우 난포란의 (卵胞卵) 체외성숙에 관한 연구 Ⅰ. 난포란의 회수 및 체외배양

윤산현(S . H . Yoon),고대환(D . H . Ko),박세필(S . P . Park),박태균(T . G . Park),정길생(K . S . Chung) 한국축산학회 1989 한국축산학회지 Vol.31 No.4

These experiments were carried out to develop the effective recovering method of ovarian follicular oocytes and to investigate the optimum culture conditions for in vitro maturation of recovered follicular oocytes. Bovine follicular oocytes with various configurations of cumulus cell mass were collected by aspirating follicular fluid from the ovarian follicles of various size, and then cultured in various media for 28h in a incubator with 5% C0₂ in air at 38℃. The results obtained in these experiments were summarized as follow: 1. The average number of the follicular oocytes recovered from a cow was 46.4 and the highest recovery rate was obtained from middle size follicles, 3-5㎜ in diameter. 2. The majority of the follicular oocytes with compacted cumulus cells existed in GV stage While those with dispersed cumulus cells or without cumulus cells existed between GVBD and MⅡ stage. 3. The extrusion rate of Ist polar body of the follicular oocytes cultured in Ham`s F-10, m-KRB and BMOC-3 containing hormones was not higher than those of oocytes cultured in those media without hormones. However, in case of m-TALP medium, the highest extrusion rate of Ist polar body, 77.7% was obtained by adding hormones such as FSH, HCG and Estradiol. 4. The maturation rate of the follicular oocytes cultured in basic medium without or with matured cumulus cell was 40% or 51.1%, respectively, with significant difference(p$lt;0.05).

Cryopreservation of Human Multi-Pronuclear (PN) Zygote by Ultra-Rapid Freezing

김은영,이봉경,남화경,이금실,윤산현,박세필,정길생,임진호,Kim, E.Y.,Yi, B.K.,Nam, H.K.,Lee, K.S.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.2

본 연구는 인간 전핵기 수정란의 동결시 election microscope grid론 사용하는 초급속 동결방법이 유용하는지 여부를 조사하고자 실시하였다. 본 실험에서는 인간 IVF 시술시 생성되는 다-전핵기 (>2PN) 수정란을 정상 2PN 수정란 대신 사용하였으며, 또한 이들은 3PN과 $\geq4PN$ 수정란으로 나누어 전핵의 수에 따른 냉해와 융해 후 체외 배발달율을 조사하였다. 동해제는 30% ethylene glycol, 18% ficoll, 0.5 M sucrose와 10% FBS 등이 D-PBS에 첨가되어 제작된 EFS30을 사용하였다. 본 연구에서 얻어진 결과는 다음과 같다. 초급속동결-융해 후, 인간 다-전핵기 수정란의 생존율은 85.5%였다. 대조군과 동결군의 난할율을 진핵의 수에 따가 니누어 비교하였을 때, 각 군간에는 유의한 차이를 나타내지 않았다 (3PN; 81.3%와 85.4%, $\geq4PN$; 90.0%와 95.7%). 또한, 융해 후 체외발달율을 조사하였던바, $\geq4PN$수정란에서는 동결군 (4.5%)의 체외발달이 대조군 (44.4%)보다 유의하게 낮게 나타났던 반면, 3PN 수정란에서는 동결군 (22.0%)의 결과가 대조군 (38.5%)에 비해 유의한 차이를 나타내지 않았다. (p<0.05). 따라서 인간 다-전핵기 수정란은 EM grid와 EFS30 동결액을 이용한 초급속 동결로 배발달을 유도할 수 있다는 것을 알 수 있었다. The objective of this study was to test whether the developmental capacity of human multi-pronuclear (PN) zygotes after ultra-rapid freezing using EM grid can be maintained. For this experiment, multi-PN zygotes which produced in human IVF program were used as an alternatives of normal 2PN zygotes, and they were separated into 3PN or $\geq4PN$ zygotes to compare their in vitro development and cryoinjury according to PN number. As freezing solution, EFS30 which consisted of 30% ethylene glycol, 18% bcoll, 0.5 M sucrose and 10% FBS added D-PBS was used. The result obtained in this experiment was summarized as follows; When the multi..PN zygotes were ultrarapidly frozen and thawed, the high mean percentages (85.5%) were survived. Also when the cleavage rates between control and freezing group were compared with PN number, there were not significantly different in each group (3PN; 81.3% & 85.4% and $\geq4PN$; 90.0% & 95.7%). When the in vitro development rates after thawing were examined, freezing 3PN group (22.0%) was not differed to control 3PN group (38.5%), although the development result of freezing $\geq4PN$ group (45%) was significantly lower than that of control $\geq4PN$ group (44.4%) (p<0.05). These results demonstrate that developmental capacity of human zygote can be obtained by ultra-rapid freezing method using EM grid and EFS30.

