Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage.
The transition between dependence on maternal transcripts and proteins inherited in the oocyte and embryonic gene expression in the human preimplantation embryo occurs at the four- to eight-cell stage. Recently, studies using reverse transcriptase polymerase chain reaction (RT-PCR) have detected paternal transcripts for the Y-linked genes, ZFY and SRY, and the myotonic dystrophy associated protein kinase gene, DK, as early as the late pronucleate one-cell stage. However, expression at the protein level has not been demonstrated and its function at these early stages is unknown. Using coding sequence polymorphisms to distinguish maternal and paternal transcripts, we have examined the transcription of two ubiquitously expressed genes: X-linked glucose-6-phosphate dehydrogenase (G6PD) and adenosine deaminase (ADA). Both G6PD and ADA are housekeeping genes with TATA-less promoters which, because of their roles in metabolism and ubiquitous expression, may provide a more reliable indication of the timing of activation of the embryonic genome. They also each have biallelic polymorphisms with a high heterozygosity ratio which can be detected by restriction digestion. Couples undergoing in vitro fertilization (IVF) were screened for these polymorphisms. Individual spare oocytes and embryos at different stages of preimplantation development were analyzed by RT-PCR and appropriate restriction digestion in those cases in which the male partner carried a different allele to the female partner. In addition, since only female embryos inherit the paternal allele of X-linked G6PD, cDNA was also analyzed for ZFX/ZFY transcripts to identify the sex of each embryo. One hundred and twenty three individual oocytes and embryos were analyzed by RT-PCR and restriction digestion to detect the paternal transcripts from the polymorphic alleles. Maternal transcripts for G6PD, ADA, and ZFX were detected in all oocytes and embryos and at all stages. Following restriction digestion, paternal G6PD and ZFY transcripts were first detected at the four-cell stage and paternal ADA transcripts in an embryo at the three-cell stage coinciding with the onset of dependency on transcription from the embryonic genome. This approach should be widely applicable to other genes since similar polymorphisms exist in the coding regions of many genes.
Taylor
D M
DM
Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Ray
P F
PF
Ao
A
A
Winston
R M
RM
Handyside
A H
AH
D000243:Adenosine Deaminase / Q000235:genetics*; D001483:Base Sequence; D001755:Blastocyst / Q000201:enzymology*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005220:Fathers; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D008040:Genetic Linkage; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics*; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008297:Male; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D014157:Transcription Factors; D014158:Transcription, Genetic; D014960:X Chromosome / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; C054430:ZFY protein, human; C062076:zinc finger protein, X-linked; D005954:Glucosephosphate Dehydrogenase; D000243:Adenosine Deaminase