PARENT SESSION
SLIDE SESSION 5: REGULATION OF MEIOSIS
Chairs: Stephen Downs, Sue Varmuza, Michele Calder (Trainee)
Univ Ottawa-Monpetit 202
2:30 PM-4:30 PM

CHARACTERIZATION OF PROTEIN KINASE C-DELTA (PKC-) IN MOUSE OOCYTES DURING MEIOTIC MATURATION.

Viveiros, Maria¹, O'Brien, Marilyn¹, Eppig, John¹, ¹

ABSTRACT- Protein kinase C (PKC) is a family of serine/threonine kinases which play a major role in multiple cellular processes including the regulation of critical cell cycle transitions. PKC- is a "novel" isoform of the PKC family expressed in mouse oocytes. We recently demonstrated that PKC- co-localizes with the meiotic spindle during the anaphase I to telophase transition then progressively associates with the chromosomes at metaphase II (MII) in oocytes from LTXBO mice; however, these oocytes exhibit defects in meiosis I. To determine the role of PKC- during normal meiotic maturation, the dynamics and sub-cellular distribution pattern of this kinase were examined in normal (C57BL/6JxSJL/J) F₁ oocytes. We confirmed previous observations that PKC- is expressed as a doublet (74 and 76 kDa) in oocytes arrested at prophase I with an intact germinal vesicle (GV). In addition, significant expression of a 47 kDa carboxy-terminal (catalytic domain) fragment of PKC- was also detected. Both the holoenzyme and catalytic fragment became phosphorylated coincident with GV breakdown (GVBD) and remained phosphorylated during progression to MII. Suppression of GVBD with a phosphodiesterase inhibitor (milrinone) or a p34cdc2 inhibitor (roscovitine) prevented phosphorylation of PKC-. Moreover, when MII eggs were fertilized or parthenogenetically activated PKC- became dephosphorylated within 1 to 2 hours. Confocal microscopy analysis demonstrated that PKC- was distributed diffusely throughout the cytoplasm of GV-stage oocytes, then localized with the meiotic spindle at MI and eventually associated with the chromosomes at MII. These results indicate that a fast migrating unphosphorylated form of PKC- is characteristic of interphase, while a phosphorylated form is maintained during metaphase and may depend on M-phase promoting factor (MPF) activity. The change in phosphorylation status and subcellular distribution of PKC- suggest that this kinase plays a role during meiotic maturation and might be involved in M-phase entry or exit. Supported by a grant from The National Cancer Institute (CA62392).

KEY WORDS: meiosis, protein kinase C, spindle, oocyte