• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.115-115
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• 2002

To evaluate the correlations between the expression of cyclin B1 mRNA and protein after stimulation and oocyte activation and development of nuclear transferred mouse embryos, this study was performed. 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 (CH). Cyclin B1 mRNA and protein in mouse oocytes was evaluated by PCR and western blot. The activation and blastocyst development in both single (P<0.05) and combined (P<0.01) stimulation was higher than in non-activated group. The cyclin B1 mRNA and protein levels were significantly reduced in both single and combined stimulation groups (P<0.05), respectively. Cyclin B1 mRNA expression showed a negative correlation between activation and blastocyst development in both single and combined stimulation groups. And also the expression of cyclin B1 protein showed a negative correlation with between oocyte activation and blastocysts development in both single and combined stimulation groups. In conclusion, it may suggest that single and combined stimulation increases the oocyte activation and blastocyst development of nuclear transferred embryos, because it induces the degradation of cyclin B1 mRNA and protein after activation in enucleated mouse oocytes.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.369-375
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• 2002

A sperm factor(s) for oocyte activation during fertilization has not been clearly identified. In this study to elucidate an oocyte activation factor(s), mouse sperm were sonicated and ultra-filtered with a 30 kilo-daltons (KD) cutoff membrane and the ultra-filtrate was then sequentially fractionated over Suporose 12 column and Superdex column, The recovered fractions were micro-injected into Mⅱmouse oocytes and second polar body formation (PBF) was examined. Suporose fraction RV2.10 prepared from sperm extract significantly increased PBF. Of Superdex fractions re-separated from Suporose fraction RV2.10, fraction RV2.12 also had the strongest PBF activity. By analyzing with micro-reverse phase column (URPC), the Superdex fraction RV2.12 appeared to be glutamic acid. In microinjection test, glutamic acid significantly increased PBF. This study suggests that glutamic acid should be a type of sperm factor for second polar body formation related to oocyte activation.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.63-63
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• 2004

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.124-124
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• 2003

• Journal of Animal Science and Technology
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• v.48 no.3
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• pp.353-360
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• 2006

This study was performed to investigate the effects of artificial stimulation on the increase of the oocyte activation, to evaluate the expression of cyclin B1 protein levels in enucleated mouse oocytes, and to investigate correlation between the oocyte activation and the cyclin B1 protein levels. The oocyte activation was induced by 7% ethanol (EtOH) or 10μg/ml Ca-ionophore with or without 10μg/ml cycloheximide (CH). The activation rate was significantly higher in both single (p<0.05) and combined (p<0.01) stimulated groups compared to control group. The cyclin B1 protein level was significantly reduced in both stimulated groups (p<0.05), except for EtOH+CH treatment group. The expression of cyclin B1 protein showed a higher negative correlation with activation rate in EtOH+CH (r=0.61, p<0.05) and Ca+CH (r=0.86, p<0.01) stimulation groups, but not in a both single stimulation groups. Taken together, it can be suggested that single (EtOH and Ca- ionophore) and combined (EtOH+CH and Ca+CH) stimulation increases the oocyte activation, especially combined stimulation, because it induces the degradation of cyclin B1 protein after artificial stimulation treatments in mouse oocytes.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.31-41
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• 1994

핵 이식 기술을 이용한 cloning 송아지 생산이 처음 보고(Prather et al., 1987) 된 후, 소 수정란 Cloning에 대한 많은 연구가 분자 생물학 등 여러 분야에서 꾸준히 계속되고 있다. 이 기술은 빈우의 번식 능력을 향상시켜 유전적 개량량을 증대할 수 있는 번식과 육종을 위한 도구로써 많은 잠재력을 지니고 있다. 최근 핵 이식 기술을 이용하여 유전적으로 우수한 빈우로부터 수천개의 수정란을 생산하여, 이들 수정란에게 생산된 송아지가 번식 축군으로 공시되어 있으므로, 그 결과가 주목되나 아직까지는 비용이 많이 들고 송아지 생산 효율이 저조하므로, 가까운 장래에 일반 양축가에 이용될 가능성이 낮다. 그러나 이 기술의 실용화를 위하여 선결되어야 할 많은 문제점들 중, 지난 몇 년 동안 많은 연구기관에서 수행된 활발한 연구의 결실로써, 난포란 제핵, cell fusion 과 oocyte activation의 방법등 주요 장애 요인들이 점차 극복되면서 실용화를 위한 접근이 예견되어지며, 구미의 일부 개량 기관에서는 이를 상업화 하기 위한 여건을 다지고 있다. 그러므로 이 Review에서는 fllicular dynamics, 난포란의 성숙, cell cycle, 난포란 제핵, cell fusion과 oocyte activation, 이식후 핵의 remodeling과 reprogramming에 대한 현재까지의 보고된 자료를 기초로 그 기본 원리를 재고하는데 초점을 맞추었다.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.201-206
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• 2005

This study was conducted to investigate the effects of oocyte activation after ICSI and of capacitation of insemination sperm before ICSI in Swine. There was no significant difference on cleavage rate and blastocyst developmental rate treated with ethanol, cycloheximide, or ethanol and cycloheximide jointly between treatment and control groups. However, significantly difference was found on cleavage rate and blastocyst developmental rate treated with caffeine and Ca-ionophore on capacitation of insemination sperm before ICSI (p<0.05). There was no significant difference on pronuclear formation rate and total oocyte activation rate treated with oocyte activation after ICSI between treatment and control groups, but was significant difference on pronuclear formation rate and total oocyte activation rate treated with capacitation treat of sperm (p<0.05).

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.77-82
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• 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.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.229-234
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• 2005

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ for 4 h (immediate activation after injection; IAI), or cultured in vitro for $2\~3$ h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation $2\~3$ h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of $36.6\%\;(15/41),\;39.5\%\;(17/43)\;and\;46.3\%$ (25/54), respectively. However, in the ABI group, only one embryo ($1.8\%$, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage $(4.9\%\cdot2/41)$. However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.