Xp42Mpk1 Activation Is Not Required for Germinal Vescicle Breakdown but for Raf Complete Phosphorylation in Insulin-stimulated Xenopus Oocytes

Abstract

Fully grown G 2 -arrested Xenopus oocytes resume meiosis in vitro upon exposure to hormonal stimulation. Progesterone triggers oocyte meiosis resumption through a Ras-independent pathway that involves a p39 Mpk1 activation induced by insulin is dependent upon p39Mos synthesis. Raf complete phosphorylation appears to require the MEK/MAPK pathway activation both in progesterone and insulin-stimulated oocytes.

Kicked by Mos and tuned by MPF—the initiation of the MAPK cascade inXenopusoocytes

“…Nevertheless, the cells exhibit individual concentration variation (as we will also see below). These data are consistent with previous works reporting Mos detection in MAPK inhibiting conditions such as HSP-90-inhibitor geldanamycin incubation (Fisher et al, 2000) or U0126 treatment of oocytes stimulated by insulin through a Rasdependent pathway (Baert et al, 2003). Considering that no significant differences in Mos levels appear in presence or in absence of MAPK activity, MAPK obviously does not contribute as a major component in the initiation of the cascade.…”

“…SDS-PAGE and western blotting: Proteins (the equivalent of a third of an oocyte was loaded per lane) were run on a 12,5% or a 15% modified SDS-PAGE (Baert et al, 2003, Chesnel et al, 1997 for Erk2, Rsk1, cyclin B2, and electroblotted onto nitrocellulose sheet. While the quantity of proteins remains rather constant in the Xenopus oocyte, equal loading and transfer efficiency were checked using the Ponceau red staining.…”

Oocyte Maturation, Mos and Cyclins—A Matter of Synthesis: Two Functionally Redundant Ways to Induce Meiotic Maturation

“…However the inhibition of the MAPK pathway does not block oocyte maturation induced by insulin/IGF-1 or microinjection of active H-RasV12. 29,30 Besides, Xenopus H-RasV12 appears to be a poor in ovo activator of MAPK, probably because Raf is not readily accessible. Finally a dominant negative form of H-Ras is able to block maturation induced by H-RasV12 injection but neither by insulin nor by progesterone.…”

Comparing Pig and Amphibian Oocytes: Methodologies for Aneuploidy Detection and Complementary Lessons for MAPK Involvement in Meiotic Spindle Morphogenesis

“…Once accumulated, Mos phosphorylates MEK, which in turn activates MAPK/Erk by dual phosphorylation of a TEY motif (Ferrell and Bhatt, 1997). In amphibian oocytes, Mosactivated cascade prevents DNA synthesis during meiosis and promotes spindle morphogenesis as well as cytostatic activity present in metaphase-II arrested oocytes (Baert et al, 2003;Bodart et al, 2005;Dupre et al, 2002;Sagata, 1997). In this model, physiological role of MAPKKK Raf, has been minored and Mos is thought to literally hijack the control of the MAPK cascade.…”

“…Next the oocytes are incubated in a low percentage of detergent for permeabilization purposes together with diluted antibodies and revealed as previously mentioned. Treated oocytes are bisected and animal halves are placed on standard slides with mounting medium including glycerol and Hoechst 33342, which reveals chromosomes, and observed under an epifluorescent microscope (Figure 4) (Baert et al, 2003;Bodart et al, 2005;Bodart and Duesbery, 2006).…”