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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.
Hao, Yan-Hong,Lai, Liang-Xue,Liu, Zhong-Hua,Im, Gi-Sun,Wax, David,Samuel, Melissa,Murphy, Clifton N.,Sutovsky, Peter,Prather, Randall S. Wiley Subscription Services, Inc., A Wiley Company 2006 Molecular reproduction and development Vol.73 No.1
<P>Parthenogenetically activated (PA) embryos exhibit delayed development, a lower blastocyst rate, and less successful development in vitro compared to in vitro fertilized (IVF) embryos. To investigate the possible mechanisms for unsuccessful parthenogenetic development, this study analyzed the chromosome abnormalities and developmental potential of porcine PA embryos. Mature oocytes were electrically activated and cultured in Porcine Zygote Medium-3 (PZM<SUB>3</SUB>) supplemented with 3 mg/ml BSA for 6, 7, or 8 days. The percentage of PA blastocysts was lower than that of IVF embryos on days 6 and 7 (16.4 ± 7.4 vs. 28.7 ± 3.7; 10.9 ± 2.8 vs. 21.5 ± 4.7, P < 0.05; respectively), and the PA blastocysts had significantly fewer nuclei than IVF blastocysts (23.2 ± 1.8 vs. 29.7 ± 0.8; 29.7 ± 3.3 vs. 32.0 ± 2.4, P < 0.05). The percentage of abnormal PA embryos (including embryos with condensed nuclei, arrested embryos and fragmented embryos) was higher than that of IVF embryos (PA: 52.9 ± 12.8 vs. 16.4 ± 7.4 on day 6), and increased with culture time (71.9 ± 12.1 vs. 10.9 ± 2.8. on day 7,and 75.0 ± 22.6 vs. 12.1 ± 2.3 on day 8, P < 0.05). The Day-6 PA blastocysts (n = 147) were divided into three classes according to the total number of nuclei (<20, 20–39, >40) and into three groups according to the morphological diameter (<150, 150–180, >180 µm). Of the haploid blastocysts, 56.1% had less than 20 nuclei, and 71.5% were less than 150 µm in diameter. Of all (114) blastocysts suitable for analysis, 55.5% displayed chromosomal abnormalities. Among chromosomal abnormalities in PA blastocysts, haploid blastocysts were most prevalent (43.6%), while polyploidy (4.4%) and mixoploidy (7.7%) embryos were less prevalent. Chromosomal abnormalities of porcine PA embryos might contribute to a higher rate of abnormal embryonic development. We suggest that a careful consideration should be given when using the blastocysts with smaller size, and establishing the optimum culture condition for PA embryos development in vitro. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.</P>