PARENT SESSION
Preimplantation Embryo/Fetal Development

EXPRESSION OF 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE mRNA ISOFORMS IN MOUSE OOCYTES AND EMBRYOS DEVELOPED IN VIVO AND IN VITRO.

Winger, Quinton¹, Lane, Michelle¹, Gardner, David¹, ¹

ABSTRACT- 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a bifunctional enzyme that catalyses the synthesis and degradation of fructose 2,6-bisphosphate (F2,6BP). F2,6BP is a potent activator of phosphofructokinase, a key regulatory enzyme of glycolysis. Several isozymes of PFK-2 have been detected in mammals, and they are unique based on their tissue distribution, kinase/bisphosphatase activity ratio and in their regulation. In the human and rat, Pfk-2 has been localized to at least 4 different gene regions on different chromosomes producing distinct mRNA transcripts. In addition, multiple transcripts originating from the same gene have been identified as products of alternate start sites and splicing. Interestingly, the liver and muscle mRNA isoforms are both transcribed from the same gene located on the X-chromosome. The physiology of metabolism in the mouse embryo switches from a carboxylic acid based to a glucose based metabolism around the time of compaction. It is also known that PFK-2 isozymes have different kinase/bisphosphatase activity ratios in some forms, such as the placenta/inducible form, which has a greater kinase activity. Therefore, a greater level of placenta/inducible PFK-2 in mouse embryos would increase the F2,6BP concentration which would have a significant role in the regulation of the glycolytic activity in embryos. The goal of this study was to determine which Pfk-2 isoform transcripts are present during mouse embryo development. This study compared mRNA transcripts encoding Pfk-2 in B6CBAF1 and CF1 mouse embryos grown in vivo or in G1/G2 culture media. Transcripts were detected by RT-PCR using primers designed to differentiate between the mouse liver, muscle, heart and placenta/inducible form of Pfk-2. Heart, muscle and the placenta/inducible Pfk-2 transcripts were detected in mRNA isolated from oocytes, 2-cell embryos and blastocysts for both strains of mice. The liver isoform was not detected in any of these stages from either strain. Therefore the expression of the muscle mRNA and lack of expression of the liver mRNA shows the embryonic control of expression of this isoform from the X-chromosome. Furthermore, the expression patterns detected for these transcripts were the same for embryos produced in vivo and in culture. This research is the first step in understanding the regulation of glycolysis by Pfk-2 in early embryos.

KEY WORDS: phosphofructo-2-kinase/fructose-2,6-bisphosphatase, metabolism, embryo, phophofructokinase