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 po rcine embryos. After poly(A) RNA isolation, RT-cPCR was performed on single embryos using an introduced, truncated cyclin B1 DNA competitor. Visualiza tion of embryonic cyclin B1 cDNA and competitor for each reaction allowed a ratio to be formed for use in transcript quantity calculations when compar ed to cPCR standards. Analysis of in vivo- and in vitro-derived control embryos revealed a declin e 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 wa s 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitr o-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 findin gs suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.