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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.
( Sabina Bhandari ),( Jayaswori Sharma ),( Sarbesh Rizal ),( Young-joo Yi ),( Gaurishankar Manandhar ) 한국축산학회(구 한국동물자원과학회) 2021 한국축산학회지 Vol.63 No.1
Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 μg/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.