Effect of Epidermal Growth Factor on In Vitro Maturation in Pig Immature Oocytes;IV. Effect of Epidermal Growth Factor on In Vitro Development

엄상준,김은영,김묘경,김태완,윤산현,박세필,정길생,임진호,Uhm, S.J.,Kim, E.Y.,Kim, M.K.,Kim, T.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.3

본 연구는 epidermal growth factor (EGF)가 처리된 돼지 체외성숙란의 채외수정 후 배발달을 조사하기 위하여 실시하였다. 난구세포가 치밀한 미성숙란은 TCM 199 배양액에 (1) 무처리군 (2) 10 ng/ml EGF 처리군 (3) 10 ${\mu}g/ml$ FSH와 10% FBS 처리군 (4) 10 ng/ml EGF와 10 ${\mu}g/ml$ FSH 그리고 10% FBS 처리군으로 나누어 42시간 동안 배양하였다. 실험 1은 이들 처리군을 수정 후 NCSU (0.4% BSA) 배양액에서 배양하여 후기 배발달을 조사하였다. 그 결과 (3)과 (4)처리군 (13.4, 18.3%)의 배반포로의 발달율이 (2) 처리군(5.2%, p < 0.005)보다 현저하게 높았지만, (2) 처리군의 경우 (1) 처리군(1.2%, p < 0.005)의 배반포로의 발달보다는 현저하게 높았다. 실험 2는 수정 이후 6일째 이들 처리군에서 생산된 배반포기배를 double staining (propidium iodide and bisbenzimide) 방법을 이용하여 세포수를 조사하였다. 그 결과 (4) 처리군 (58.80 ${\pm}$ 11.90)의 배반포의 total 세포수가 (2)와 (3) 처리군 (42.17 ${\pm}$ 9.97, 49.07 ${\pm}$ 9.77, p < 0.05)보다 현저하게 많았으며, 배반포의 ICM 수에 있어서도 (4) 처리군 (11.69 ${\pm}$ 5.56)이 (2)와 (3) 처리군 (5.00 ${\pm}$ 4.24, 6.77 ${\pm}$ 4.92, p < 0.05)보다 현저하게 많았다. 더 우기 (4) 처리군 (19.0 ${\pm}$ 1.6)의 total 세포수에 대한 ICM의 비율이 (2)와 (3) 처리군 (11.1 ${\pm}$ 3.0, 12.7 ${\pm}$ 2.1) 보다 높았다. 이들의 결과로서, EGF가 단독 처리된 돼지 체외성숙란은 비록 낮았지만 배반포로 발달할 수는 있으나, 체외성숙시 FSH와 FBS의 추가 첨가는 높은 배반포로의 발달과 total 세포수와 ICM의 증가를 가져올 수 있음을 알수 있었다. The objective of this study was to examine the effect of embryos development following IVF of in vitro-matured porcine oocytes treated with epidermal growth factor (EGF). When cumulus-enclosed oocytes were incubated in TCM 199 medium supplemented with (1) control group, (2) 10 ng/ml EGF, (3) 10${\mu}g$ml FSH and 10% FBS, or (4) 10 ng/ml EGF, 10 ${\mu}g$/ml FSH, and 10% FBS for 42 hr, the late developmental rates on NCSU (0.4% BSA) medium after fertilization were higher in (3) and (4) groups (13.4, 18.3%) than in (2) group (5.2%, p < 0.005), but (2) group is significantly higher than the development to blastocyst of oocytes of (1) group (1.2%). Also, when the cell number of total, ICM, and TE of those blastocysts at 6 day produced in vitro was investigated by double staining (PI and bisbenzimide), total cell number of (4) group (58.80${\pm}$ 11.90) was higher than that of (2) and (3) groups (42.17${\pm}$9.97, 49.07${\pm}$9.77, P < 0.05). ICM cell number of blastocysts of (4) group (11.69${\pm}$5.56) was higher than that of (2) and (3) groups (5.00${\pm}$4.24, 6.77${\pm}$4. 92, P < 0.05). Furthermore, the proportion of ICM in (4) group (19.0${\pm}$1.6) was higher than that in (2) and (3) groups (11.1${\pm}$3.0, 12. 7${\pm}$2.1). These results suggested that in vitromatured porcine oocytes treated with EGF alone can be developed to blastocyst, but high proportion on the development to blastocyst and number of total cell and ICM in blastocyst can be obtained when supplemented with additional FSH and FBS.

