Assessment by differential display-RT-PCR of mRNA transcript transitions and alpha-amanitin sensitivity during bovine preattachment development.
The objectives of this study were to compare patterns of mRNA expression, investigate the onset of transcription, and isolate stage-specific and alpha-amanitin-sensitive mRNAs during early bovine development by differential-display-reverse transcription-polymerase chain reaction (DD-RT-PCR). Embryos representing a preattachment developmental series from the 1-cell to the expanded/hatched blastocyst stage were cultured in synthetic oviduct fluid medium + citrate and amino acids (cSOFMaa) with and without alpha-amanitin (100 microg/mL) for 4 and 12 hr. mRNA profiles were displayed by DD-RT-PCR using 5' primers A and N. Total conserved cDNA banding patterns varied according to embryo stage with cDNA band numbers declining during early cleavage stages compared to oocyte values and then increasing in total number from the 6-8-cell stage through to the blastocyst stage. A cDNA banding pattern was established at the 8-16-cell stage that was largely unchanged through to the blastocyst stage. These findings with respect to cDNA banding patterns were conserved between oligo primer sets and experimental replicates. alpha-Amanitin sensitivity was first detected at the 2-5-cell stage but became predominant following the 6-8-cell stage of development to eventually affect the appearance of up to 40% of all cDNA bands by the blastocyst stage. A 12 hr alpha-amanitin treatment was required to effectively block (3)H-uridine incorporation into mRNA in blastocyst stage embryos. Several stage-specific and alpha-amanitin-sensitive cDNAs were isolated and they will be a focus for future studies. In conclusion, DD-RT-PCR is an effective tool for contrasting gene expression patterns and isolating uncharacterized mRNA transcripts during bovine early development. Mol. Reprod. Dev. 55:152-163, 2000.
Natale
D R
DR
Department of Obstetrics and Gynaecology, The University of Western Ontario, London, Ontario, Canada.
Kidder
G M
GM
Westhusin
M E
ME
Watson
A J
AJ
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst; D002417:Cattle; D004622:Embryo, Mammalian / Q000378:metabolism*; D047108:Embryonic Development; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D009865:Oocytes; D011247:Pregnancy; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014529:Uridine / Q000378:metabolism
D000546:Amanitins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D012319:RNA Polymerase II; D014529:Uridine