• 한국수정란이식학회지
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• 제17권3호
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• pp.195-201
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• 2002

본 실험은 원시난포의 체외배양 체계를 확립할 수 있는 가능성을 검토하기 위해 0, 5, 10, 15 및 20일령 마우스에서 분리한 난소를 20, 15, 10, 5 및 0일동안 organ culture 하여 난소의 성장률, 난자의 회수율과 성장률 및 난자의 핵성숙 단계를 조사하여, 비교 ·검토하였다. 그 결과를 요약하면 다음과 같다. 1. Organ culture 전과 후의 각 일령에 따른 난소의 면적 차이는 0일령 35.9%, 5일령 8.7%, 10 일령 1.2% 및 15일령 14.4%로 15일령을 제외 하고는 배양일령이 증가할수록 면적 차이는 감소하였다. 2. Organ culture 후 난자의 회수율과 난자의 직경은 배양일령이 증가할수록 증대되었다. 3. GV기 이상의 핵성숙은 organ culture 후 배양 일령이 증가할수록 진행되는 단계에 있는 것으로 나타났다.

• 한국수정란이식학회지
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• 제9권3호
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• pp.249-254
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• 1994

This study was undertaken to investigate effects of granulosa cells on mejotic maturation of porcine oocytes in vitro. The results obtained in this study were summarized as follows : The germinal vesicle breakdown(GVBD) rates were 91.5, 93.3 and 96.6%, respectively, when the cumulus oocy:e cornplexes(COC) in the TCM-199 medium with sodium bicarbonate, Na pyruvate, penicillin G, streptomycin sulfate and 10% FCS were cultured in the condition of FSH(0.02 Au/ml), LH(10 $\mu$g/ml) and FSH + LH added. And when the COC were co-cultured with granulosa cell (5$\times$ 106 cells /ml) in the condition of FSH, LH and FSH + LH added, GVBD rates were 94.3, 92.9 and 98.9%, respectively. However, when the COC were cultured in the condition of hormone free and co-cultured with granulosa cells in the condition of hormone free, the GVBD rates were 40.4 and 86.3%, respectively. The GVBD rates were 41.0, 62.7, 84.6, 88.1 and 93.6%, respectively, when the COC were co-cultured with granulosa cells that the concentrations are 0 cells /ml, 1 $\times$ 106 cells /ml, 5:: 106 cells /ml, 1$\times$ 107 cells /ml and 5$\times$ 107 cells /ml.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.138-138
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• 2005

• Asian-Australasian Journal of Animal Sciences
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• 제17권7호
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• pp.885-891
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• 2004

To evaluate the feasibility of using sperm-mediated gene transfer (SMGT) for carrying foreign gene into chicken oocyte, a reporter gene, CX-EGFP, was used in this study. The reporter gene was first mixed with liposome or liposome-like compound and the mixtures were further combined with ejaculated cock spermatozoa. The spermatozoa treated with liposome and CX-EGFP mixture was subsequently coincubated with DNaseI to remove the extra DNA which insured the authenticity of positive signals. The treated sperms were then subjected to transgene (reporter gene) existence analysis and artificial insemination of laying hens. Obtained results indicated that the spermatozoa were able to take-in the foreign DNA; which was confirmed by polymerase chain reaction and Southern blot analysis. In the following experiment, fresh ejaculated sperms were mixed with CX-EGFP-liposome or CX-EGFP-liposome-like complex then used for artificial insemination of each of six laying hens. Eggs laid between day-3 and day-7 post insemination were collected. Newly hatched chicks, two out of 53 from CX-EGFP/liposome treated group and two out of 21 from CXEGFP/liposome-like treated group, were proven to be transgenic. This study suggests that SMGT is a powerful method for generating transgenic chickens.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.407-414
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• 2011

The development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is dependent upon numerous factors. Central to development is the quality and developmental competence of the recipient cytoplast and the type of the donor nucleus. Typically metaphase of the second meiotic division (MII) has become the cytoplast of choice. Production of a cytoplast requires removal of the recipient genetic material, however, it may remove proteins which are essential for development or reduce the levels of cytoplasmic proteins to influence subsequent reprogramming of the donor nucleus. In this study, enucleation at MII did not affect the activities of either MPF or MAPK kinases. Immunocytochemical staining showed that both Cyclin B1 (MPF) and Erk1/2 (MAPK) were associated with the meiotic spindle of AI/TI oocytes with little staining in the cytoplasm, however, at MII association of both proteins with the spindle had reduced and a greater degree of cytoplasmic distribution was observed. The analysis of oocyte proteins removed during enucleation is a difficult approach to the identification of factors which may be depleted in the cytoplast. This is primarily due to the large numbers of aspirated karyoplasts which would be required for the analysis.

