Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro.
We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.
Sun
Qing-Yuan
QY
Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Wu
Guang-Ming
GM
Lai
Liangxue
L
Bonk
Arron
A
Cabot
Ryan
R
Park
Kwang-Wook
KW
Day
Billy N
BN
Prather
Randall S
RS
Schatten
Heide
H
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002453:Cell Cycle; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure; D002970:Cleavage Stage, Ovum / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D016200:Maturation-Promoting Factor / Q000037:antagonists & inhibitors; D008540:Meiosis; D008677:Metaphase; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009685:Nuclear Envelope / Q000187:drug effects / Q000648:ultrastructure; D019319:Okadaic Acid / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D008941:Spindle Apparatus / Q000187:drug effects; D013552:Swine
D002843:Chromatin; D004791:Enzyme Inhibitors; D019319:Okadaic Acid; C082868:butyrolactone I; D016200:Maturation-Promoting Factor; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D015107:4-Butyrolactone