Parthenogenetic cells display decreased global methylation and low DNA methyltransferase transcription

The diploid status of a parthenote is generally obtained combining the activation of metaphase 2 oocytes with exposure to an actin polymerization inhibitor. The consequent microfilaments disruption effectively inhibits the extrusion of the second polar body. The embryos obtained with this approach are characterized by a high level of homozygosity, since the diploid status is obtained after the segregation of sister chromatids. Furthermore, because of their parthenogenetic origin, they contain only maternally inherited imprinted genes. Parthenogenetic embryos and derived cells are therefore very exciting and relevant experimental models in order to investigate both the effect of asymmetric imprinting as well as high homozygosity. Based on these observations we analyzed epigenetic mechanisms involved in the control of gene expression in cell lines obtained from parthenogenetic embryos. In particular we studied the transcription of the three active DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. These molecules are known to specifically methylate DNA CpG sequences and modulate gene transcriptional accessibility in mammals. To this purpose total RNA was extracted from human parthenogenetic cell lines (HP1 and HP3) and biparental human ESC lines (HES 7, HES I-3 and HES I-6). The expression levels of the DNMT1, DNMT3A, and DNMT3B genes were analyzed by Real- Time PCR. The results showed a significantly lower expression of the three genes in parthenogenetic cells when compared with their bi-parental counterpart. To further investigate the epigenetic profile of parthenogenetic cells, global methylation studies were carried out using a specific anti-5-methylcytosine (5-MeC) antibody. Immuno-staining results were in agreement with the molecular data and showed low methylation levels in parthenogenetic cells. We suggest that the decreased percentage of methylation detected in parthenogenetic cells is likely to reflect the extensive DNA demethylation process that takes place in the maternal genome during early embryo development (Reik et al., 2003).