Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase

We have developed a competitive reverse transcription-polymerase chain reac tion (RT-PCR) sensitive enough to detect and quantify as little as 2-fold d ifferences in gene expression in individual oocytes and embryos throughout human preimplantation development, This RT-PCR assay can be tailored for th e examination of any specific gene and so will give a unique insight into h uman preimplantation development. This technique was used to quantify the l evel of hypoxanthine phosphoribosyl transferase (HPRT) expression during pr eimplantation development and to correlate this with embryo sex. The amount of HPRT transcripts present in the unfertilized oocyte was equivalent to 7 .7 fg of competitor cDNA. At the 4-cell stage there is a significant drop ( P = 0.0006) to similar to1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro cu lture, In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts through out each stage of human preimplantation development and it indicates that t he accumulated HPRT transcripts present in the unfertilized human oocyte un dergo extensive destruction following fertilization. This work also suggest s that X-inactivation occurs beyond the 8-cell stage of human preimplantati on development.