Molecular Human Reproduction, Vol. 7, No. 2, 147-154,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase
1 Department of Reproductive Sciences and Medicine, Division of Obstetrics and Gynaecology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, 2 School of Biology, University of Leeds, Leeds LS2 9JT, UK, 3 Centre Génétique, U393 Institut Necker, Hôpital des Enfants Malades, Paris, France and 4 Department of Obstetrics, Gynaecology and Human Genetics, Royal Victoria Hospital, McGill University, Montreal, QC H3A 1A1, Canada
Abstract
We have developed a competitive reverse transcription-polymerase chain reaction (RT–PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT–PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation 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 ~1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro culture. In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts throughout each stage of human preimplantation development and it indicates that the accumulated HPRT transcripts present in the unfertilized human oocyte undergo extensive destruction following fertilization. This work also suggests that X-inactivation occurs beyond the 8-cell stage of human preimplantation development.
HPRT/human preimplantation embryo/oocyte/quantification/RT-PCR
Notes
5 To whom correspondence should be addressed at: Department of Reproductive Sciences and Medicine, Division of Obstetrics and Gynaecology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: deborah.taylor{at}ic.ac.uk
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