Specific maternal transcripts in bovine oocytes and cleavaged embryos: identification with novel DDRT-PCR methods.
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.
Hwang
Kyu-Chan
KC
Department of Animal Science, Chungbuk National University, Gaesin-dong, Cheongju, Chungbuk, South Korea.
Park
Sae-Young
SY
Park
Se-Pill
SP
Lim
Jin Ho
JH
Cui
Xiang-Shun
XS
Kim
Nam-Hyung
NH
D000818:Animals; D002417:Cattle; D002970:Cleavage Stage, Ovum / Q000378:metabolism*; D004527:Egg Proteins / Q000235:genetics; D020224:Expressed Sequence Tags / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D051379:Mice; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism*
D004527:Egg Proteins; D012333:RNA, Messenger