Submitted March 8, 2007
Returned for revision May 26, 2007
Accepted October 8, 2007
Gamete Biology
Proteolytic Activity of the 26S Proteasome Is Required for the Meiotic Resumption, Germinal Vesicle Breakdown, and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes Matured In Vitro
Young-Joo Yi ,
Eva Nagyova ,
Gaurishankar Manandhar ,
Radek Procházka ,
Miriam Sutovsky ,
Chang-Sik Park ,
and
Peter Sutovsky *
* To whom correspondence should be addressed. E-mail: sutovskyp{at}missouri.edu.
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 (IVM), 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 while 83.7% of control ova reached metaphase II (0% metaphase II in MG132). 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 1st 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 to 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid (HA) and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin-C-terminal hydrolase L3 (UCHL3), 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.
Key words:
Proteasome •
cumulus expansion •
germinal vesicle breakdown •
oocyte maturation •
ubiquitin
