Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Abstract

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturationpromoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturationpromoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These MATERIALS AND METHODSOocyte Collection, Culture, and Microinjection. B6C3 F1 female mice were injected intraperitoneally with 5 units of pregnant mare serum gonadotropin. Cumulus-enclosed oocytes were isolated 45-48 hr later and cultured in modified Whitten's medium (21) containing 0.4% BSA and 100 ,uM IBMX. For microinjection, oocytes were transferred to modified Whitten's media with Hepes (PGC Scientific, Gaithersburg, MD; specialty media) containing 5% fetal calf serum and 100 ,tM IBMX. Oocytes were injected in the cytoplasm with '10 pl of RNA (1 gg/,ul) in Dulbecco's PBS. Injected or uninjected oocytes were washed in modified Whitten's medium containing IBMX and incubated at 38.5°C in humidified 5% CO2 in air.RNA was synthesized in vitro from the mos-containing plasmid, PHTX, with T7 RNA polymerase and the RNA cap analog, 7-methyl-G(5')ppp(5')G (22). Gain-of-function mek* RNA (17) was synthesized in vitro using the same method. The RNA was precipitated twice in ethanol, then resuspended in Dulbecco's PBS.Immunofluorescence and Confocal Microscopic Analysis.Oocytes were stripped of their zonae pellucidae with acidic Tyrode's solution (pH 2.5), fixed with 1.8% paraformaldehyde in PBS (fixing solution) for 40 min at room temperature, and made permeable with 1% Triton X-100 in fixing solution for 40 min. Oocytes were washed with 0.1% Tween-20 in PBS for 20 min and incubated with 3% BSA, 10% goat serum, andAbbreviations: MAPK, mitogen-activated protein kinase; MT, microtubule; MEK, MAPK kinase; MPF, maturation-promoting factor;IBMX, 3-isobutyl-1-methylxanthine; GV, germinal vesicle; DAPI, 4',6-diamino-2-phenylindol; GVBD, GV breakdown. 4730The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S C. §1734 solely to indicate this fact.

Activation of the Mitogen-activated Protein Kinase ERK1 during Meiotic Progression of Mouse Pachytene Spermatocytes

“…When cAMP levels drop there is an increase in the activity of MPF and MAPKs resulting in the germinal vesicle breakdown and progression through the first meiotic division. On the contrary of what has been observed in Xenopus oocyte maturation, activity of Mos is dispensable for the G 2 /M transition after prophase I in both female and male mice (17,18), even though Mos might participate in some aspects of meiotic maturation in mouse oocytes (19,20). The oocyte proceeds to the second meiotic division and arrests at metaphase II with the sister chromatids aligned on the spindle, due to the concerted action of Mos and MAPKs, which, at this stage, prevent cyclin destruction and consequent MPF inactivation (5).…”

Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase II arrest

“…In mos -/-mouse oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex. As a result, in these oocytes the first meiotic division resembles mitotic cleavage and produces two cells of the same size [22]. Inhibition of protein synthesis frequently results in a similar phenotype [23] in starfish oocytes.…”

MOS, aneuploidy and the ploidy cycle of cancer cells

“…At early meiosis I, Mos induces nucleation of multiple centrosomes in the vicinity of condensing bivalents. Further activation of mitosis-promoting factor Cdk1/cyclin B by Mos triggers contraction of bivalents and signals the multiple centrosomal microtubule arrays to form a bipolar prespindle (Choi et al, 1996). Here, the bipolar spindle of the first meiotic division is responsible for segregation of the paired homologs to two opposite poles along with the cohesed sister chromatidsthe latter a condition of somatic reduction as well (see above; Kondrashov, 1994).…”