• 한국가축번식학회지
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• 제17권4호
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• pp.287-294
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• 1994

This study was to investigate the effects of electrofusion, activation and developmental stage of donor embryos on in vitro development of nuclear transplant bovine embryos. A single blastomere nucleus from 8-cell to morula stage embryos produced by in vitro fertilization(IVF) was transferred into a recipient oocyte enucleated at 23∼25 h after in vitro maturation(IVM) or into a recipient oocyte enucleated and cultured for 14∼15 h. In one experiment the nuclear transplant embryos were subjected to additional activation treatments. Fusion rate of nuclear transplant eggs was high at direct current(D.C) voltages of 1.0 and 1.5 kV/cm 991.5 and 93.3%, respectively), but decreased at 2.0kV/cm (81.8%). Additional activation treatments by electric pulases or 7% ethanol did not affect the cleavage and development of nuclear transplant embryos. Development of nuclear transplant embryos slightly increased by delayed nuclear transfer and fusion (42∼43 h after IVM). With this system, blastocysts were obtained from transfer of 8-cell to morula stage donor nuclei (9.6%∼2.4%). The result of this study suggests that nucleo-cytoplasmic interactins, expecially activation of ooplast are very important for the development of nuclear transplant embryos, and donor cell stage does not affect the development of nuclear transplant embryos.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.99-110
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• 1994

One consequence of fertilization in mammals is an increased resistance of the zona pellucida (ZP) to proteases and various chemical reagents. This phenomenon has been called 'zona pellucida hardening' (ZPH), and it is generally accepted that it is caused by the secretory products of cortical granules released by the egg at fertilization. ZP of mouse oocytes maturing in vitro in a chemically defined medium becomes progressively more resistant to solubilization by chymotrypsin ("Spontaneous" ZP hardening). In the present study, it was aimed to find the specificity of spontaneous ZPH in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy. When a maturation inhibitors, cAMP analog(dbcAMP) and phosphodiesterase inhibitor (IBMX) was added to culture medium, it prevent spontaneous ZPH of mouse oocyte during in vitro culture. Thus spontaneous ZPH requires GVBD, since it is prevented by those agents, which inhibit GVBD in vitro. However, culture for 3 hours in the presence of PMA(lOng/ml), a protein kinase C activator, resulted in ZPH without GVBD, thus suggesting that ZPH may be regulated independently apart from the event of GVBD. Pretreatment of mouse oocyte with FBS result in partially inhibitory effect on subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhibitory effect on the spontaneous ZPH, but subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhinbitory effect on the spontaneous ZPII, but had no inhibitory effect on PMA-induced ZPH. Treatment with a microfilament formation blocker(cytochalasin-B) at 1${\mu}g$/ml concentration, resulted in the excellent inhibitory effect on spontaneous ZPH. However cytochalasin-B did not inhibit PMA-induced ZPH. Thus this suggesting that spontaneuse ZPH had a different mechanism from PMA-induced ZPH.

• 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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• 제16권3호
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• pp.173-182
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• 2001

본 연구는 돼지 난포란의 체외성숙배양액에 superoxide dismutase(SOD)를 첨가배양하여 체외성숙과 수성이후 배발달에 미치는 영향과 산소농도 및 SOD가 체외생산된 돼지초기수정란치 배발달에 미치는 영향을 규명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지 난포란을 체외성숙배양액인 NCSU-23에 SOD를 각각 0, 100,500 및 1,000uni1s/m1 첨가하여 성숙시킨 다음, 체외수정을 실시한 결과, 핵성숙률, 정자침입율, 다정자침입률 및 평균침입정자수는 처리구와 대조구간 유의적(P<0.05)인 차이가 인정되지 않았고, 웅성전핵형성률은 처리구(90.9∼81.2%)보다 오히려 대조구(95.0%)가 유의적으로 높은 결과를 나타냈다(P<0.05). 2. 체외수정 후, 배발달배양액인 NCSU-23에 7일 동안 배양한 결과, 배반포형성률과 총세포수는 처리구가 대조구보다 유의적(P>0.05) 으고 낮은 결과를 나타냈다. 3. 배발달배양액인 NCSU-23에 SOD를 각각 0, 100, 500 및 1,000uni1s/m1을 첨가하고 5% 및 20% 산소조건하에서 7일동안 배발달을 유기한 결과, 배양 7일째 산소조건에 따른 처리구간 배반포형성률 및 총세포수는 유의차가 없었으며, SOD농도별 배반포형성률은 100 units/ml 처리구에서 0, 500 및 1,000uni1s/m1 처리구 보다 유의적으로 높은 배반포형성률을 나타냈으나, 총세포수에서는 처리구(27.6∼37.3개)가 대조구(45.6개)보다 유의적(P<0.05) 로 낮은 결과를 나타내었다. 따라서 NCSU-23배양액에 SOD의 첨가는 돼지난포란의 체외성숙과 초기배발달에 영향을 미치지 않는 것으로 조사되었으며, 5% 및 20% 산소농도에 따른 영향도 없는 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제31권9호
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• pp.1420-1430
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• 2018

Objective: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and $200{\mu}M$), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the $50{\mu}M$ EGCG-treated group compared with the control group. Adding $50{\mu}M$ EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the $50{\mu}M$ EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the $50{\mu}M$ EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the $50{\mu}M$ EGCG-treated oocytes. Conclusion: In conclusion, $50{\mu}M$ EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

