Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo- and in vitro-derived 4-cell porcine embryos.
Using reverse transcription-competitive polymerase chain reaction (RT-cPCR), the quantity of cyclin B1 transcript present over the maternal to zygotic transition was determined for both in vivo- and in vitro-derived 4-cell porcine embryos. After poly(A) RNA isolation, RT-cPCR was performed on single embryos using an introduced, truncated cyclin B1 DNA competitor. Visualization of embryonic cyclin B1 cDNA and competitor for each reaction allowed a ratio to be formed for use in transcript quantity calculations when compared to cPCR standards. Analysis of in vivo- and in vitro-derived control embryos revealed a decline in cyclin B1 transcripts from 5 to 33 h post-4-cell cleavage (P4CC). The quantity of cyclin B1 for the in vivo-derived embryos at 5 and 33 h P4CC was 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitro-derived embryos had 20.18 and 7.52 attomol/embryo, respectively (P < 0.03). Treatment with alpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cyclin B1 quantities that did not differ from those in the 33-h control embryos, irrespective of time spent in the inhibitor. These findings suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.
Anderson
J E
JE
Department of Animal Science, University of Missouri, Columbia, Missouri 65211 Agricultural Research Service, United States Department of Agriculture, Columbia, Missouri 65211, USA.
Matteri
R L
RL
Abeydeera
L R
LR
Day
B N
BN
Prather
R S
RS
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D019926:Cyclin B / Q000235:genetics*; D056744:Cyclin B1; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013094:Spermatozoa / Q000737:chemistry; D013552:Swine / Q000196:embryology*; D015053:Zygote / Q000737:chemistry*
D000546:Amanitins; D019926:Cyclin B; D056744:Cyclin B1; D018076:DNA, Complementary; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger