Effects of different post-activation conditions on the developmental competence of in vitro matured porcine oocytes

Pantu Kumar Roy,Xun Fang,Bahia MS Hassan,Jong Ki Cho 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

The objective of this study was to establish the effect of post-activation treatment with cytoskeletal regulators of CB, CB+CHX, CB+DC, CB+6’DMAP on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT embryos were produced by using in vitro matured pig oocytes and treated for 4 h after electric activation with cytochalasins B (7.5 μg/ml), CB+cycloheximide (10 μg/ml), CB+demecolcine (0.4 μg/ml), and CB+2mM 6-Dimethylaminopurine. Post-activation treatment of PA oocytes with CB, CB+CHX, CB+DC and CB+6’DMAP no significant differences were found in embryo cleavage (83.2~91.5%), mean cell number of blastocysts (40.6~ 42.3% cells/blastocyst) but significantly (P<0.05) differences blastocyst formation (28.6~36.4%). When PA oocytes were treated with CB, CB+CHX, CB+DC, CB+6’DMAP, blastocyst formation was significantly (P<0.05) improved by CB (36.6%) compared to CB+CHX (30.9%), CB+DC (28.6%) and CB+6’DMAP (35.2%). In SCNT, was not significantly (P<0.05) increased by post-activation treatment with CB+CHX (81.3%), CB+DC (83.9%) and CB+6’DMAP (90.0%) compared to CB (84.5%) on embryo cleavage, blastocyst formation (19.1%~23.6%) and blastocyst cell number (39.6~41.4% cells/blastocyst) also were not influenced. But increased tendency in CB+6’DMAP. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-4 cell stage SCNT embryos were significantly higher 6’DMAP treated group than other treatment groups of SCNT embryos. These observations suggested that 2-4 cell cleaving embryos at have high developmental competence, and which may be influenced by survivin expression in porcine SCNT embryos. Our results demonstrate that post-activation treatment with CB, CB+CHX, CB+DC, CB+6’DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

Effect of the Treatment of Cytochalasin-B and the Loading Improvement of In-Vitro Porcine Embryos for Vitrification

Mi Hyun Ahn,In Duck Kim,Ho Bong Seok 한국동물생명공학회(구 한국동물번식학회) 2004 발생공학 국제심포지엄 및 학술대회 Vol.2004 No.1

Post-thaw development of in vitro produced buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification

B. M. Manjunatha,J. P. Ravindra,P. S. P. Gupta,M. Devaraj,T. G. Honnappa,A. Krishnaswamy 대한수의학회 2009 JOURNAL OF VETERINARY SCIENCE Vol.10 No.2

The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO2 incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 μl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the nonvitrified control. However, the hatching rate of cyto-btreated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.

Development of a Microdilution Device with One-step Dilution of Cytochalasin-B for Treating ORL-48 Cancer Microtissues

Chin Fhong Soon,Sargunan A/L Sundra,Nurfarina Zainal,Farshid Sefat,Mohd Khairul Ahmad,Nafarizal Nayan,Kian Sek Tee,Sok Ching Cheong 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.5

Mixing and dilution are essential procedures in pharmaceutical operation to aliquot two or more components serially to produce less concentrated and well mixed solutions. However, conventional serial dilution method used in laboratory is tedious and utilized large quantity of plasticwares. In this study, a two-tier microdilution device with two inlets and four outlets was designed, simulated, and prototyped to dilute Cytochalasin-B (CB). Using the microdilution device, CB in linear concentration gradients were produced based on one-step dilution method. The different concentrations of CB were applied to treat monolayer and microtissues of ORL-48 cells. The morphological responses, cell viability and cell proliferation of ORL-48 monolayer cells (2D) and microtissues (3D) treated in four different CB concentrations were investigated via phase contrast microscopy, live/dead stainings, and Alamar Blue® assay, respectively. The results showed that 2D ORL-48 cells were morphologically affected but 3D ORL-48 cells stayed viable and proliferative after treated with similar concentrations of CB. The microdilution device enables serial dilutions to produce reagents in linear concentration gradients via a one-step dilution method.

