Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo- and in vitro-derived 4-cell porcine embryos

• Published in: Biology of reproduction 1999-12, Vol.61 (6), p.1460-1467
• Main Authors: Anderson, J.E, Matteri, R.L, Abeydeera, L.R, Day, B.N, Prather, R.S
• Format: Article
• Language: English
• Subjects: alpha-amanitin; Amanitins - pharmacology; Animals; Biological and medical sciences; cell cycle; Cyclin B - genetics; Cyclin B1; DNA, Complementary - analysis; Early stages. Segmentation. Gastrulation. Neurulation; embryo (animal); embryo culture; embryogenesis; Embryology: invertebrates and vertebrates. Teratology; Female; Fundamental and applied biological sciences. Psychology; Male; messenger RNA; Nucleic Acid Synthesis Inhibitors - pharmacology; Polymerase Chain Reaction; Pregnancy; proteins; quantitative analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; Spermatozoa - chemistry; swine; Swine - embryology; transcription (genetics); Zygote - chemistry
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod61.6.1460

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.