[학위논문]인간 배아 생산을 위한 하이브리드 마우스 체외수정 조건 확립에 관한 연구 Study on hybrid mice in vitro fertilization conditions established for the production of human embryo production원문보기
본 연구는 B6D2F1/CrljOri(모계: C57BL/6NCrIBR. 부계: DBA/2CrIBR) 교잡종 마우스를 이용하여 체외수정 및 체외 발달에서 최적의 조건을 찾기 위해 4가지로 설계하여 실시하였다. 1. 정소상체미부의 수송온도(4℃ vs. 37℃)가 정자의 생존율과 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 정자의 생존율(59.0%±0.1 vs. 47.6%±0.1), 체외 수정율(90.7%±0.1 vs. 90.7%±0.1), 발달율(90.0%±0.1 vs. 90.0%±0.1) 및 포배아 형성율(53.1%±0.2 vs. 52.3%±0.2)은 두 군 간에 유의적인 차이가 없었다(...
본 연구는 B6D2F1/CrljOri(모계: C57BL/6NCrIBR. 부계: DBA/2CrIBR) 교잡종 마우스를 이용하여 체외수정 및 체외 발달에서 최적의 조건을 찾기 위해 4가지로 설계하여 실시하였다. 1. 정소상체미부의 수송온도(4℃ vs. 37℃)가 정자의 생존율과 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 정자의 생존율(59.0%±0.1 vs. 47.6%±0.1), 체외 수정율(90.7%±0.1 vs. 90.7%±0.1), 발달율(90.0%±0.1 vs. 90.0%±0.1) 및 포배아 형성율(53.1%±0.2 vs. 52.3%±0.2)은 두 군 간에 유의적인 차이가 없었다(NS). 그러나 투명대 탈출율은 4℃군이 37℃군 보다 (47.8%±0.1 vs. 25.6%±0.2) 유의하게 높았다(p < .05). 2) 포배아의 세포수를 살펴보면, % ICM(/total cells)은 4℃군이 37℃군(27.0%±0.1 vs. 18.3%±0.1) 보다 유의하게 높았다(p < .05). 그러나 총 세포 수(72.7±31.6 vs. 62.0±36.6), ICM 세포 수(17.0±7.8 vs. 14.6±8.6), TE 세포 수(55.8±29.8 vs. 64.0±24.4), ICM:TE 비(1:4.2±4.1 vs. 1:6.4±7.2)는 두군 간에 유의한 차이가 없었다. 2. 난구세포의 유무(COC vs. O)가 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 체외 수정율(94.4%±0.1 vs. 86.7%±0.1)과 발달율(94.4%±0.1 vs. 84.4%±0.2) 및 투명대 탈출율(52.0%±0.1 vs. 51.4%±0.1)은 두군 간에 유의적인 차이가 없었다. 그러나 포배아 형성율은 COC군이 O군(61.1%±0.1 vs. 42.2%±0.1) 보다 유의하게 높았다(p < .05). 2) 포배아의 세포수를 살펴보면, 총 세포 수(85.3±29.5 vs. 60.6±35.2)와 ICM 세포 수(15.8±8.3 vs. 10.9±9.3)는 COC군이 O군보다 유의하게 높았다(p < .05). 그러나 TE 세포 수(69.6±28.0 vs. 49.6±33.4), % ICM(20.5%±0.1 vs. 20.6%±0.2), ICM:TE 비(1:6.5±6.6 vs. 1:6.9±5.5)는 두군 간에 유의한 차이가 없었다. 3. 오일의 종류(OVOIL vs. OIL)가 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 난구세포의 확장율(82.0%±0.2 vs 78.0%±0.1), 체외 수정율(92.0%±0.1 vs. 88.0%±0.1), 발달율(91.0%±0.1 vs. 87.0%±0.2), 포배아 형성율(56.0%±0.1 vs. 57.0%±0.1) 및 투명대 탈출율(41.4%±0.2 vs. 24.0%±0.1)은 두군 간에 유의차가 없었다. 