Journal
BIOLOGY OF REPRODUCTION
Volume 78, Issue 1, Pages 115-126Publisher
OXFORD UNIV PRESS INC
DOI: 10.1095/biolreprod.107.061366
Keywords
cumulus expansion; FSH; germinal vesicle breakdown; LH; MG132; oocyte; oocyte maturation; porcine; proteasome; ubiquitin
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The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 mu M MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 mu M MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase 1, and 16.2% in metaphase 1, whereas 83.7% of control ova reached metaphase 11 (0% of MG132 reached metaphase 11). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 mu M). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 mu M MG132 and 10 mu M CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 mu M MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (I alpha I) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin-C-terminal hydrolase L3, an important regulator of UPP. RAC1, a UPP-controlled regulator of actin polymerization was maintained at steady levels throughout cumulus expansion. We conclude that proteasomal proteolysis has multiple functions in the progression of oocyte meiosis beyond GV and metaphase I stage, polar body extrusion, and cumulus expansion.
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