• 한국가축번식학회지
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• 제23권3호
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• pp.263-270
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• 1999

본 연구는 체외성숙배 양액에 L-ascorbic acid 와 selenium을 첨가 배양, 돼지 미성숙 난포란의 체외성숙, 체외수정 및 체외배발달에 미치는 영향을 검토코자 수행하였다. 배양액내 L-ascorbic acid를 0. 62.5. 100 그리고 30 $\mu$Ml 첨가 40~44시간동안 배양한 성적은 난핵포 붕괴율이 각각 86.8%, 92.9%, 91.7%, 그리고 92.6%였으며 핵성숙율은 각각 44.7%. 57.1%, 52.8%, 그리고 53.7%로 첨가구에서 유의적으로 높았다 (p＜0.05). 체외성숙배양액내 L-ascorbic acid와 selenium을 첨가 배양 후 체외 수정 유기 결과, 웅성전핵 형성율은 유의적으로 높았고 (p＜0.05) 다정자 침입율은 유의적으로 낮게 나타났으며 (p＜0.05), 체외수정 후 난할율, 상실배와 배반포배 발달율도 첨가구에서 유의적으로 높게 나타났다 (p＜0.05). 이러한 결과는 체외성숙 배양액내 L-ascorbic acid와 selenium을 첨가 배양했을 때 돼지 미성숙난포란의 체외 성숙율, 웅성 전핵 형성율 그리고 돼지 체외수정란의 배발달율을 향상 시킬 수 있으리라 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제40권1호
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• pp.7-11
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• 2013

Objective: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. Methods: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. Results: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. Conclusion: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

• 한국수정란이식학회지
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• 제19권2호
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• pp.165-172
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• 2004

본 연구는 보다 안정된 돼지 체외수정란 생산시스템 확립을 목적으로 체외성숙배양액 mNCSU-37, mNCSU-23 및 TCM-199 가 각각 미성숙 난포란의 체외성숙, 체외수정, 체외발달에 미치는 영향을 구명하기 위하여 수행하였다. 도축돼지의 난소에서 채취한 COCs를 10% pFF가 포함된 각각의 성숙배양액에서 최종동도가 1${\times}10^5/m{\ell}$ 의 농도로 체외수정 시킨 다음, NCSU-23 에서 체외발달을 유도한 결과는 다음과 같다. 1. TCM-199에서 성숙시킨 난자가 mNCSU-37 이나 mNCSU-23에서 성숙시킨 것보다 난핵포 붕괴율과 핵 성숙율에서 다소 낮은 경향을 보였으나 유의적인 차이는 아니었다. 2. 정자 침투율은 성숙배양액간 유의적인 차이를 나타내지 않았으나 웅성정핵 형성율은 mNCSU-37 에서 성숙시킨 난자가 88.0%로 TCM-199 에서 성숙시킨 것의 71.1%보다 유의적으로 높았다.(p<0.05). 3. 난분할율은 mNCSU-37(52.3%)이나 mNCSU-23(53.7%)에서 성숙시킨 난자가 TCM-199(43.1%)에서 성숙시킨 난자보다 유의적으로 높았다(p<0.05). 배반포발달율도 mNCSU-37이나 mNCSU-23에서 성숙시킨 것이 TCM-199에서 성숙시킨 것보다 다소 높았지만 유의적인 차이는 아니었다. 이상의 결과로 보아 돼지 미성숙 난포란 유래의 체외수정란 생산을 위한 체외성숙배양액으로 TCM-199보다 mNCSU-37이나 mNCSU-23이 적합한 것으로 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.31-31
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• 2001

This study was to establish in uitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, This study was to establish in vitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, $\geq$morula: 4.8%) and 7 hrs ($\geq$2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.morula: 4.8%) and 7 hrs (2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.

• 한국발생생물학회지:발생과생식
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• 제10권4호
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• pp.239-245
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• 2006

본 연구는 소의 미성숙 난포란의 체외성숙시 ${\beta}-mercaptoethanol({\beta}-ME)$의 첨가가 체외성숙, 체외수정 후 웅성전핵의 형성 및 세포질 내의 GSH 수준에 미치는 영향을 알아보고자 실시하였다. 체외성숙시 $25\;{\mu}M$과 $50\;{\mu}M$의 ${\beta}-ME$를 첨가한 경우 대조구에 비하여 성숙율이 증가하는 것으로 나타났으며(p<0.05), 모든 실험구에 있어서 12시간 체외성숙보다 24시간 체외성숙에서 높은 성숙율을 나타냈다(p<0.05). 체외수정 후 웅성전핵 형성에 있어서는 $25\;{\mu}M$와 $50\;{\mu}M$ 농도의 ${\beta}-ME$ 첨가구에서 대조구에 비하여 높게 나타났으나(p<0.05), $25\;{\mu}M$과 $50\;{\mu}M$ 농도구와의 유의적인 차이는 없었다. GSH의 수준은 체외성숙 후 $50\;{\mu}M$의 ${\beta}-ME$ 첨가구가 다른 처리구에 비교하여 높게 나타났으며(p<0.05), 체외수정 후 웅성전핵이 형성된 다음 세포질 내 GSH 수준 역시 $50\;{\mu}M$의 ${\beta}-ME$ 첨가구에서 가장 높은 결과를 나타냈다(p<0.05).