PARENT SESSION
SLIDE SESSION 17: PREIMPLANTATION DEVELOPMENT
Chairs: Richard Leach, Carol Brenner, David Natale (Trainee)
Arts Hall 026
1:30 PM-3:30 PM

p38 RELATED/ACTIVATED KINASE (PRAK) TRANSLOCATES FROM NUCLEAR TO PERI-NUCLEAR REGIONS JUST PRIOR TO BLASTOCYST FORMATION IN THE MOUSE AND COW.

Natale, David¹, Watson, Andrew ¹, ¹

ABSTRACT- We have isolated an embryonic cDNA (470bp designated C13) following the application of a Differential Display-Reverse Transcription-Polymerase Chain Reaction analysis applied to 1-cell and blastocyst stage bovine pre-implantation embryos. Our initial selection of C13 was based upon its apparent specificity to the 1-cell zygote. Subsequent cloning and sequencing of C13 indicated a high sequence similarity (85-90% over 380bp) to human sequence of a p38-signaling pathway member, PRAK. p38 is a member of the MAP kinase superfamily and its activation in response to cellular stress leads to the direct phosphorylation of PRAK resulting in the activation of the small heat shock protein, hsp27. The role of this signaling pathway is unexplored during preimplantation development. Expression of PRAK mRNA in bovine and murine preimplantation staged embryos was confirmed by RT-PCR with primers designed against the human nucleotide sequence and to partially overlap with C13. In contrast to initial DD-RT-PCR results, mRNA encoding PRAK were detected in bovine and murine embryos in all cleavage stages from the mature oocyte through to the blastocyst stage. PRAK protein localization in early murine and bovine embryos was investigated by whole-mount indirect immunofluorescence using antiserum against recombinant PRAK (gift from J. Han). PRAK staining was undetectable in mature oocytes but was observed in the nuclei of blastomeres at the 2-cell, 4-cell, 8-cell and compacting morula stages. This nuclear localization pattern was not maintained at the blastocyst stage, as PRAK was primarily confined to perinuclear regions of each blastomere at this stage. Future experiments include characterization of expression and activation of the p38-signaling pathway in embryos to investigate the functional significance of this dramatic shift in PRAK cellular localization during preimplantation development. Research supported by NSERC Canada.

KEY WORDS: preimplantation, embryo, signaling, gene expression