MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes

Marie Hélène Verlhac, Henri De Pennart, Bernard Maro, Melante H. Cobb, Hugh J. Clarke

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Abstract

Using antisera generated against sequences conserved between the ERK1- and the ERK2-encoded species of mitogen-activated protein (MAP) kinases of the rat, species of approximate Mr 42 and 44 kDa were identified in mouse oocytes. When oocytes underwent meiotic maturation, both species displayed a retarded electrophoretic mobility, consistent with modification by phosphorylation. The slow-migrating forms first appeared after the oocytes had entered metaphase, and their appearance was sensitive to inhibitors of protein synthesis or phosphorylation. These forms remained throughout maturation and in oocytes arrested at metaphase II. Following oocyte activation, which induces a transition to interphase, the slow-migrating forms were replaced by the fast-migrating forms observed in prophase oocytes. MAP kinase activity also increased after oocytes entered metaphase, and this increase required protein synthesis and phosphorylation. To investigate the intracellular distribution of the immunoreactive species, spindles were purified from metaphase II eggs. Both the 42- and the 44-kDa species were detected in immunoblots, and bright staining of the spindle poles was observed by immunofluorescence. When intact oocytes undergoing maturation were examined by immunofluorescence, foci of staining were initially detected on opposing sides of the condensing chromosomes and then became congregated at each pole of the first meiotic spindle. No localized staining was observed during the first meiotic division, but stained foci were present at the poles of the second meiotic spindle. In addition, several cytoplasmic foci of staining often could be seen. When oo-cytes were exposed to taxol, which permits nonspindle microtubule-organizing centers (MTOCs) present in the cytoplasm to nucleate micro tubule assembly, the cytoplasmic foci labeled by the MAP kinase antibodies were found to contain tubulin. We conclude that mouse oocytes contain 42- and 44-kDa species of MAP kinase and that, after maturing oocytes enter metaphase, MAP kinase activity is stimulated by means of a process requiring protein synthesis and phosphorylation. MAP kinase is present in the spindle and is specifically associated with the MTOCs present at the spindle poles and in the cytoplasm. Evidence from cell-free systems suggests that the alterations in MTOC activity that normally occur at metaphase in oocytes may be regulated by MAP kinase. The association of MAP kinase with MTOCs provides a potential structural basis for this cell cycle-dependent change in MTOC activity.

Original languageEnglish (US)
Pages (from-to)330-340
Number of pages11
JournalDevelopmental Biology
Volume158
Issue number2
StatePublished - 1993

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Microtubule-Organizing Center
Metaphase
Mitogen-Activated Protein Kinases
Oocytes
Phosphorylation
Spindle Poles
Staining and Labeling
Spindle Apparatus
Fluorescent Antibody Technique
Cytoplasm
Prophase
Protein Synthesis Inhibitors
Cell-Free System
Conserved Sequence
Interphase
Tubulin
Paclitaxel
Eggs
Immune Sera
Cell Cycle

ASJC Scopus subject areas

  • Developmental Biology

Cite this

MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. / Verlhac, Marie Hélène; De Pennart, Henri; Maro, Bernard; Cobb, Melante H.; Clarke, Hugh J.

In: Developmental Biology, Vol. 158, No. 2, 1993, p. 330-340.

Research output: Contribution to journalArticle

Verlhac, Marie Hélène ; De Pennart, Henri ; Maro, Bernard ; Cobb, Melante H. ; Clarke, Hugh J. / MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. In: Developmental Biology. 1993 ; Vol. 158, No. 2. pp. 330-340.
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abstract = "Using antisera generated against sequences conserved between the ERK1- and the ERK2-encoded species of mitogen-activated protein (MAP) kinases of the rat, species of approximate Mr 42 and 44 kDa were identified in mouse oocytes. When oocytes underwent meiotic maturation, both species displayed a retarded electrophoretic mobility, consistent with modification by phosphorylation. The slow-migrating forms first appeared after the oocytes had entered metaphase, and their appearance was sensitive to inhibitors of protein synthesis or phosphorylation. These forms remained throughout maturation and in oocytes arrested at metaphase II. Following oocyte activation, which induces a transition to interphase, the slow-migrating forms were replaced by the fast-migrating forms observed in prophase oocytes. MAP kinase activity also increased after oocytes entered metaphase, and this increase required protein synthesis and phosphorylation. To investigate the intracellular distribution of the immunoreactive species, spindles were purified from metaphase II eggs. Both the 42- and the 44-kDa species were detected in immunoblots, and bright staining of the spindle poles was observed by immunofluorescence. When intact oocytes undergoing maturation were examined by immunofluorescence, foci of staining were initially detected on opposing sides of the condensing chromosomes and then became congregated at each pole of the first meiotic spindle. No localized staining was observed during the first meiotic division, but stained foci were present at the poles of the second meiotic spindle. In addition, several cytoplasmic foci of staining often could be seen. When oo-cytes were exposed to taxol, which permits nonspindle microtubule-organizing centers (MTOCs) present in the cytoplasm to nucleate micro tubule assembly, the cytoplasmic foci labeled by the MAP kinase antibodies were found to contain tubulin. We conclude that mouse oocytes contain 42- and 44-kDa species of MAP kinase and that, after maturing oocytes enter metaphase, MAP kinase activity is stimulated by means of a process requiring protein synthesis and phosphorylation. MAP kinase is present in the spindle and is specifically associated with the MTOCs present at the spindle poles and in the cytoplasm. Evidence from cell-free systems suggests that the alterations in MTOC activity that normally occur at metaphase in oocytes may be regulated by MAP kinase. The association of MAP kinase with MTOCs provides a potential structural basis for this cell cycle-dependent change in MTOC activity.",
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T1 - MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes

AU - Verlhac, Marie Hélène

AU - De Pennart, Henri

AU - Maro, Bernard

AU - Cobb, Melante H.

