• Reproductive and Developmental Biology
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• 제29권2호
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• pp.117-120
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• 2005

This study was conducted to establish the optimal temperature condition before oocyte activation in B6m F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB $(56.2\%\;vs\;81.0\%\;p<0.01)$. Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM $(22.0\%\;versus\;8.8\%\;p<0.05)$. In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at $37^{\circ}C$, Group B: pre-incubation at $37^{\circ}C$ for 90 min then at $25^{\circ}C$ for 30 min, Group C: pre-incubation at $37^{\circ}C$ for 60 min then at $25^{\circ}C$for 60 min, Group D: pre-incubation at $37^{\circ}C$ for 30 min then at $25^{\circ}C$ for 90 min, and Group E: pre-incubation at $25^{\circ}C$ for 120 min before activation. Group A $(67.6\%)$ and B $(66.7\%)$ showed better development to the blastocyst stage than other groups $(Group\;C:\;50.0\%\;Group \;D:\;49.2\%\;Group\;E:\;33.3\%,\;p<0.05)$. The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

• 한국가축번식학회지
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• 제22권3호
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• pp.221-227
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• 1998

This study was carried out to determine the effects of ionomycin and 6-dimethylaminopurine (6-DMAP) treatments on parthenogenesis of rabbit oocytes. The oocytes were randomly assigned to the activation treatments with either ionomycin plus 6-DMAP or electric stimulation. The oocytes were colected from the oviducts of superovulated rabbits at 13~14 hours and 19~20 hours post hCG injection and were activated with 5$\mu$M ionomycin for 5 min and 2 hours incubation in 2mM 6-DMAP. The other oocytes were stimulated by three pulses of 3.6kV/cm for 60 $\mu$sec each 30 min apart, starting 19 hours post in hCG in 0.28M mannitol solution with 100$\mu$M Ca2+ and Mg2+. The results obtained were summarized as follows: 1. Following treatment of the oocytes with ionomycin plus 6-DMAP, the cleavage rate and in vitro developmental rate to blastocyst were significantly(P<0.01) higher in the oocytes collected bet ween 19~20 hours than between 13~14 hours after hCG injection. 2. When the oocytes were treated with ionomycin plus 6-DMAP, 85(98.8%) of 86 treated oocytes extruded the second polar bodies, with the entire chromatin complements outside ooplasm. However when the oocytes were restored during subsequent incubation in the drug-free medium, the cytoplasts regain their full capacity for parthenogenetic activation and nuclear remodelling. 3. The cleavage rate and the in vitro developmental rate to blastocyst were not significantly different in the oocytes activated by ionomycin plus 6-DMAP treatment(91.2 and 45.6%) or electrical stimulation(89.6 and 34.3%).

• 대한수의학회지
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• 제42권1호
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• pp.29-33
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• 2002

This study was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 24 hrs post onset of maturation, the oocytes were cultured $3{\sim}13{\mu}M\;Ca^{2+}$ for 5 min., $5-8{\mu}g/ml$ cytoclacin for 6 hrs, 0.5~2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs alone or combination. The activated oocytes were cultured in TCM-199 media at 5% $CO_2$, 95% air, $38^{\circ}C$. The cleavage rate after 48 hrs culture of oocytes treated with $3-13{\mu}M\;Ca^{2+}$, $5-8{\mu}g/ml$ cytoclacin and 0.5~2.0 mM DMAP for 5 min., 6 hrs and 3 hrs were 9.6%~20.0%, 0.0%~7.3% and 9.4%~21.8%, 0.0%~7.3% and 9.1%~21.8% and 0.0%~7.3%, respectively. When oocyte were treated with $10{\mu}M\;Ca^{2+}$, $10{\mu}g/ml$ cytoclacin and 2.0 mM DMAP the blastocyst formation rate was significantly higher than other group. The cleavage rate after 48 hrs culture of oocytes treated with $Ca^{2+}$ + cytoclacin, $Ca^{2+}$ + DMAP, cytoclacin + DMAP were 75.9%~93.5% and 9.7%~19.0%, respectively. When oocytes were treated with $Ca^{2+}$ followed by DMAP, the blastocyst formation rate was significantly higher than other group(p<0.05). When necleus transferred embryos co-cultured with bovine serum albumin(BSA), epithemal growth factor(EGF) and calf serum(CS), the developmental rate to blastocyst were higher than control group.

• 한국가축번식학회지
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• 제23권1호
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• pp.19-27
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• 1999

본 연구는 사람정자 추출물을 마우스 난모세포의 세포질내에 주입하여 단위발생에 미치는 영향을 구명하기 위하여 실시하였으며, 그 얻어진 결과는 다음과 같았다. 1. 마우스 난모세포가 0, 1.7 그리고 5mM의 칼슘농도를 가지는 PBS 배지로 각각 10 pl 씩 주입되었을 때, 전핵을 형성하고 제2극체를 방출하는 난모세포의 활성율은 각각 14.5, 9.8 그리고 14.9%였다. 칼슘농도별 난모세포의 활성율은 유의성이 인정되지 않았다 (P＞0.05). 2. 마우스난모세포에 가열하지 않은 사람정자 추출물 10 pl을 주입후 0, 1.7 그리고 5mM의 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 1.7 mM 칼슘 농도에서의 난모세포 활성율(51.8%)은 0 과 5mM 칼슘 농도에서의 난모세포 활성율보다 유의하게 높았다. 3. 마우스 난모세포에 가열처리한 사랍정자 추출물 10 pl을 주입 후 0, 1.7 그리고 5mM의 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 칼슘 농도별 난모세포 활성율은 유의성이 인정되지 않았다. 난모세포 활성율은 11.8∼17.0%의 범위에 있었다. 4. 마우스 난모세포에 가열하지 않은 사람 정자 추출물, 가열 처리한 사람 정자 추출물, 그리고 PBS 배지를 각각 l0 pl 씩 주입 후 1.7 mM 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 가열하지 않은 사람 정자 추출물을 주입한 난모세포의 활성율이 54.5% 로 제일 높았다. 처리별 난모세포의 활성율은 유의성이 인정되었다(P＜0.05), 5. 마우스 난모세포에 가열하지 않은 l일령과 6일령 정자 추출물을 각각 l0 pl 씩 주입 후 1.7mM 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 1일령 정자 추출물을 주입한 난모세포의 활성율은 60.0%로 6일령 정자 추출물을 주입한 난모세포의 활성율 11.1% 보다 유의하게 높았다. 이상의 결과를 종합해 보면 가열하지 않은 사람정자 추출물에는 oscillin 과 같은 난모세포 활성인자가 존재한다는 것이 입증되었다.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.241-245
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• 2004

This study was conducted to investigate nuclear remodeling and developmental rate following nuclear transfer of fetal fibroblast cells, ear skin cells and oviduct epithelial cells into porcine recipient oocytes. To test par-thenogenetic activation, oocytes were treated with a 6-dimethylaminopurine (6-DMAP), a single DC-pulse (DC), calcium ionomycin (ionomycin), DC+6-DMAP and ionomycin + 6-DMAP after in vitro maturation. For nuclear transfer, in vitro matured oocytes were enucleated, and donor cells were transferred into oocytes. Cloned embryos were fused and stimulated with 6-DMAP for 4 h and cultured in vitro for 6 days. Among treatments for parthenogenesis, the activation rate of DC +6-DMAP treatment was significantly higher than that of single treatment roups (p<0.01), except for DC treatment group. However, the difference was not significant in activation rate compared to other complex treatment groups. Nuclear swelling of the cloned embryos was initiated at 60 min after stimulation and increased afterwards. Fusion rates were not different among different donor cells. Cleavage rates of DC treatment groups were significantly higher than those of DC+6-DMAP treatment groups (p<0.05) in case that fetal fibroblast and ear cells were used for nuclear donor. The cloned embryos from developed to blastocysts in oviduct epithelial cell nuclear transfer with DC+6-DMAP treatment was significantly higher compared to those with DC only treatment (p<0.05). However, no blastocyst was developed from nuclear transfer of fetal fibroblast and ear cells regardless of activation treatments. Based on these results, a proper activation stimulation may be necessary to increase the activation rate and the development to blastocyst in cloned porcine embryos.

