 5-methylcytosine and 5-hydroxylmethylcytosine.Blastocyst rates were observed in IVF,Parthenogenetic and cloned embryos after Tet3 was knocked down by RNAi.Results1.During early pronuclear development stages,the fluorescence intensity of 5-hydroxymethylcytosine(5hmC)in the paternal pronucleus increased from PN3 to PN5,while 5hmC staining of maternal pronucleus exhibited no significantdifference.The same phenomenon was observed in the dual pronuclear clone embryos.We did not observe this phenomenon in the dual pronuclear parthenogenetic.2.By the BSP we found the total genomic methylation level in SINE,POU5F1 and Satelite Region decreased from PN2 to PN5 in fertilized zygotes.3.By th GluMS-PCR we found the ratio of 5hmC/5mC in SINE and POU5F1 increased from PN2 to PN5 in fertilized zygotes.4.Tet3 mRNA was specifically detected in oocytes and zygotes.At the zygotic stage,the Tet3 protein was concentrated in the male pronucleus.The unique expression pattern of Tet3 suggests its possible role in modifying the zygotic paternal genome.Control staining shows Dnmtl in the cortical cytoplasm and Dnmt3a(red)in both pronuclei and the cytoplasmat.The nucleolus had no staining signal.5.The qRT-PCR results showed that there was moderate expression of Tetl and Tet2 in the early pronuclear stage embryos.However in fertilized embryos,Tet3 showed high expression levels in at the Pronuclear stage in IVF,Parthenogenetic and cloned embryos.The expression of Dnmt1 increased from PN2 to PN5.6.Tet3 was knocked down by RNAi to determine the role of Tet family in the development of embryo development.IF in fertilized zygotes results showed that during early pronuclear embryonic stages,the fluorescence intensity of 5-hydroxymethylcytosine(5hmC)in the paternal and maternal pronucleus exhibited no obvious differences from PN1 to PN5.Similarly no changes were observed in the embryo development competence.Conclusions1.The active demethylation from 5mC to 5hmC happened during early pronuclear embryonic stages in zygotes.The conversion from 5mC to 5hmC is mainly catalyzed by Tet3.2.Tet3 mediated formation of 5-hydroxymethylcytosine from 5-methylcytosine in active DNA demethylation in zygotes may be derived from oocytes.3.Tet3 may mediate formation of 5-hydroxymethylcytosine from 5-methylcytosine in active DNA demethylation in parthenogenetic and cloned embryos.