Characterization of a bovine cDNA encoding citrate synthase, and presence of citrate synthase mRNA during bovine pre-attachment development

Citrate synthase is a key regulatory metabolic enzyme that catalyzes the fi rst step in the tricarboxylic acid (TCA) cycle, the synthesis of citrate fr om acetyl coenzyme A and oxaloacetate. Aerobic metabolism via the TCA cycle is high in bovine embryos at the 4-cell stage then decreases until the com pact morula stage before increasing at the expanded blastocyst stage. This study characterizes the presence of citrate synthase mRNA in bovine preatta chment embryos to determine if a variation in mRNA transcript expression pa tterns is associated with previous reports of the patterns of TCA cycle act ivity. The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect citrate synthase mRNA from the 1-cell to blastocyst stag e of bovine embryo development, and in embryos cultured under either an atm osphere of 5% CO2 in air or 5% CO2/5% O-2/90%N-2. The nucleotide sequence e ncoding citrate synthase was determined from bovine heart cDNA by the rapid amplification of cDNA ends (RACE) technique. This 1455-bp nucleotide fragm ent contained an open reading frame that encoded a deduced protein of 466 a mino acids. The bovine nucleotide sequence was 92.1% and 93.8% identical to the human and porcine coding sequence, respectively. The amino acid sequen ce predicted from the bovine sequence is 95.1% identical to the human seque nce and 96.3% identical to the porcine sequence. The porcine sequence conta ins a stop codon that results in a peptide truncated by 2 amino acids. The detection of citrate synthase transcripts from the 1-cell to blastocyst sta ge demonstrates that the decrease in TCA cycle activity observed following the 4-cell stage is not associated with an absence of citrate synthase mRNA . (C) 2000 Wiley Liss, Inc.