그러나 난자의 성숙율은 OVOIL군이 OIL군(96.0%±0.1 vs. 87.0%±0.1) 보다 유의하게 높았다(p < .05). 2) 포배아의 세포수를 살펴보면, 총 세포 수(83.9±26.1 vs. 56.9±23.9), ICM 세포 수(15.7±8.8 vs. 6.3±3.5), TE 세포 수(68.3±25.7 vs. 50.7±24.1), % ICM(21.6%±0.1 vs. 12.7%±0.1) 및 ICM:TE 비(1:6.2±6.5 vs. 1:10.3±7.0)는 OVOIL군이 OIL군 보다 유의하게 높았다(p < .05). 4. 배양액 용량(25 ㎕ vs. 50 ㎕ vs. 100 ㎕)이 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 체외 수정율(82.7%±0.1 vs. 91.8%±0.1 vs. 90.1%±0.1), 발달율(79.1%±0.1 vs. 90.1%±0.1 vs. 87.3%±0.1) 및 투명대 탈출율(50.0%±0.4 vs. 43.4%±0.3 vs. 36.6%±0.2)은 실험군간 유의한 차이가 없었다. 그러나 포배아 형성율(33.6%±0.1 vs. 50.0%±0.1 vs. 48.2%±0.2)은 25 ㎕군 보다 50 ㎕군과 100 ㎕ 군이 유의하게 높았다(p < .05). 2) 포배아의 세포 수를 살펴보면, 총 세포 수 (73.7±35.1 vs. 71.1±31.9 vs. 75.6±35.6), ICM 세포 수 (16.7±12.2 vs. 14.5±10.7 vs. 12.0±8.2), TE 세포 수 (57.5±30.7 vs. 56.5±28.9 vs. 63.4±33.1), % ICM(22.9%±0.1 vs. 21.0%±0.1 vs. 17.9%±0.1) 및 ICM:TE 비(1:4.3±2.5 vs. 1:6.2±5.6 vs. 1:6.9±7.8)는 실험군 간에 유의차가 없었다. 결론적으로, 정소상체미부의 수송온도는 4℃, 수정 시 난자는 COC 상태, 오일은 OVOIL로 하고, 배양액 용량 50 ㎕ 또는 100 ㎕로 했을 때 난자와 배아의 발생능력이 가장 우수하였다. 본 실험 결과는 인간 체외 수정술에서 최적의 배양 환경을 간접적으로 알아보기 위해 실시하였으며, 응용이 가능할 것으로 사료된다.
본 연구는 B6D2F1/CrljOri(모계: C57BL/6NCrIBR. 부계: DBA/2CrIBR) 교잡종 마우스를 이용하여 체외수정 및 체외 발달에서 최적의 조건을 찾기 위해 4가지로 설계하여 실시하였다. 1. 정소상체미부의 수송온도(4℃ vs. 37℃)가 정자의 생존율과 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 정자의 생존율(59.0%±0.1 vs. 47.6%±0.1), 체외 수정율(90.7%±0.1 vs. 90.7%±0.1), 발달율(90.0%±0.1 vs. 90.0%±0.1) 및 포배아 형성율(53.1%±0.2 vs. 52.3%±0.2)은 두 군 간에 유의적인 차이가 없었다(NS). 그러나 투명대 탈출율은 4℃군이 37℃군 보다 (47.8%±0.1 vs. 25.6%±0.2) 유의하게 높았다(p < .05). 2) 포배아의 세포수를 살펴보면, % ICM(/total cells)은 4℃군이 37℃군(27.0%±0.1 vs. 18.3%±0.1) 보다 유의하게 높았다(p < .05). 그러나 총 세포 수(72.7±31.6 vs. 62.0±36.6), ICM 세포 수(17.0±7.8 vs. 14.6±8.6), TE 세포 수(55.8±29.8 vs. 64.0±24.4), ICM:TE 비(1:4.2±4.1 vs. 1:6.4±7.2)는 두군 간에 유의한 차이가 없었다. 2. 난구세포의 유무(COC vs. O)가 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 체외 수정율(94.4%±0.1 vs. 86.7%±0.1)과 발달율(94.4%±0.1 vs. 84.4%±0.2) 및 투명대 탈출율(52.0%±0.1 vs. 51.4%±0.1)은 두군 간에 유의적인 차이가 없었다. 그러나 포배아 형성율은 COC군이 O군(61.1%±0.1 vs. 42.2%±0.1) 보다 유의하게 높았다(p < .05). 2) 포배아의 세포수를 살펴보면, 총 세포 수(85.3±29.5 vs. 60.6±35.2)와 ICM 세포 수(15.8±8.3 vs. 10.9±9.3)는 COC군이 O군보다 유의하게 높았다(p < .05). 그러나 TE 세포 수(69.6±28.0 vs. 49.6±33.4), % ICM(20.5%±0.1 vs. 20.6%±0.2), ICM:TE 비(1:6.5±6.6 vs. 1:6.9±5.5)는 두군 간에 유의한 차이가 없었다. 3. 오일의 종류(OVOIL vs. OIL)가 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 난구세포의 확장율(82.0%±0.2 vs 78.0%±0.1), 체외 수정율(92.0%±0.1 vs. 88.0%±0.1), 발달율(91.0%±0.1 vs. 87.0%±0.2), 포배아 형성율(56.0%±0.1 vs. 57.0%±0.1) 및 투명대 탈출율(41.4%±0.2 vs. 24.0%±0.1)은 두군 간에 유의차가 없었다. 그러나 난자의 성숙율은 OVOIL군이 OIL군(96.0%±0.1 vs. 87.0%±0.1) 보다 유의하게 높았다(p < .05). 2) 포배아의 세포수를 살펴보면, 총 세포 수(83.9±26.1 vs. 56.9±23.9), ICM 세포 수(15.7±8.8 vs. 6.3±3.5), TE 세포 수(68.3±25.7 vs. 50.7±24.1), % ICM(21.6%±0.1 vs. 12.7%±0.1) 및 ICM:TE 비(1:6.2±6.5 vs. 1:10.3±7.0)는 OVOIL군이 OIL군 보다 유의하게 높았다(p < .05). 4. 배양액 용량(25 ㎕ vs. 50 ㎕ vs. 100 ㎕)이 난자의 발생능력에 미치는 영향은 다음과 같다. 1) 체외 수정율(82.7%±0.1 vs. 91.8%±0.1 vs. 90.1%±0.1), 발달율(79.1%±0.1 vs. 90.1%±0.1 vs. 87.3%±0.1) 및 투명대 탈출율(50.0%±0.4 vs. 43.4%±0.3 vs. 36.6%±0.2)은 실험군간 유의한 차이가 없었다. 그러나 포배아 형성율(33.6%±0.1 vs. 50.0%±0.1 vs. 48.2%±0.2)은 25 ㎕군 보다 50 ㎕군과 100 ㎕ 군이 유의하게 높았다(p < .05). 2) 포배아의 세포 수를 살펴보면, 총 세포 수 (73.7±35.1 vs. 71.1±31.9 vs. 75.6±35.6), ICM 세포 수 (16.7±12.2 vs. 14.5±10.7 vs. 12.0±8.2), TE 세포 수 (57.5±30.7 vs. 56.5±28.9 vs. 63.4±33.1), % ICM(22.9%±0.1 vs. 21.0%±0.1 vs. 17.9%±0.1) 및 ICM:TE 비(1:4.3±2.5 vs. 1:6.2±5.6 vs. 1:6.9±7.8)는 실험군 간에 유의차가 없었다. 결론적으로, 정소상체미부의 수송온도는 4℃, 수정 시 난자는 COC 상태, 오일은 OVOIL로 하고, 배양액 용량 50 ㎕ 또는 100 ㎕로 했을 때 난자와 배아의 발생능력이 가장 우수하였다. 본 실험 결과는 인간 체외 수정술에서 최적의 배양 환경을 간접적으로 알아보기 위해 실시하였으며, 응용이 가능할 것으로 사료된다.
In the present study, 4 different study designs were tested in order to find optimal conditions for in vitro fertilization and in vitro development utilizing a hybrid mice model, B6D2F1/CrljOri (Maternal line: C57BL/6NCrIBR, Paternal line: DBA/2CrIBR). 1. Effects of transfer temperature of epididymi...
In the present study, 4 different study designs were tested in order to find optimal conditions for in vitro fertilization and in vitro development utilizing a hybrid mice model, B6D2F1/CrljOri (Maternal line: C57BL/6NCrIBR, Paternal line: DBA/2CrIBR). 1. Effects of transfer temperature of epididymis on survival rate of semen and development ability of oocyte 1) No significant differences were noted in the survival rate of semen (59.0% ± 0.1 vs. 47.6% ± 0.1), in vitro fertilization rate (90.7% ± 0.1 vs. 90.7% ± 0.1), developmental rate (90.0% ± 0.1 vs. 90.0% ± 0.1), and blastulation rate (53.1% ± 0.2 vs. 52.3% ± 0.2) between groups. However, the zona hatched rate of blastocysts (47.8% ± 0.1 vs. 25.6% ± 0.2) was significantly higher in the 4°C group compared to those of the 37°C group (p<0.05). 2) Looking at the cell number in the embryo, % ICM(27.0%±0.1 vs. 18.3%±0.1) was significantly higher in the group of 4°C compared to the 37°C (p<0.05). However there were no differences in total cell numbers (72.7 ± 31.6 vs. 62.0 ± 36.6), ICM cell numbers (17.0 ± 7.8 vs. 14.6 ± 8.6), TE cell numbers (55.8 ± 29.8 vs. 64.0 ± 24.4), and ICM:TE ratio (1:4.2 ± 4.1 vs. 1:6.4 ± 7.2) between two groups. 2. Effects of cumulus cells (COC vs. O) on the oogenesis 1) No significant differences were noted on in vitro fertilization rate (94.4% ± 0.1 vs. 86.7% ± 0.1), clevage rate (94.4% ± 0.1 vs. 84.4% ± 0.2), and zona hatched rate (52.0% ± 0.1 vs. 51.4% ± 0.1) between groups. But blastulation rate (61.1% ± 0.1 vs. 42.2%) was higher in the COC group compared to the O group (P<0.05). 2) Looking at the cell number in the embryo, the total cell numbers (85.3 ± 29.5 vs. 60.6 ± 35.2) and ICM cell numbers (15.8 ± 8.3 vs. 10.9 ± 9.3) were higher in the COC group compared to the O group (P<0.05).But no further difference was not shown between two groups in the TC cell numbers (69.6 ± 28.0 vs. 49.6 ± 33.4), % ICM (20.5% ± 0.1 vs. 20.6% ± 0.2), and ICM:TE ratio (1:6.5 ± 6.6 vs. 1:6.9 ± 5.5). 3. Effect of types of oil (OVOIL vs. OIL) on the oogenesis 1) The expansion rate of cumulus cells (82.0% ± 0.2 vs 78.0% ± 0.1), in vitro fertilization rate (92.0% ± 0.1 vs. 88.0% ± 0.1), clevage rate (91.0% ± 0.1 vs. 87.0% ± 0.2), blastulation rate (56.0%±0.1 vs. 57.0%±0.1), and zona hatched rate (41.4% ± 0.2 vs. 24.0% ± 0.1) were not different between experimental groups. However, the OVOIL group showed higher maturation rate compared to that of the OIL group (96.0% ± 0.1 vs. 87.0% ± 0.1; P<0.05). 