Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase.
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 approximately 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.
Taylor
D M
DM
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. deborah.taylor@ic.ac.uk
Handyside
A H
AH
Ray
P F
PF
Dibb
N J
NJ
Winston
R M
RM
Ao
A
A
D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D009865:Oocytes / Q000378:metabolism*; D011247:Pregnancy; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012727:Sex Characteristics
D017931:DNA Primers; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase