The use of amplified cDNA to investigate the expression of seven imprintedgenes in human oocytes and preimplantation embryos

Imprinted genes are characterized by expression of only one of the two alle les according to its inheritance from the mother or the father. This mono-a llelic expression must arise from primary differential epigenetic modificat ion of the parental alleles of the imprinted gene in the spermatozoon and t he oocyte. Most of the information on the onset of imprinted gene expressio n, and on the molecular mechanisms regulating mono-allelic expression, have been derived from studies in the mouse. In this paper, we investigate the expression of seven imprinted genes in human preimplantation development. D ue to limitations imposed by the rarity of human embryos available for rese arch, our approach has been to screen amplified cDNA preparations prepared from human unfertilized oocytes and individual embryos at each of the 4-cel l, 8-cell and blastocyst stages. Gene-specific primers were used to investi gate expression of the imprinted genes by polymerase chain reaction (PCR) a nalysis of these amplified cDNA. We found that expression is inherently var iable in the amplified cDNA from embryo to embryo but the use of several sa mples at each stage showed that the SNRPN, UBE3A and PEG1 genes are express ed throughout human preimplantation development. This was confirmed by dire ct analysis by gene-specific reverse transcription-PCR on a limited number of lysed embryos (one gene analysed per embryo). Thus, the amplified cDNA m ay be used to rapidly identify those imprinted genes expressed in preimplan tation development and, hence, those genes amenable to investigation of the epigenetic mechanisms regulating mono-allelic expression. Confirmation of preimplantation expression also identifies those imprinted diseases amenabl e to preimplantation diagnosis, and the imprinted genes which may be used i n assessment of possible perturbations of imprinting following new procedur es in assisted reproduction. Our series of single embryo amplified cDNA are established as a valuable resource for comparative studies of gene express ion within one embryo and between embryos throughout early human developmen t. The amplified cDNA thus circumvent the need for a continuous supply of h uman embryos for studies on embryonic gene expression.