Cytochalasin B의 사람심방 K+ 통로 활성 차단 효과

백승우 ( Seung Woo Baik ),김태균 ( Tae Kyun Kim ),곽용근 ( Yong Geun Kwak ) 전북대학교 의과학연구소 2001 全北醫大論文集 Vol.25 No.2

본 연구에서는 cytochalasin B의 hKv1.5통로 차단기전을 명확히 구명하고 나아가 선택적 차단약물의 개발에 기여하고자, hKv1.5통로를 선택적으로 발현하는 세포주에서 cytochalasin B가 hKv1.5통로에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. Cytochalasin B is a widely used-actin disrupting agent. Accidently, we found the non-specific channel blocking effect of cytochalasin B. In addition, voltage-gated K` channels represent the most complex group of ion channel genes expressed in cardiovascular system. The human Kv1.5 channel (hKv1.5) represents the IKur repolarizing current in human atial myocytes. Thus hKv1.5 is thought to be an unique target for atrial fibrillation. In present study, we examined the effects of cytochalasin B on hKv1.5 current stably expressed in Ltk~ cells. Cytochalasin B inhibited the hKv1.5 currents in a dose-dependent manner in the cells transfected with hKv1.5, whereas a actin polymerizing agent phalloidin did not affect the cytochalasin B-induced block. Additionally, cytochalasin B reduced the tail current amplitude recorded at -50 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a ‘crossover’ phenomenon when the tail currents recorded under control conditions and in the presence of cytochalasin B were superimposed. These results indicate that cytochalasin B primarily block activated hKv1.5 channels in a concentration-dependent manner regardless of cytoskeleton, thereby possibly being a leading compound to develop a antiarrhythmic drug selective for atrial fibrillation.

배양된 간세포 담세관 및 세포질소기관의 미세구조 연구 : Phalloidin과 Cytochalasin D의 영향 Effects of Phalloidin and Cytochalasin D

김진국,양남길,안의태,고정식,박경호,김주원 순천향의학연구소 1995 Journal of Soonchunhyang Medical Science Vol.1 No.2

간세포 담세관의 주위세포질에는 미세사가 풍부하게 분포하는데, 이 실험에서는 배양 간세포의 미세사형성을 촉진 또는 억제시킨 후에 담세관 및 세포질소기관의 미세구조를 관찰하여 미세사의 형태학적 및 기능적 의의를 연구하였다. Sprague Dawley계 숫흰쥐의 간조직에 IV형 collagenaseㄹ를 처리하여 간세포를 분리한 후 배양하며 phalloidin(미세사 중합 촉진제)이나 cytochalasin D(미세사 중합 억제제)를 배지에 투여하고 전자현미경으로 관찰하여 다음과 같은 결과를 얻었다. 1. 분리된 간세포 세포질에는 골지복합체와 소포 및 용해소체들이 세포의 일부분에 분포되어 생체의 간세포와 같이 세포질소기관 분포의 극성을 나타냈다. 2. Phalloidin와 cytochalasin D 투여군이 담세관은 확장되고 미세융모의 소실이 뚜렷하였다. Phalloidin투여군에서는 미세사가 증식되고 외형질층이 두터워져 담세관은 경직된 모습이었고, cytochalasin D투여군에서는 미세사가 감소되고 담세관의 확장은 더욱 심했다. 3. Phalloidin이나 cytochalasin D 투여군 모두에서 간세포 표면에는 세포질이 돌출되었다. Phalloidin투여군에서는 돌출부의 연결부분이 잘록하였고 외형질에 미세사가 증식되어 돌출부와 세포질을 분리시키고 있었다. 반면에 cytochalasin D투여군에서는 외형질층에서 미세사가 뚜렷한 감소되었고 각종 세포질소기관들이 포함된 채로 세포질 일부를 밀고나와 반구형의 돌출부를 이루었고 연결부는 넓었다. 4. 세포질에는 많은 소포와 공포들이 융합되어 세포의 주변부분이나 담세관주위에 세포내공간이 형성되었는데, 이 공간은 세포바깥이나 담세관과 교통되었다. 이상의 결과를 보면, 미세사가 증식 혹은 감소되면 배양간세포의 모습이 변화되며, 담즙의 이동에도 장애가 생겨 담즙정체를 일으킨다. 따라서 미세사는 간세포의 형태유지 및 담즙분비에 중요하게 관여하는 것으로 생각된다. The cytoplasmic microfilaments of hepatocytes are abundant beneath the plasma membrane, especially in the pericanalicular ectoplasm. In this study, the ultrastructural changes of bile canaliculus and cytoplasmic organelles induced by alteration of the microfilaments on the cultured rat hepatocytes were examined. Sprague-Dawley rats(male, about 200gm) were used. The isolated hepatocytes obtained by perfusion of 0.05% collagenase type IV through the portal vein, were cultred in the L-15 medium containing phalloidin(stabilizer of microfilaments) or cytochalasin D(destabilizer of microfilaments) for 10 min, 30 min, 1 hour, 2 hours, 4 hours, 10 hours and 20 hours, respecitvely. The hepatocytes on the cultured dish were fixed in 2.5% glutaradehyde -1.5% paraformaldehyde and 1% osmium tetroside. After alcohol dehydration, the cells were embedded in the araldite mixture. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and observed with JEM100 CX-II electron microscope. The results were as follow: 1. Isolated hepatocytes maintained the typical architectionic relationships of secretory organelles, i.e., Golgi apparatus, vesicles, lysosomes, etc., in the vicinity of bile canalicular region. 2. In the phalloidin or cytochalasin D treated groups, bile canaliculi were dilated and devoid of microvilli. In phalloidin treated group, the pericanalicular ectoplasm containing the microfilaments microfilaments was thicker than that of cytochalasin D treated group. Whereas, the dilation of bile canaliculus was more marked in cytochalasin D group. 3. Both drugs, phalloidin or cytochalasin D, produced the alteration of cell shaper to form cytoplasmic protrusions at the cell surface. In the phalloidin treated group, protusions were pedunculated, and the microfilaments were accumulatd at the narrow neck region. In cytochalasin D treated group, in contrast, no microfilament barrier was seen at the broad base of protrusion which exhibit direct continuity with the internal cytoplasm. 4. Numerous vesicles and vacuoles were formed near the cell surface and perianalicular cytoplasm in the treated groups, and later in culture they fused each other to form large intracellular space. Eventurally. this space was connected to the extracellular space or bile canaliculus. This experiment demonstrated that excessive accumulation or depletion of microfilaments induced by phallyoidein or cytochalasin D altered the cell shape and disturbed the vesicular transport of bile components into bile canaliculi. The results suggest that dysfunction of microfilaments may play an imprtant role in the impairement of canalicular contraction and the integrity of microfilaments is necessary for the billiary secretin as well as for the maintainance of the cell shape of hepatocytes.

Cytochalasin B와 Colchicine이 생쥐 간장의 담세관과 간세포의 미세구조에 미치는 영향

박창현,장병준,엄창섭,엄창섭 대한해부학회 1998 Anatomy & Cell Biology Vol.31 No.2

Tetraploidy Induction of Mouse Embryos by In Vitro Culture with Cytochalasin B

진동일 한국수정란이식학회 1999 한국동물생명공학회지 Vol.14 No.2

효율적인 homozygous동물을 생산하기 위한 실험의 단계로 염색체가 4배체인 수정란의 이용성을 타진하기 위해 생쥐 수정란과 cytochalasin B를 사용하여 4배체 유도에 관한 실험을 수행하였다. 생쥐 2-세포기 수정란을 10 ㎍/ml 농도의 cytochalasin B로 약 20시간 배양하였을 때 모든 수정란은 발육을 거의 멈추었으나, 이 수정란을 cytochalasin B-free medium에 체외배양 하였을 때 발육이 재개되어 48시간 후 상실기나 배반포기까지 약 74%의 발육율을 나타내었다. 그러나 발육된 수정란의 세포수는 대조구에 비해 훨씬 적은 것으로 나타났다. 염색체 분석결과 cytochalasin B로 처리한 대부분의 수정란은 4배체인 것으로 나타났고 약간의 수정란은 mosaicism과 다배체를 나타내기도 하였다. 그러므로 cytochalasin B를 이용하여 효과적으로 4배체의 수정란을 유도할 수 있는 것으로 나타났다.

Phalloidin 및 Cytochalasin D에 의한 흰쥐 배양된 간세포의 형태학적 변화

박경호,김종봉,안의태,고정식,김진국 대한해부학회 2000 Anatomy & Cell Biology Vol.33 No.6

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Minjung Kim, M.D.,송경,진은정,손종경 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.9

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.