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Yi, Young-Joo,Nagyova, Eva,Manandhar, Gaurishankar,Proch?yka, Radek,Sutovsky, Miriam,Park, Chang-Sik,Sutovsky, Peter 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
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 5 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 10 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 (lVM), followed by 22 h of culture with or without MG132. Treatment with 10 μM MGI32 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 15 increased progressively to >90% with increased concentration of MG132 (20-200μM). 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 10μM CE underwent GVBD 20 despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10μM 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 (IαI) 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.
Glasa, Miroslav,Predajna, Lukas,Soltys, Katarina,Sihelska, Nina,Nagyova, Alzbeta,Wetzel, Thierry,Sabanadzovic, Sead The Korean Society of Plant Pathology 2017 Plant Pathology Journal Vol.33 No.1
Grapevine rupestris stem pitting-associated virus (GRSPaV) is a worldwide-distributed pathogen in grapevines with a high genetic variability. Our study revealed differences in the complexity of GRSPaV population in a single host. A single-variant GRSPaV infection was detected from the SK30 grapevine plant. On the contrary, SK704 grapevine was infected by three different GRSPaV variants. Variant-specific RT-PCR detection protocols have been developed in this work to study distribution of the three different variants in the same plant during the season. This study showed their randomized distribution in the infected SK704 grapevine plant. Comparative analysis of full-length genome sequences of four Slovak GRSPaV isolates determined in this work and 14 database sequences showed that population of the virus cluster into four major phylogenetic lineages. Moreover, our analyses suggest that genetic recombination along with point mutations could play a significant role in shaping evolutionary history of GRSPaV and contributed to its extant genetic diversification.