• 한국가축번식학회지
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• 제13권2호
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• pp.70-78
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• 1989

Mouse eggs recovered from oviducts at one hourly intervals between 10 and 20 hours after administration of hCG were fixed, stained and then investigated the rate of in vitro fertilization and nuclear maturation. In case of out- bred ICR mice, ovulations were occured between 11 and 13 hours after hCG injection. The stages of in vitro maturation of eggs recovered from female mice at various times after hCG injection were metaphase I, anaphase I, telophase I and metaphase II. However the majority was metaphase I(17.6 to 44.4%) and metaphase II(42.9 to 80.0%) stage. When the eggs were inseminated with epididymal spermatozoa, the fertilization rate was declined as the egg recovery time after hCG administration was delayed. That is, the proportion of eggs undergoing fertilization became higher(68.1 to 77.4%) in the eggs at 12 to 15hr after injection of hCG than those(17.5 to 56.4) at 16 to 20 hr after injection of hCG. Also, when nuclear maturation of the unfertilized eggs were observed at 8 hours after insemination, the majority was in metaphase I and metaphase II and no anaphase I and telophase I were observed.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.235-240
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• 2004

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.193-197
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• 2013

In order to investigate the effects of antioxidants on the culture of mouse preantral follicles in vitro, we examined the effects of taurine, glutathione and catalase on their growth and maturation. Addition of taurine was not effective on the survival of preantral follicles. However, metaphase II rates of oocytes within preantral follicles were significantly higher in 1 mM treated group than in control and 10 mM treated group (p<0.05). Glutathione did not improved the rates of survival and metaphase II oocytes. However, metaphase II rates of oocytes progressively decreased with increasing glutathione concentration. Catalase also showed that the rates of survival and metaphase II oocytes progressively decreased with increasing concentration. Especially, all of preantral follicles cultured in medium containing 100 IU/ml catalase were degenerated. These results suggest that low concentraion of taurine, as an antioxidant, have positive effect on the culture of mouse preantral follicles in vitro.

• 한국가축번식학회지
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• 제24권4호
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• pp.375-383
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• 2000

본 연구는 공여난소의 황체 형태가 소 미성숙난자의 체외 성숙과 발생에 미치는 영향을 감수분열과 수정란 생산을 비교하여 조사하였다. 공여난소를 황체의 형태에 따라 다음과 같이 4 group 으로 분류하였다. Group 1 ; 최근에 배란한 흔적으로 보이는 혈포가 있는 난소. Group 2 ; 황체는 적색 또는 갈색이며, 혈관이 황체의 가장자리에 국한되어 분포하고 있는 난소. Group 3 ; 황체는 오렌지 또는 황갈색이며, 혈관이 황체의 정상부 까지 분포한 난소. Group 4 ; 황체는 잃은 황색 또는 백색이며, 황체 정상부의 혈관분포가 사라진 난소. 미성숙난자의 체외성숙과 배발생을 위하여 각각 TCM 199과 TLP-PVA을 기본배지로 사용하였다. 각 group에서 채취한 미성숙 난자를 4, 14, 24시간 체외성숙시켰을 때 metaphase I과 metaphase II에 도달한 난자의 비율은 각 group 사이에 차이가 없었으며, 각 group 에서 metaphaseII난자는 체외성숙 후 14시간에 나타나기 시작하여 24시간에 성숙이 종료하였다. 체외 배발생 후 2세포기와 8세포기까지의 발생에는 공여난소 황체의 형태가 영향을 미치지 않았으나, 상실배와 배반포로 발생한 수정란의 비율은 group 1과 3에서 채취한 난자에서 group 2와 4에서 채란한 난자에 비하여 유의적으로 증가하였다 (p＜0.05). 이러한 결과는 공여난소의 번식상태가 체외 수정란생산에 유의적인 영향을 미치는 것으로 암시하고 있다.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.71-76
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• 2007

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM This is approximately 500$\sim$1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (${\sim}50{\mu}M$) and butyrolactone (${\sim}100{\mu}M$). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone HI kinase and MAP kinase in mouse oocytes.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.301-305
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• 2006

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48hr of culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%, 51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.

