Specific maternal transcripts in bovine oocytes and cleavaged embryos: identification with novel DDRT-PCR methods

Abstract

We used annealing control primer (ACP)-based differential display reverse transcription polymerase chain reaction (DDRT-PCR) to isolate differentially expressed amplicons in bovine germinal vesicle (GV) stage oocytes, 8-cell stage embryos produced in vitro, and blastocyst stage embryos produced in vitro. Four expressed sequence tags (ESTs) of genes that were specifically and predominantly expressed in GV oocytes were cloned and sequenced. We have used a fluorescence monitored real-time quantitative PCR (qPCR) to quantify and analyzed the temporal expression of the target differentially expressed transcripts throughout the preimplantation stages from oocytes to blastocysts. The cloned genes or ESTs all exhibited significant sequence similarity with known bovine genes (98%-100%; DNCL1 and ZP2) or ESTs (81%-97%; FANK1 and GTL3) of other species. As revealed by real-time qRT-PCR, DNCL1, FANK1, GTL3, and ZP2 transcripts were observed in the GV stage oocytes and expression gradually decreased up to the 8-cell stage embryo and the transcripts were not detected in later stages. Similarly, upregulation was observed in GV stage mouse oocytes and metaphase II, suggesting that these four differentially expressed orthologous genes play important roles in early preimplantation, as maternally-derived transcripts.