Quantification of hexokinase mRNA in mouse blastocysts by competitive reverse transcriptase polymerase chain reaction.
Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription-nested polymerase chain reaction (RT-nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT-nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95 x 10(-18) g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation.
Johnson
M D
MD
Department of Gynecology and Obstetrics, Stanford University School of Medicine, CA 94305-5317, USA.
Batey
D W
DW
Behr
B
B
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000201:enzymology* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D006593:Hexokinase / Q000235:genetics*; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D006593:Hexokinase; D005947:Glucose