The Expression And Methylation Patterns Analysis Of Imprinted Genes In Porcine Parthenogenetic Fetuses

Parthenogenesis is a form of reproduction without being fertilized by aspermatozoon. It is a process that an oocyte is able to develop into an embryo alone.Natural parthenogenesis exists in invertebrate and lower vertebrate, but not inmammals. Previous studies have shown that MII artificially activated oocytes is ableto develop to somites stage after implantation in the recipients. However,parthenogenetic embryo cannot develop to term in mammals due to the lack ofpaternal gene expression and failed X chromosome inactivation. Previous studiessuggested that the expression of imprinted genes is different in different species ofanimals. So far, the research of imprinted genes expression in porcine parthenogeneticembryos has not been reported yet. To further characterize porcine parthenogenesis,the expression of18imprinted genes was compared between parthenogeneticembryos and normally fertilized embryos using quantitative real-time PCR (q-PCR).The results revealed that maternally expressed genes were over-expressed, whereaspaternally expressed genes were significantly reduced in PA fetuses and placentas.Bisulfite sequencing PCR (BSP) test demonstrated that PRE-1and Satellite werehypermethylated in both normal and parthenegentic fetuses and placentas, while XISTDMRs were hypomethylated only in PA samples. These results suggested that theaberrant methylation of XIST DMRs and abnormal imprinted gene expression may beresponsible for developmental failure and impaired growth in porcineparthenogenesis.The somatic cells could be reprogrammed through somatic cell nuclear transfer(SCNT). The genomic imprint of parthenogenetic embryos could be erased duringdevelopment after implantation in a recipient. Thus, we hypothesized thatdevelopmental potential of the SCNT parthenogenetic embryos might be improved in vivo. In this study, we used parthenogenetic somatic cells as nuclear donors to performSCNT. The development term of porcine parthenogenetic fetus was extended to aslong as39days after transferred into the oviduct of a surrogate sow. Parthenogenticsomatic nuclear transfer (PSCNT) fetuses were smaller and developmentally retardedwhen compared to normally fertilized controls, judged by weight and morphologicalchanges. Quantitative gene expression analysis indicated that in PSCNT fetuses, H19was overexpressed, whereas IGF2was significantly reduced (p <0.05) comparedwith their respective controls. In addition, BSP test demonstrated that H19DMRs(differentially DNA methylated regions) were hypomethylated in PSCNT fetuses,while IGF2DMRs were hypermethylated in both PSCNT and control fetuses.Our results suggest that in vivo development of the porcine parthenogentic fetus canbe extended using PSCNT, but is still limited due to abnormal DNA methylation ofH19DMRs.To verify if the genomic imprinting status is altered in cell level during theprocess of SCNT, the gene expression and methylation patterns of H19, IGF2, NNATand MEST were analyzed by qRT-PCR in fetal fibroblasts isolated from normalfetuses, parthenogenetic fetuses, cloned fetuses derived from normal somatic cells andcloned fetuses from parthenogenetic somatic cells respectively. The results revealedthat there is no significant difference for expression of imprinted genes among thedifferent donor cells. The methylation status study by BSP and COBRA assay showedgenomic imprinting status is not different in the donor cells and the cloned embryocells. Taken together, these results suggest faithful expression of imprinted genesbetween donor cells and SCNT cloned embryo cells.In conclusion, the results of the present study demonstrated that the expression ofimprinted genes and the DNA methylation pattern was abnormal, especially for XIST,which might be responsible for developmental failure associated with porcineparthenogenesis. The extent of developmental ability of parthenogenetically clonedporcine embryos is still limited. The expression of imprinted genes was faithfulbetween donor cells and SCNT cells. The data achieved in this report provides useful information for future study of embryo development of pigs.