• 한국수정란이식학회지
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• 제15권1호
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• pp.15-21
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• 2000

These studies were undertaken to evaluate morphological normality and development competence in vitro of hyman oocytes following vitrificatioin using ethylene glycol and electron microscopic grid. Human immature oocytes retrieved from natural and stimulated cycles was vitrified at 0 or 48 h and 0, 8 to 15 or 24 to 28 h after maturation culture, respectively. In oocytes retrieved from unstimulated cycle, no signifciant differences were found in morphological normality (56 to 63%) and fertilization (31 to 37%) rates between the times of vitrification. In stimulated patients, however, more oocytes were morphologically normal when vitrified at 24 to 28 h than when vitrified at 0 or 8 to 15 h after maturation culture. Regardlesss of the hormonal stimulation, high cleavage rates(83 to 100%) were obtained in all treatment groups but did not differ significantly. Twenty to 43% of cleaved oocytes developed to the blastocyst stage at 6 days after IVF. These results suggest that vitrified oocytes from unstimulated and stimulated cycles could develop to the blastocyst stage, regardless of the stages of vitrification.

• 한국수정란이식학회지
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• 제16권1호
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• pp.1-5
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• 2001

A total of 92 unfertilized human oocytes were treated with ethanol (EtOH), calcium ionophore A23187 (CI) or electric pulse (EP) for activating pronuclear formation and subsequent development. In Experiment 1, there was a significant (P=0.0001) treatment effect on the activation of unfertilized oocytes. No spontaneous activation was occurred in the control, but activation treatments induced PN formation with various efficacy. More unfertilized oocytes (UFOs) were activated after EtOH or EP treatment than after CI treatment. EP was as effective (63.6 %) as EtOH, but fragmentation was observed in 43% of UFOs activated by EP. Proportion of UFOs that formed presumptive haploid PN (2 PNs+1 PB or 1 PN +2 PBs) was 33.3, 0 and 28.6% after EtOH, CI and EP treatments, respectively. In Experiment 2, a significant (P=0.0362) effect of immature oocytes (IOs) status on activation was fecund. IOs at the GVBD-MI oocytes had higher potential to form PN than those at the GV stage or with abnormal morphology (25 vs. 77.8%). The results of this study clearly demonstrated that the treatment of 10% ethanol for 5 min effectively induced the activation of UFOs. IOs could form pronucleus with high efficacy by ethanol treatment, as long as they grew beyond the GVBD stage.

• 한국수정란이식학회지
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• 제32권4호
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• pp.267-274
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• 2017

Alpha lipoic acid (ALA) is a biological membranes compound. As the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E, and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM ALA increased significantly (P < 0.05) and exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50 uM of ALA during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the cytoplasmic maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

• 대한수의학회지
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• 제38권1호
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• pp.209-216
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• 1998

The purpose of present study is to improve the efficiency of fusion and the developmental rates of nuclear transplanted embryos to produce genetically identical twins from Korean native cattle. The diameter of aspirated follicles had no significant effect on the recovery rates of oocytes collected by ovum pick-up technique. The fusion rates of nuclear transplanted embryos were significantly higher in 50 and $100{\mu}s$ DC duration groups(73.3 and 72.0% ; respectively) than that in $30{\mu}s$ group(55.6% ; p<0.05). The cleavage rates of nuclear transplanted embryos appeared to be significantly higher in donor nuclei derived from in vivo (65.0%) than in those from in vitro (50.5% ; p<0.01), but the developmental rates to morulae and blastocysts were not significantly different between them(13.7 vs 10.9%, respectively).

• 한국수정란이식학회지
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• 제33권4호
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• pp.229-235
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• 2018

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus.