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정범식,전익수,박수봉,최광수 韓國受精卵移植學會 1994 한국동물생명공학회지 Vol.9 No.3
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 g/ml) and FSH + LH added. And when the COC were co-cultured with granulosa cell (5 106 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, 1 106 cells /ml, 5:: 106 cells /ml, 1 107 cells /ml and 5 107 cells /ml.
Glucose와 Phosphate가 제거된 M-TALP 배지에서의 난구세포 공배양에 의한 임신율 향상에 관한 연구
정범식,장우현,이문희,김지연,방지호,김규현,서태광,Chung, B.S.,Chang, W.H.,Lee, M.H.,Kim, J.Y.,Bang, J.H.,Kim, K.H.,Suh, T.K. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.1
The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.
性腺자극호르몬과 牛胎兒血淸첨가가 돼지卵胞卵의 體外成熟 및 體外受精에 미치는 영향
정범식,박수봉,박항균,김규현 慶北大學校農業科學技術硏究所 1990 慶北大農學誌 Vol.8 No.-
본 실험은 性腺자극호르몬과 牛胎兒血淸첨가가 돼지 卵胞卵의 體外成熟 및 體外受精에 미치는 영향을 조사하기 위하여 실시하였다. 末經産豚(체중 80~90㎏)의 卵巢를 도살된 직후에 절취하여 37~39℃의 보온병에 담아 실험실로 운반하여 卵胞 직경이 3~5㎜되는 것만을 골라 卵胞를 찔러서 卵胞卵을 채취하였다. 成熟牡豚(체중 130~150㎏)의 精巢上體尾部精子를 ?? 농도로 희석하여 體外受精에 이용하였다. 본 실험의 결과를 요약하면 다음과 같다. 1. m-KRB와 10% FCS를 m-KRB에 첨가한 경우 成熟率은 82.37%이며, 10% FCS가 첨가된 배양액에 PMSG, hCG 그리고 PMSG와 hCG를 각각 10IU/㎖ 첨가한 경우 66, 58, 68%로서 成熟率이 향상되었다. 2. 卵丘細胞의 膨化는 m-KRB와 10% FCS가 첨가된 배양액에서 일어나지 않았으나 10% FCS가 첨가된 배양액에 PMSG, hCG 그리고 PMSG와 hCG를 각각 10IU/㎖ 첨가시 92, 13, 91%로서 膨化率이 향상되었다. 3. m-KRB에서 성숙된 卵胞卵의 體外受精에서 정자침입율과 雄性前核 형성율은 각각 93.7%였으나 FCS와 性腺자극호르몬을 첨가한 경우 각각 100, 80%로서 雄性前核 형성이 향상되었다. This study was carried out of investigate the effects of fetal calf serum (FCS) and gonadotropins supplemented to the medium on maturation and fertilization in vitro of porcine follcular oocytes. Ovaries were obtained from gilts at local slaughter-house. Oocyte-cumulus complexes were recovered by puncturing the ovarian follicles(3~5 ㎜ in diameter). The complexes from individual ovaries were pooled in a o.4㎖ droplet of medium covered with paraffin oil, then washed twice in fresh droplet and cultured for 36hrs in culture media according to experimental conditions. Boar epididymal spermatozoa were capacitated by preincubation for 4hrs in m-KRB medium and the preincubated spermatozoa were insemenated in the fertilization medium containing the cultured oocytes. The results obtained in this study are summarized as follows: 1. The maturation rates of oocytes cultured in m-KRB and m-KRB supplemented to 10% FCS were 82 and 37%, respectively. When PMSG, hCG. and PMSGt hcG(10Iu/㎖) were added to the media supplemented to 10% FCS, the maturation rates were 66, 58, and 68%, respectively. 2. Expansion of cumulus cells was not occured in m-KRB and m-KRB supplemented to 10% FCS. However, when PMSG, hCG and PMSG+hCG(10Iu/㎖) were added to m-KRB supplemented to 10% FCS, the expansion rates of cumulus cell layers were 92, 13, and 91%, respectively. 3. When oocytes were mltured in m-KRB, the rates of penetration and formation of male pronucle: were 93 and 7%, respectively By adding FCS and gonadotropin to m-KRB, the penetration and formation of male pronuclei were 100 80%, respectively.