• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.66-66
• /
• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2002년도 국제심포지엄
• /
• pp.116-116
• /
• 2002

Further development of reconstructed embryos may be dependent upon the synchronization of donor nucleus and recipient cytoplasm at cell fusion, To control the synchronization of donor and recipient cells, the enucleated MII arrested oocytes are artificially stimulated prior to embryo reconstruction. Destruction of cyclin B results in the exit of cells from M-phase of cell cycle. This study was designed to investigate the effects of single or combined stimulation affected cyclin B1 mRNA and protein levels in mouse oocytes. The oocyte activation was induced by 7% ethanol or 10$\mu\textrm{g}$/$m\ell$ Ca-ionophore without (single) or with (combined) 10$\mu\textrm{g}$/$m\ell$ cycloheximide. Competitive quantitative PCR for cyclin Bl mRNA and western blot analysis for cyclin B1 protein was preformed in mouse oocytes. Cyclin B1 mRNA level was significantly reduced in single (P<0.05) and combined (P<0.05) stimulation groups. However, this level did not change in non-activated group and increased in intact group. Cyclin B1 protein level was also significantly reduced in both single (P<0.05) and combined (P<0.05) stimulation groups. In conclusion, single and combined stimulation induces the degradation of cyclin B1 mRNA and protein after activation in enucleated mouse oocytes.

• 한국수정란이식학회지
• /
• 제28권2호
• /
• pp.127-132
• /
• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• Reproductive and Developmental Biology
• /
• 제31권2호
• /
• pp.77-82
• /
• 2007

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for $30{\mu}sec$; ES), 2) double electric stimulations (1.5 kV/cm for $30{\mu}sec$, followed by 1.0 kV/cm for $50{\mu}sec$ after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6-7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

• 한국수정란이식학회지
• /
• 제9권1호
• /
• pp.117-125
• /
• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

• 한국가축번식학회지
• /
• 제26권3호
• /
• pp.245-252
• /
• 2002

본 연구는 체세포 핵치환에 탈핵 후 통격한 소미수정란을 사용함에 있어서, MVC 초자화 동결방법과 탈핵난자의 활성화시기가 융해 후 생존율과 핵치환 이후 체외 발달에 미치는 영향을 조사하고자 실시하였다. 체외에서 20시간 동안 체외성숙된 소 미수정란은 수핵란으로 사용하기 위하여 5$\mu\textrm{g}$/$m\ell$ hoechst 처리 후, 형광현미경하에서 핵을 제거하였다. 본 실험은 세 그룹으로 나누어 실행되었다 Group I은 동결하지 않고 핵치환을 한 대조군이며, group III와 group II는 핵이 제거된 난자를 MVC 방법으로 동결하기 전과 후에 활성화 처리 (5$\mu\textrm{m}$의 ionomycin에 의해서 5분간 처리) 한 군이다. 초자화 동결을 위해서는 group II와 group III의 탈핵란은 EG10에서 5~10분간 전처리하고 EG30에서 30초간 노출하여 액체 질소에 침지하였다. 융해는 37$^{\circ}C$에서 4단계로 이루어졌다. 실험군은 모두 소 귀세포를 이용하여 핵치환을 실시하였으며, 전핵을 유도하기 위한 활성화를 위해서는 10$\mu\textrm{g}$/$m\ell$ cycloheximide와 2.5$\mu\textrm{g}$/$m\ell$ cytochalasin D)가 첨가된 CRlaa 배양액에서 1시간, 이후 10 $\mu\textrm{g}$/$m\ell$ cycloheximide가 들어있는 CRlaa 배양액에서 4시간동안 배양하였다. 활성화 처리가 끝난 난자들은 CRlaa 배양액에서 2일간 배양하여 난할이 유도된 난자만을 선별하여 난구세포와 7일 동안 공배양하였다. 동결 융해 이후 group II와 group III의 탈핵된 소 미수정란의 체외 생존율은 81.0%와 84.9%로 유의적인 차이가 없었다. 체세포와 수핵란과의 융합율도 각각 69.0%와 70.0%로 대조군 (75.2%) 과도 유의적인 차이를 나타내지 않았다. 난할율은 53.4%와 58.4%로 group II와 group III간에 유의적인 차이를 나타내지 않았지만 group II의 분할된 세포질을 가진 이상난자의 비율이 group III보다 유의하게 높게 나타났다 (P<0.05). 또한, morula 이상으로 발달율도 group II (8.6%) 에서 group III (15.6%)보다 낮은 결과를 얻었다 하지 만 group III (15.6%)의 체외 발달율은 대조군 (24.8%)과 유의한 차이를 없었다. 따라서, MVC 동결 방법은 탈핵된 소 미수정란을 동결하기에 적합한 방법이며, 탈핵 후 activation을 유도하고 초자화 동결한 난자는 동결하지 않은 신선란과 동일하게 체세포 핵치환에 유용하게 이용될 수 있으리라 사료된다.

• 한국수정란이식학회지
• /
• 제28권2호
• /
• pp.133-139
• /
• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with 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.

• 한국가축번식학회지
• /
• 제20권3호
• /
• pp.239-250
• /
• 1996

The objective of the present experiments were to determine whether micromanipulative and electro-stimulation conditions for blastomere survival overlapped those for oocyte activation in porcine. Eggs selected for in vitro development potential of blastomeres isolated from 4-cell embryos and oocyte activation by electrostimulation were equilibrated for 5~10 min, in 0.3M sucrose solution containing 7.5$\mu\textrm{g}$/ml cytochalasin B, and then electrostimulated for 30$\mu$sec using one pulse of 100, 120, 150 or 180 volts DC with electrodes 0.2mm apart. Single blastomeres were inserted into empty zona pellucida prior to electrostimulaticn. Then they were cultured in 20${mu}ell$ drops of fresh BECM to observe their developmental ability in vitro in a humidified incubat or at 38.5$^{\circ}C$. The results obtained from these experiments are as follows : 1. When one pulse of 100, 120, 150 or 180 volts DC for 30$\mu$sec were applied to porcine oocytes having the slit formed on zona pellucida for activation, activation rates were 65.1, 66.7, 70.7 and 91.7%, respectively. Higher activation rate was observed in 180V. 2. Infact oocytes incubated for 30 min, in 0.3M sucrose solution after electrostimulation were significantally different from control group with increasing of voltages(p<0.05). When voltages used for electrostimulation were increased, activation rates of oocytes were improved in all treatment groups. 3. When zona punctured-oocytes were only electrostimulated, or incubated in 0.3M sucrose solution for 30 min. after electrostimulation at 180 volt DC, activation rates were 90.5 and 95.5%, respectively. And activation rates of zona punctured-oocytes were significantly different from the groups for which zona pellucida was not punctured(P<0.05). 4. When single blastomeres form 4-cell transferred into empty zona pellucida were incubated for 0, 15 and 30 min. in 0.3M sucrose solution after electrostimulation using one pulse of 180 volt DC for 30 $\mu$sec, developmental rates of electrostimulated-single blastomeres to blastocyst were 72.5, 59.0 and 51.2%, respectively, and the ratio of control group developed to blastocyst were 80.0%. 5. The average cell number in electrostimulated-blastomeres developed to blastocyst were 7.9~10.8, and reduced than the cell number in diploid control ; Also cell number decreased with increasing of voltages. The results of these experiments indicate that the optimal condition for achieving in vitro developmental ability of single 4-cell blastomeres and oocyte activatin is 1 pulse, duration 30 $\mu$sec. in 180 volt, and incubation of blastomeres and oocytes in 0.3M sucrose solution after electrostimulation was not significantally different from another treatment groups. The results also show that this condition is suitable for nuclear transplantation using porcine eggs.

• Animal cells and systems
• /
• 제2권1호
• /
• pp.87-91
• /
• 1998

The present study investigated the involvement of the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways during progesteroneinduced meiotic maturation in amphibian (Rana dybowskii) oocytes. Prosesterone-induced germinal vesicle breakdown (GVBD) of oocytes was significantly inhibited by a PKC inhibitor, staurosporine and a PLC inhibitor, U73122, in a dose-dependent manner. In contrast, U73343, an inactive analogue of U73122, was ineffective in suppressing GVBD. PKC activity in oocytes reached a maximum level at 30 min after progesterone stimulation and this elevated PKC activity was effectively suppressed by U73122 or staurosporine, suggesting that the activation of PKC enzyme is closely linked to PLC signaling during oocyte maturation. In addition, these inhib itors blocked the maturation promoting factor (MPF) activity which appeared in oocytes in response to progesterone, suggesting that PKC activation is an important signal for MPF activity. Therefore, this study demonstrates that the activation of PKC via PLC signaling is directly linked to an intracellular protein kinase cascade related to the appearance of MPF activity during meiotic maturation in amphibian (Rana dybowskii) oocytes.

• Reproductive and Developmental Biology
• /
• 제30권1호
• /
• pp.41-45
• /
• 2006

Electrical treatment has been widely used for porcine oocytes activation. However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide $(10{\mu}g/ml)$ for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to $6.25{\mu}M$ calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to $10{\mu}g/ml$ cycloheximide and electrical pulse treatment was significantly increased (P<0.05) than in the control group. And exposure to $6.25{\mu}M$ calcium ionophore for 2 min with $10{\mu}g/ml$ cycloheximide for 10min and electrical pulse treatment significantly increased (P<0.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development.