Characterization of a bovine cDNA encoding citrate synthase, and presence of citrate synthase mRNA during bovine pre-attachment development
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abstract
Citrate synthase is a key regulatory metabolic enzyme that catalyzes the first step in the tricarboxylic acid (TCA) cycle, the synthesis of citrate from 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 compact morula stage before increasing at the expanded blastocyst stage. This study characterizes the presence of citrate synthase mRNA in bovine preattachment embryos to determine if a variation in mRNA transcript expression patterns is associated with previous reports of the patterns of TCA cycle activity. The reverse transcription-polymerase chain reaction (RT- PCR) method was used to detect citrate synthase mRNA from the 1-cell to blastocyst stage of bovine embryo development, and in embryos cultured under either an atmosphere of 5% CO2 in air or 5% CO2/5% O2/90%N2. The nucleotide sequence encoding citrate synthase was determined from bovine heart cDNA by the rapid amplification of cDNA ends (RACE) technique. This 1455-bp nucleotide fragment contained an open reading frame that encoded a deduced protein of 466 amino acids. The bovine nucleotide sequence was 92.1% and 93.8% identical to the human and porcine coding sequence, respectively. The amino acid sequence predicted from the bovine sequence is 95.1% identical to the human sequence and 96.3% identical to the porcine sequence. The porcine sequence contains 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 stage demonstrates that the decrease in TCA cycle activity observed following the 4-cell stage is not associated with an absence of citrate synthase mRNA.
