|Anderson, J - UNIVERSITY OF MISSOURI|
|Abeydeera, L - UNIVERSITY OF MISSOURI|
|Day, B - UNIVERSITY OF MISSOURI|
|Prather, R - UNIVERSITY OF MISSOURI|
Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 15, 1999
Publication Date: N/A
Interpretive Summary: The use of porcine eggs for in vitro fertilization and maturation offers tremendous potential for advances in reproductive efficiency and dissemination of superior genetic material. Approximately 75% of in vitro fertilized eggs do not develop normally, possibly resulting from delayed or inefficient production of critical embryonic proteins. Suboptimal production of such proteins also may contribute to a natural major loss of pig embryos (approx. 30%) that occurs during very early normal pregnancies. The production of the protein called cyclin B1 may be critical for the embryo's survival as early as the 4-cell stage in pigs. The production of cyclin B1 is controlled by its RNA (cyclin B1 RNA). The present study evaluated the 4-cell pig embryo's ability to produce cyclin B1 RNA. Natural and in vitro-produced embyros were compared. The results showed that the 4-cell pig embryo does not produce its own cyclin B1 RNA regardless of natural or in vitro origins. These findings indicate that embryonic events important for survival may occur at later stages of development and/or involve other regulatory proteins. This information is of use to the scientific community conducting research on early embryonic development.
Technical Abstract: By 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. Following 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 attomoles/embryo, respectively (p<0.03), while the in vitro derived embryos had 20.18 and 7.52 attomoles/embryo, respectively (p<0.03). Treatment with halpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cycli B1 quantities which did not differ from 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.