Comparative effects of insulin on the activation of the Raf/Mos-dependent MAP kinase cascade in vitellogenic versus postvitellogenic Xenopus oocytes

Xenopus postvitellogenic oocytes resume meiosis in vitro upon exposure to insulin or insulin like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage TV (600 mu m < diameter < 1000 mu m) oocytes cannot, The aim of the present study was to determine which event(s) of the transduction pathway ham 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 an pp39(mos) synthesis 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 active raf mRNA 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 TV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the ''Mos synthesis machinery.'' The present results show that incompetence of Xenopus stage TV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39(mos) synthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation. (C) 1997 Academic Press.