Comparative Effects of Insulin on the Activation of the Raf/Mos-Dependent MAP Kinase Cascade in Vitellogenic versus PostvitellogenicXenopusOocytes

Abstract

Xenopuspostvitellogenic oocytes resume meiosisin vitroupon exposure to insulin or insulin-like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage IV (600 μm < diameter < 1000 μm) oocytes cannot. The aim of the present study was to determine which event(s) of the transduction pathway from IGF-1 receptor to maturation-promoting factor (MPF) activation is deficient in the small, vitellogenic, oocytes to explain their inability to undergo germinal vesicle breakdown (GVB) after insulin treatment. We thus analyzed the effect of insulin on the Ras/Raf-dependent mitogen-activated protein kinase cascade because of its crucial role prior to MPF activation. The effect of insulin on pp39mossynthesis in stage IV oocytes was also studied since this protein kinase participates in the mitogen-activated protein kinase (MAPK) pathway as a MAPKK kinase like Raf. Contrary to what is observed in postvitellogenic oocytes, MAPK was not activated in insulin-treated stage IV oocytes even 20 hr after the stimulation. This was not caused by the absence of MAPK activators like MEK (MAPKK), Raf, or Ras, but rather by the inability of insulin to activate Ras. Interestingly, injection of constitutively activerafmRNA as well as oncogenic Ras protein, Ha-Ras lys12, in stage IV oocytes resulted in MAPK activation, whereas neither Mos accumulation nor GVB occurred, suggesting that the Ras → Raf → MAPKK → MAPK cascade was functional but that MAPK activation alone was not sufficient for the mitogenic signal to proceed further down in the pathway leading to MPF activation. Treatment of stage IV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the “Mos synthesis machinery.” The present results show that incompetence ofXenopusstage IV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39mossynthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation.