Functional assessment for elimination of mismatches in nuclear and whole cell extracts obtained from mouse and human blastocysts

<p>Preimplantation embryos may have an increased risk of having mismatches due to the rates of cell proliferation and DNA replication. Elimination of mismatches in human gametes and embryos has not been investigated. In this study we developed a sensitive functional assay to examine the repair or elimination of mismatches in both commercially available cell extracts and extracts obtained from preimplantation embryos. Heteroduplex molecules were constructed using synthetic oligonucleotides. Efficiency of the repair of mismatches was semi-quantitatively analysed by exposure to nuclear/whole cell extracts (as little as 2.5 µg) and extracts obtained from pooled mouse and human blastocysts to investigate the repair capacity in human embryos. A cell free <i>in vitro</i> assay was successfully developed to analyze the repair of mismatches using heteroduplex complexes. The assay was further optimized to analyze repair of mismatches in cell extracts obtained from oocytes and blastocysts using minute amounts of protein. The efficiency of mismatch repair was examined in both mouse and human blastocysts (2.5 µg). The blastocysts were observed to have a lower repair efficiency compared to commercially available nuclear and whole cell extracts. In conclusion, a sensitive, easy, and fast <i>in vitro</i> technique was developed to detect the repair of mismatch efficiency in embryos.</p

Differential expression of parental alleles of BRCA1 in human preimplantation embryos

Gene expression from both parental genomes is required for completion of embryogenesis. Differential methylation of each parental genome has been observed in mouse and human preimplantation embryos. It is possible that these differences in methylation affect the level of gene transcripts from each parental genome in early developing embryos. The aim of this study was to investigate if there is a parent-specific pattern of BRCA1 expression in human embryos and to examine if this affects embryo development when the embryo carries a BRCA1 or BRCA2 pathogenic mutation. Differential parental expression of ACTB, SNRPN, H19 and BRCA1 was semi-quantitatively analysed by minisequencing in 95 human preimplantation embryos obtained from 15 couples undergoing preimplantation genetic diagnosis. BRCA1 was shown to be differentially expressed favouring the paternal transcript in early developing embryos. Methylation-specific PCR showed a variable methylation profile of BRCA1 promoter region at different stages of embryonic development. Embryos carrying paternally inherited BRCA1 or 2 pathogenic variants were shown to develop more slowly compared with the embryos with maternally inherited BRCA1 or 2 pathogenic mutations. This study suggests that differential demethylation of the parental genomes can influence the early development of preimplantation embryos. Expression of maternal and paternal genes is required for the completion of embryogenesis