Abstract:
Multiple signaling pathways are initiated during egg activation and fertilization in mammals. In this project we sought to
determine the dynamics of active src family kinases (SFK). The relative distribution of SFK potential target substrates was
studied by localizing tyrosine phosphorylated proteins (P-Tyr) in mouse eggs undergoing fertilization during progression from meiosis (MII) through to extrusion of the second polar body. Female CF1 mice were superovulated with PMSG and hCG and mated to B6D2F1 males and oviductal oocytes were collected 14-16 hours post-hCG and fixed in microtubule stabilization
buffer with 2% formalin. Eggs were labeled for indirect immunofluoresence detection with appropriate primary antibodies
against activated SFK or P-Tyr and all samples were counter stained for detection of f-actin (phalloidin), microtubules (alpha-tubulin) and chromatin (Hoechst). Activated SFK labeling was evenly distributed throughout the cytoplasm of eggs from MII through second polar body extrusion with no apparent changes in distribution during the completion of meiosis. In contrast, P-Tyr labeled proteins were enriched in the egg cortex and appeared to be polarized to the region overlying the meiotic spindle in most oocytes. There was pronounced labeling of activated SFK to the MII spindle that persisted in mid-body microtubules
during polar body extrusion, while P-Tyr was restricted to a small region of the spindle poles. These findings suggest that
active SFKs are sequestered within the meiotic spindle components of mouse oocytes and further studies are ongoing to
determine the role of SFKs in mouse fertilization and early embryo development.
top