ABSTRACT
Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.
Subject(s)
Abortion, Spontaneous/genetics , Embryo, Mammalian/metabolism , Fetus/metabolism , Nuclear Transfer Techniques , RNA, Long Noncoding/genetics , Animals , DNA Methylation/genetics , Female , Gene Expression Regulation , Genotype , Male , Pregnancy , RNA, Long Noncoding/metabolism , Sus scrofa , Transcription, GeneticABSTRACT
PURPOSE: To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. METHODS: RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). RESULTS: The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. CONCLUSIONS: Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.
Subject(s)
Abortion, Veterinary/genetics , DNA Methylation , Genomic Imprinting , Swine/genetics , Animals , Cloning, Organism , DNA, Satellite/chemistry , Epigenesis, Genetic , Female , Gene Expression Profiling , Nuclear Transfer Techniques , Placenta/metabolism , PregnancyABSTRACT
Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.
Subject(s)
DNA Methylation , Fetus/metabolism , Genomic Imprinting , Parthenogenesis/genetics , Placenta/metabolism , Animals , Female , Male , Pregnancy , Swine , Transcription, GeneticABSTRACT
To gain insight into parthenogenesis in pigs, we report for the first time that using parthenogenetic somatic cells as nuclear donors (PSCNT), the porcine parthenogenetic fetus can develop to gestational day 39. Weight and morphological analysis revealed that PSCNT fetuses were smaller and developmentally retarded when compared to normally fertilized controls. Quantitative gene expression analysis indicated that in PSCNT fetuses, H19 was over-expressed, whereas Igf2 was significantly reduced (p < 0.05) compared with their controls. In addition, bisulfite-sequencing PCR results demonstrated that H19 differentially DNA methylated regions (DMRs) were hypomethylated in PSCNT fetuses, while Igf2 DMRs were hypermethylated in both PSCNT and control fetuses. Our results suggest that extended development of the porcine parthenogenetic fetus can be accomplished using PSCNT and that abnormal DNA methylation of H19 DMRs might contribute to the critical barrier of parthenogenesis in pigs.
