|Matteri, Robert - Bob|
Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/4/2001
Publication Date: N/A
Citation: Interpretive Summary: The use of porcine eggs for in vitro fertilization (IVF) offers tremendous potential for advances in reproductive efficiency and dissemination of superior genetic material. A major obstacle of porcine IVF is the abnormal development of approximately 75% of in vitro fertilized eggs, 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 cdc25c may be critical for the embryo's survival. The production of cdc25c is controlled by its RNA (cdc25c RNA). The present study evaluated the 4-cell pig embryo's ability to produce cdc25c RNA. Natural and in vitro-produced embryos were compared. The results indicated that the pig embryo produces its own cdc25c RNA between 10 and 18 hours after entering the 4-cell stage regardless of natural or in vitro origins. These findings indicate that embryonic events important for survival of in vitro-derived embryos 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: To gain a better understanding of the molecular differences that may contribute to cleavage arrest and the poorer development associated with laboratory produced embryos, a series of experiments were conducted to quantitate the message levels of the cell cycle controller cdc25c, over the maternal to zygotic transition (MZT) in 4-cell in vivo- and in vitro-derived porcine embryos. The experiments were designed to measure both maternal and embryonic derived cdc25c transcripts by quantitative reverse transcription-competitive polymerase chain reaction (RT-cPCR), determine the point of the transition to zygotic genome activation, and study the interaction between initial embryonic transcription and maternal cdc25c degradation. Analysis of in vivo- and in vitro-derived embryos revealed no difference in cdc25c message level for any of the times P4CC (P>0.05). Comparison of control embryos from 5- to 33-h P4CC revealed a reduction in transcript quantities in the 10-h P4CC group that was maintained at later time points (P>0.05). Embryos cultured in the RNA polymerase inhibitor, alpha-amanitin, from cleavage to 5-, 10-, 18-, 25-, or 33-h P4CC displayed no difference in cdc25c levels when compared to controls at similar time points (P>0.05). However, if embryos were first exposed to alpha-amanitin after 18-h P4CC with this treatment continuing to 33-h, the levels of cdc25c transcript were reduced (P<0.04) when compared to those embryos that were first exposed to the inhibitor at either 5- or 10-h P4CC. This finding and the comparison of these same embryos to the 0-33-h alpha-amanitin and control groups allowed us to conclude that cdc25c transcription began between 10- and 18-h P4CC, with the degradation of maternal cdc25c message dependent on transcriptional initiation.