AU - Clarke, Hugh J.

PY - 1993

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N2 - Using antisera generated against sequences conserved between the ERK1- and the ERK2-encoded species of mitogen-activated protein (MAP) kinases of the rat, species of approximate Mr 42 and 44 kDa were identified in mouse oocytes. When oocytes underwent meiotic maturation, both species displayed a retarded electrophoretic mobility, consistent with modification by phosphorylation. The slow-migrating forms first appeared after the oocytes had entered metaphase, and their appearance was sensitive to inhibitors of protein synthesis or phosphorylation. These forms remained throughout maturation and in oocytes arrested at metaphase II. Following oocyte activation, which induces a transition to interphase, the slow-migrating forms were replaced by the fast-migrating forms observed in prophase oocytes. MAP kinase activity also increased after oocytes entered metaphase, and this increase required protein synthesis and phosphorylation. To investigate the intracellular distribution of the immunoreactive species, spindles were purified from metaphase II eggs. Both the 42- and the 44-kDa species were detected in immunoblots, and bright staining of the spindle poles was observed by immunofluorescence. When intact oocytes undergoing maturation were examined by immunofluorescence, foci of staining were initially detected on opposing sides of the condensing chromosomes and then became congregated at each pole of the first meiotic spindle. No localized staining was observed during the first meiotic division, but stained foci were present at the poles of the second meiotic spindle. In addition, several cytoplasmic foci of staining often could be seen. When oo-cytes were exposed to taxol, which permits nonspindle microtubule-organizing centers (MTOCs) present in the cytoplasm to nucleate micro tubule assembly, the cytoplasmic foci labeled by the MAP kinase antibodies were found to contain tubulin. We conclude that mouse oocytes contain 42- and 44-kDa species of MAP kinase and that, after maturing oocytes enter metaphase, MAP kinase activity is stimulated by means of a process requiring protein synthesis and phosphorylation. MAP kinase is present in the spindle and is specifically associated with the MTOCs present at the spindle poles and in the cytoplasm. Evidence from cell-free systems suggests that the alterations in MTOC activity that normally occur at metaphase in oocytes may be regulated by MAP kinase. The association of MAP kinase with MTOCs provides a potential structural basis for this cell cycle-dependent change in MTOC activity.

AB - Using antisera generated against sequences conserved between the ERK1- and the ERK2-encoded species of mitogen-activated protein (MAP) kinases of the rat, species of approximate Mr 42 and 44 kDa were identified in mouse oocytes. When oocytes underwent meiotic maturation, both species displayed a retarded electrophoretic mobility, consistent with modification by phosphorylation. The slow-migrating forms first appeared after the oocytes had entered metaphase, and their appearance was sensitive to inhibitors of protein synthesis or phosphorylation. These forms remained throughout maturation and in oocytes arrested at metaphase II. Following oocyte activation, which induces a transition to interphase, the slow-migrating forms were replaced by the fast-migrating forms observed in prophase oocytes. MAP kinase activity also increased after oocytes entered metaphase, and this increase required protein synthesis and phosphorylation. To investigate the intracellular distribution of the immunoreactive species, spindles were purified from metaphase II eggs. Both the 42- and the 44-kDa species were detected in immunoblots, and bright staining of the spindle poles was observed by immunofluorescence. When intact oocytes undergoing maturation were examined by immunofluorescence, foci of staining were initially detected on opposing sides of the condensing chromosomes and then became congregated at each pole of the first meiotic spindle. No localized staining was observed during the first meiotic division, but stained foci were present at the poles of the second meiotic spindle. In addition, several cytoplasmic foci of staining often could be seen. When oo-cytes were exposed to taxol, which permits nonspindle microtubule-organizing centers (MTOCs) present in the cytoplasm to nucleate micro tubule assembly, the cytoplasmic foci labeled by the MAP kinase antibodies were found to contain tubulin. We conclude that mouse oocytes contain 42- and 44-kDa species of MAP kinase and that, after maturing oocytes enter metaphase, MAP kinase activity is stimulated by means of a process requiring protein synthesis and phosphorylation. MAP kinase is present in the spindle and is specifically associated with the MTOCs present at the spindle poles and in the cytoplasm. Evidence from cell-free systems suggests that the alterations in MTOC activity that normally occur at metaphase in oocytes may be regulated by MAP kinase. The association of MAP kinase with MTOCs provides a potential structural basis for this cell cycle-dependent change in MTOC activity.

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