• 한국가축번식학회지
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• 제18권2호
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• pp.121-126
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• 1994

This study was conducted to examine the condition of activation of the nuclear transplant bovine embryos. In vitro fertilized(IVF) and nuclear transplant embryos(NTs) were co-cultured with bovine oviduct epithelial tissue(BOET). NTs were treated with cycloheximide(CHXM) for 0 to 6 h after electrofusion to investigate the activation conditin of recipient ooplast. Then, the infljence of the CHXM treatment timing on the cleavage and development of NTs were investigated in relation to the nuclear transplant time. The cleavage rates of NTs were increased with the increasing time of the CHXM treatment from 0 to 6 h (54.7 to 91.3%, P<0.01). Similar trend was shown in the development into the morula or blastocyst stage, but very limitted. Activation of enucleated oocytes prior to fusion enhanced development of NTs compared with that post fustion. This result suggests that the frequency of activation of NTs can be greatly enhanced by treating with CHXM for 6 h. The result also suggests that if blastomeres of unknown cell cycle stage are used, activation of enucleated oocytes prior to fusion enhances development of NTs.

• 한국수정란이식학회지
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• 제26권4호
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

• Clinical and Experimental Reproductive Medicine
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• 제41권2호
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• pp.47-61
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• 2014

Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.111-120
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• 2001

Objective: To verify the expression of leptin receptor (OB-R) in oocytes and preimplantation embryos, the involvement of mitogen activated protein kinase (MAPK or Erk1/2) in the leptin signaling, and effect of leptin on the oocyte maturation in mice. Method: RT-PCR analysis of OB-R was conducted in germinal vesicle (GV)-intact and MII stage oocytes, and 1, 2, 8-cell embryos and blastocysts. Germinal vesicle breakdown (GVB), polar body extrusion, monitored in the presence or absence of leptin ($1{\mu}M$). Following the leptin treatment, temporal changes in MAPK activity were verified by immunoprecipitation and in vitro kinase assay in MII oocytes. Results: The expression of OB-R mRNA was found in GV and MII oocyte but not in the embryos. MAPK activity of the MII oocytes was significantly increased by brief incubation in the HTF supplemented with leptin ($1{\mu}M$). Priming of PD098059, a MEK inhibitor to leptin treatment attenuated the activation of MAPK by leptin in MII oocytes. Following 24 hrs of culture of the GV oocytes, leptin significant increased the GVB and 1 st polar body extrusion. Conclusion: This result suggested that functional interaction between leptin and OB-R resulted in potentiation of MAPK (Erk1/2) activity in MII oocytes through MEK activation and that leptin might be a local regulator of meiotic maturation of the mouse oocytes.

• 한국가축번식학회지
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• 제22권3호
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• pp.277-286
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• 1998

The effect of MAP kinase activity on maturation of porcine oocytes was investigated. MAP kinase was detected by immunofluorescence staining in nucleus of oocytes just before entering GVBD (germinal vesicle breakdown)stage. In a Western blot with GV (germinal vesicle) from these oocytes (cultured for 25 hours), a shift of MAP kinase band a, pp.ared, suggesting an activated stage of the kinase. No activity was shown in the blot with GV isolated from, oocytes cultured for 0 hour. To confirm that activation of MAP kinase induce GVBD, we microinjected MAP kinase purified from matured oocytes starfish into the cytoplasm of oocytes in GV stage (cultured for 0 hour). The injected MAP kinase did not cause early a, pp.arance of GVBD. No oocytes showed GVBD state until 20 hours of culture. Activity of MAP kinase did not increase significantly after the injection. When the exogenous MAP kinase was injected into GV, GVBD was induced in about 20% of oocytes cultured for 5 to 10 hours. These results suggest that MAP kinase is traslocated to nucleus and function as a factor inducing GVBD.