• 한국산학기술학회논문지
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• 제11권6호
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• pp.2124-2128
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• 2010

본 연구에서는 MAPK 저해제인 U0126이 난자성숙과정에서 특히 감수분열, 미세소관 형성 그리고 액틴 필 라먼트 형성에 미치는 영향을 조사하였다. 그 결과 MAPK 단백질은 12시간째에 인산화되기 시작하여, 24시간째에 대부분 인산화 되었고 metaphase II에 이르기 까지 유지되었다. 배포단계(GV)에 있는 난자를 U0126의 $20{\mu}M$ 농도로 처리하였을 때 MAPK의 인산화가 완전히 억제되었으나 배포의 파열 단계(GVBD)로의 성숙에는 진행하였으나, metaphase I까지는 발달하지 못하였다. 또한 MAPK 저해제로 인해 비정상적인 방추사의 형성을 초래하였다. 난자를 배포의 파열단계(GVBD) 이후에 U0126을 처리하였을 때 극체의 방출은 정상 이였으나 중기 판의 배열과 염색체의 분열은 비정상적 이였다. 결론적으로, 유사분열 활성화 효소단백질인 MAPK의 활성은 돼지 난자의 체외성숙과정에서 배포단계(GV)의 염색체의 배열과 감수분열의 완성에 중요한 조절 인자임을 이번 연구를 통해 알 수 있었다.

• 한국수정란이식학회지
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• 제7권1호
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• pp.41-47
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• 1992

1. In vitro system, LR and FSR accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus. 2. Caffeine (2mM) in the fetilization medium was required not only for inducing zona penetrating ability of boar also for developing to the male pronucleus of the penetrat- ing spermatozoa in vitro. 3. The germinal vesicle (GV)stage was observed for the first 17.6 hr;germinal vesicle break-down (GVBD)stage between 17.6~26.4 hr ;metaphase I (M-I)from 26.4 - 30. 9hr;anaphase I(A-I)ranged from 30. 9~33.4hr;telophase I(T-I) at 33.4~34.4hr; and metaphase II(M-II) at 34.4-48hr. 4. The addition of 10%(v /v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (p<0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. 5. The presence of a primary culture of POEC promotes in vitro development of early cleavage stage pig embryos.

• 한국수정란이식학회지
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• 제19권3호
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• pp.283-289
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• 2004

동결 과정 중 필수적인 단계중 하나인 냉각(cooling)과 냉각 후 배양시간이 생쥐 난자의 방추체의 형태와 염색체의 배열에 미치는 영향을 알아봄으로서 냉각 후 손상되었던 난자의 방추체와 염색체가 정상적으로 회복하는데 필요한 최적의 배양시간을 알아보기 위해 본 실험을 실시하였다. 생후 4-6주령의 암컷 B6C3Fl 생쥐를 과배란 처리하여 metaphase II상태의 난자를 회수하여 다음과 같이 처리하였다. 대조군은 난자를 냉각처리하지 않았으며 실험군은 난자를 $0^{\circ}C$에서 30분간 냉각한 후 37$^{\circ}C$에서 가온하여 즉시 일부 난자는 면역형광 염색을 실시하고 나머지 난자는 5% $CO_2$ 37$^{\circ}C$가 유지된 배양기내에서 Ml6 배지에 각각 5분, 15분, 30분, 60분, 120분간 배양한 후 면역 형광염색을 실시하였다. 난자의 방추체와 염색체를 평가하기 위한 면역형광염색은 Zenes 등의 방법(2001)에 준하여 실시하였다. 냉각처리하지 않은 생쥐 난자를 면역형광 염색하여 방추체와 염색체를 관찰한 결과 생쥐 metaphase II 상태의 난자는 대칭성의 원통모양의 방추체 형태를 보였으며 염색체는 metaphase plate위에 분리된 다발모양으로 밀집되어 보였다. 냉각 직후 미세관의 소실에 의한 방추체 형태의 이상과 형광성의 소실이 나타났으며 염색체는 다발모양의 밀집된 형상에서 벗어나 비정상적인 배열상을 보였다. 냉각 처리된 난자를 37$^{\circ}C$에서 가온하고 배양하였을 때 미세관의 재중합이 일어나 미세관의 형광성을 회복하기 시작하였고 방추체는 정상적인 배열상으로 회복되었다. 생쥐 난자를 냉각처리한 후 배양시간에 따른 방추체 미세관의 형광성(FIS), 염색체의 배열, 방추체의 형태를 비교하였다. 배양 5분에서 60분까지 FIS, 정상 염색체 배열을 보인 난자의 비율, 정상 방추체의 형태를 보인 난자의 비율이 점진적으로 증가하였으나 120분 배양에서는 감소하였다(P<0.05). 위의 세 가지 평가를 기준으로 하여 냉각 후 난자의 회복율을 관찰하였을 때 배양 60분에서 최상의 회복율을 나타냈다.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.147-152
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• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.