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Division of Animal Sciences,3 University of Missouri-Columbia, Columbia, Missouri 65211
Research Center for Transgenic Cloned Pigs,4 Chungnam National University, Daejeon, Korea
Institute of Animal Physiology & Genetics,5 Czech Academy of Sciences, 277 21 Lib
chov, Czech Republic
Department of Obstetrics & Gynecology,6 University of Missouri-Columbia, Columbia, Missouri 65211
ABSTRACT
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 µM 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 µM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20–200 µM). 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 µM MG132 and 10 µM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 µM 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
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.
cumulus expansion, FSH, germinal vesicle breakdown, LH, MG132, oocyte, oocyte maturation, porcine, proteasome, ubiquitin
1Supported by National Research Initiative (NRI) Competitive Grants #2002-35203-12237 and 2007-35203-18274 from the US Department of Agriculture (USDA) Cooperative State Research, Education, and Extension Service, USDA-NRI Discovery Award 2005, and seed funding from the Food for the 21st Century Program of the University of Missouri-Columbia. Y.-J.Y. and C.-S.P. were supported by grant R11-2002-100-00000-0 from ERC Program of Korea Science & Engineering Foundation (KOSEF). E.N. was supported by award 305/05/0960 from the Grant Agency of the Czech Republic.
Correspondence: 2Peter Sutovsky, Associate Professor, University of Missouri-Columbia, S141 ASRC, 920 East Campus Dr., Columbia, MO 65211-5300. FAX: 573 884 5540; e-mail: SutovskyP{at}missouri.edu
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