Gamete Biology

Regulation of Ubiquitin-Proteasome Pathway on Pig Oocyte Meiotic Maturation and Fertilization

Abstract
Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays essential roles in eukaryotic cell cycle. The main aim of this study was to analyze the functional roles and regulatory mechanisms of UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug-treatment, Western blot, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. MEK inhibitor U0126 and MPF inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of mitogen-activated protein kinase (MAPK) and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro maturation culture. U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk and the synthesis of cyclin B1 stimulated proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited and the dephosphorylation of MAPK and p90rsk and the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also for the first time investigated the subcellular localization of 20 S proteasome alpha subunits at different stages of oocyte and early embryo development. 20 S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, associated with spindle at metaphase I and II, the region between the separating chromosomes and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization and early embryonic mitosis, and it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

Key words: Gamete Biology • Fertilization • Kinases • Meiosis • Ovum

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