2) In the blastocysts cell numbers, the total cell numbers (83.9 ± 26.1 vs. 56.9 ± 23.9), ICM cell numbers (15.7 ± 8.8 vs. 6.3 ± 3.5), TE cell numbers (68.3 ± 25.7 vs. 50.7 ± 24.1), % ICM (21.6% ± 0.1 vs. 12.7% ± 0.1), and ICM:TE ratio (1:6.2±6.5 vs. 1:10.3±7.0) were significantly higher in the OVOIL group than the OIL group (p<0.05 for all). 4. Effect of culture media volume (25 μL vs. 50 μL vs. 100 μL) on the oogenesis 1) No differences were shown in in vitro fertilization rate (82.7% ± 0.1 vs. 91.8% ± 0.1 vs. 90.1% ± 0.1), clevage rate (79.1% ± 0.1 vs. 90.1% ± 0.1 vs. 87.3% ± 0.1), and zona hatched rate (50.0% ± 0.4 vs. 43.4% ± 0.3 vs. 36.6% ± 0.2) amongst tested groups. However, blastulation rate(33.6%±0.1 vs. 50.0%±0.1 vs. 48.2%±0.2) was found to be the higher in the 50 μL group and 100 μL group compared to that of 25 μL group (p<0.05). 2) Looking at the cell number in the embryo, the total cell numbers (73.7 ± 35.1 vs. 71.1 ± 31.9 vs. 75.6 ± 35.6), ICM cell numbers (16.7 ± 12.2 vs. 14.5 ± 10.7 vs. 12.0 ± 8.2), TE cell numbers (57.5 ± 30.7 vs. 56.5 ± 28.9 vs. 63.4 ± 33.1), % ICM (22.9% ± 0.1 vs. 21.0% ± 0.1 vs. 17.9% ± 0.1) and ICM:TE ratio (1:4.3 ± 2.5 vs. 1:6.2 ± 5.6 vs. 1:6.9 ± 7.8) were not different between groups. Taken altogether, it is expected to achieve the best developmental ability of embryos in which following conditions are given: 1) the transfer temperature of epididymis: 4°C, 2) cumulus cell-oocyte complex condition, 3) OVOIL, 4) the culture medium volume: either 50 μL or 100 μL. These experiments were carried out in order to determine optimal culture conditions for in vitro fertilization and human embryo culture. In future, therefore, it is expected that results herein might be applied for in vitro culture of human embryos.
In the present study, 4 different study designs were tested in order to find optimal conditions for in vitro fertilization and in vitro development utilizing a hybrid mice model, B6D2F1/CrljOri (Maternal line: C57BL/6NCrIBR, Paternal line: DBA/2CrIBR). 1. Effects of transfer temperature of epididymis on survival rate of semen and development ability of oocyte 1) No significant differences were noted in the survival rate of semen (59.0% ± 0.1 vs. 47.6% ± 0.1), in vitro fertilization rate (90.7% ± 0.1 vs. 90.7% ± 0.1), developmental rate (90.0% ± 0.1 vs. 90.0% ± 0.1), and blastulation rate (53.1% ± 0.2 vs. 52.3% ± 0.2) between groups. However, the zona hatched rate of blastocysts (47.8% ± 0.1 vs. 25.6% ± 0.2) was significantly higher in the 4°C group compared to those of the 37°C group (p<0.05). 2) Looking at the cell number in the embryo, % ICM(27.0%±0.1 vs. 18.3%±0.1) was significantly higher in the group of 4°C compared to the 37°C (p<0.05). However there were no differences in total cell numbers (72.7 ± 31.6 vs. 62.0 ± 36.6), ICM cell numbers (17.0 ± 7.8 vs. 14.6 ± 8.6), TE cell numbers (55.8 ± 29.8 vs. 64.0 ± 24.4), and ICM:TE ratio (1:4.2 ± 4.1 vs. 1:6.4 ± 7.2) between two groups. 2. Effects of cumulus cells (COC vs. O) on the oogenesis 1) No significant differences were noted on in vitro fertilization rate (94.4% ± 0.1 vs. 86.7% ± 0.1), clevage rate (94.4% ± 0.1 vs. 84.4% ± 0.2), and zona hatched rate (52.0% ± 0.1 vs. 51.4% ± 0.1) between groups. But blastulation rate (61.1% ± 0.1 vs. 42.2%) was higher in the COC group compared to the O group (P<0.05). 2) Looking at the cell number in the embryo, the total cell numbers (85.3 ± 29.5 vs. 60.6 ± 35.2) and ICM cell numbers (15.8 ± 8.3 vs. 10.9 ± 9.3) were higher in the COC group compared to the O group (P<0.05).But no further difference was not shown between two groups in the TC cell numbers (69.6 ± 28.0 vs. 49.6 ± 33.4), % ICM (20.5% ± 0.1 vs. 20.6% ± 0.2), and ICM:TE ratio (1:6.5 ± 6.6 vs. 1:6.9 ± 5.5). 3. Effect of types of oil (OVOIL vs. OIL) on the oogenesis 1) The expansion rate of cumulus cells (82.0% ± 0.2 vs 78.0% ± 0.1), in vitro fertilization rate (92.0% ± 0.1 vs. 88.0% ± 0.1), clevage rate (91.0% ± 0.1 vs. 87.0% ± 0.2), blastulation rate (56.0%±0.1 vs. 57.0%±0.1), and zona hatched rate (41.4% ± 0.2 vs. 24.0% ± 0.1) were not different between experimental groups. However, the OVOIL group showed higher maturation rate compared to that of the OIL group (96.0% ± 0.1 vs. 87.0% ± 0.1; P<0.05). 2) In the blastocysts cell numbers, the total cell numbers (83.9 ± 26.1 vs. 56.9 ± 23.9), ICM cell numbers (15.7 ± 8.8 vs. 6.3 ± 3.5), TE cell numbers (68.3 ± 25.7 vs. 50.7 ± 24.1), % ICM (21.6% ± 0.1 vs. 12.7% ± 0.1), and ICM:TE ratio (1:6.2±6.5 vs. 1:10.3±7.0) were significantly higher in the OVOIL group than the OIL group (p<0.05 for all). 4. Effect of culture media volume (25 μL vs. 50 μL vs. 100 μL) on the oogenesis 1) No differences were shown in in vitro fertilization rate (82.7% ± 0.1 vs. 91.8% ± 0.1 vs. 90.1% ± 0.1), clevage rate (79.1% ± 0.1 vs. 90.1% ± 0.1 vs. 87.3% ± 0.1), and zona hatched rate (50.0% ± 0.4 vs. 43.4% ± 0.3 vs. 36.6% ± 0.2) amongst tested groups. However, blastulation rate(33.6%±0.1 vs. 50.0%±0.1 vs. 48.2%±0.2) was found to be the higher in the 50 μL group and 100 μL group compared to that of 25 μL group (p<0.05). 2) Looking at the cell number in the embryo, the total cell numbers (73.7 ± 35.1 vs. 71.1 ± 31.9 vs. 75.6 ± 35.6), ICM cell numbers (16.7 ± 12.2 vs. 14.5 ± 10.7 vs. 12.0 ± 8.2), TE cell numbers (57.5 ± 30.7 vs. 56.5 ± 28.9 vs. 63.4 ± 33.1), % ICM (22.9% ± 0.1 vs. 21.0% ± 0.1 vs. 17.9% ± 0.1) and ICM:TE ratio (1:4.3 ± 2.5 vs. 1:6.2 ± 5.6 vs. 1:6.9 ± 7.8) were not different between groups. Taken altogether, it is expected to achieve the best developmental ability of embryos in which following conditions are given: 1) the transfer temperature of epididymis: 4°C, 2) cumulus cell-oocyte complex condition, 3) OVOIL, 4) the culture medium volume: either 50 μL or 100 μL. These experiments were carried out in order to determine optimal culture conditions for in vitro fertilization and human embryo culture. In future, therefore, it is expected that results herein might be applied for in vitro culture of human embryos.
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