In Vitro/In Vivo Development after Thawing of Vitrified Mouse Blastocysts by Culture Condition and Embryo Transfer Method

김묘경,김은영,이봉경,윤산현,박세필,정길생,임진호,Kim, M.K.,Kim, E.Y.,Yi, B.K.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.3

본 실험은 초자화 동결된 생쥐 배반포기배의 융해 후 배양조건 및 이식방법이 난자의 생존에 미치는 효과를 조사하고자 실시하였다. 체외수정후, M16배양액에서 4일동안 배양하여 얻어진 생쥐 배반포기배는 EFS40 (40% ethylene glycol, 18% Ficoll, 0.5 M sucrose가 함유된 PBS)으로 초자화동결하였다. 실험 I에서는 융해 후 배양조건에 따른 난자들의 체외/체내 생존율을 조사하였다. 융해된 난자가 M16과 4 mg/ml 소혈청알부민과 20 가지 아미노산이 함유된 m-CR1 (2% BME 아미노산 용액, 1% MEM 아미노산 용액) 및 단층배양이 유도된 난구세포 (10% FBS가 함유된 m-CR1배양액)에서 각각 배양되었을 때, 융해 후 24시간째 체외 생존율은 배양조건에 따라 차이가 없었다(75.6, 83.1, 82.4%). 그러나 체내 발달율에 있어서 임신 15일째 생존 산자율은 39.0, 49.0, 38.1%로서 유사한 성적을 나타냈으나, 전체 착상율에 있어서는 m-CR1 (80.4%)에 배양되었을 때, M16 (51.2%), 난구세포와 공배양시 (57.1%)보다 유의하게 높은 생존율을 보였다(p<0.05). 실험 II에서는 수정란 이식 방법에 따른 체내 발달율을 조사하였다. 배반포기배를 융해 후 체외배양없이 곧바로 가임신 2, 3일째 대리모에 이식을 실시하였을 때, 가임신 2일째 대리모에서는 임신징후를 얻지 못하였고, 가임신 3일째 대리모에서는 50.0%의 착상율과 15.4%의 정상산자율을 얻었다. 그러나, 이러한 결과는 융해 후, 16시간 배양하여 가임신 3일째 대리모에 이식 (73.5, 57.1%)하는 경우보다 유의하게 낮은 결과였다(p<0.05). 실험 III에서는 초자화 동결된 배반포기배의 융해 후 배양시 발달이 늦어진 수정란의 이용효율을 극대화시키기 위해 융해한 4일째 초기, 5일째 초기, 5일째 팽창 배반포기배의 체외/체내 생존율을 조사하였다. 가장 높은 체외 생존율은 5일째 팽창 배반포기배 (78.3%)에서 얻었으나, 체내 발달율 (산자율, 착상율)에 있어서는 4일째 초기 배반포기배 (33.3, 66.7%)의 경우가, 5일째 팽창 배반포기배(29.0, 38.7%)의 경우보다 높았다(p<0.05). 따라서 본 연구의 결과는 배양조건과 수정란 이식방법에 따라 초자화 동결된 배아의 체외/체내 발달율을 높일 수 있으며, 발달이 늦은 배반포기배의 체내 발달율은 체외 배양시간이 길어질수록 낮아짐으로, 5일째 팽창 배반포기배보다 4일째 초기 배반포기배를 동결하는 것이 더 유용하다는 것을 알 수 있었다. This study was to test whether in vitro/in vivo survival of vitrified mouse blastocysts was influenced by culture conditions and ET method. Mouse blastocysts were obtained from in vitro fertilization and cultured for 4 days in M16 medium, and they were vitrified in EFS40 which contained 40% ethlyene glycol, 18% Ficoll and 0.5 mol sucrose in PBS. In experiment I, in vitro and in vivo survival rate of these embryos were evaluated in different culture condition after thawing. When thawed embryos were cultured in M16 medium as a control, m-CR1 medium contained 20 amino acids (2% BME amino acis and 1% MEM non-essential amino acids solution) and 4 mg/ml BSA and cumulus monolayer cell co-cultured condition in mCR1 medium (10% FBS), their in vitro survival at 24 hr after thawing was not affected by culture condition (75.6, 83.1, 82.4%). However, in vivo survival rates of implantation in m-CR1 medium (80.4%) were significantly higher than those of M16 medium (51.2%), co-culture (57.1%) condition, although there was no difference in live fetuses rates on day 15 gestation (39.0, 49.0, 38.1%). In experiment II, the in vivo development potential of embryos by ET methods was examined. When blastocysts were transferred to the day 2, 3 pseudopregnant recipient without culture soon after thawing, no pregnant recipient was obtained on the day 2 pseudopregnancy, and 50% of pregnancy rates and 15.4% of live fetus rates were obtained on the day 3 pseudopregnant recipients. These results were significantly lower than those of transferred group (day 3 pseudopregnant recipients) after culture for 16 hr post thawing (73.5, 57.1%) (p<0.05). In experiment III, to elevate usability of delayed embryos in vitro/in vivo survival of vitrified embryos (day 4 early, day 5 early and expanding blastocyst) were examined. in vivo survival rates (live fetus, total implantation) were higher in day 4 early blastocysts (33.3, 66.7%) than in day 5 expanding blastocysts (29.0, 38.7%), although the highest in vitro survival rates were obtained in the day 5 expanding brastocysts (78.3%). Therefore, these results suggest that the in vitro/in vivo survival rates of vitrified embryos could be improve by the culture condition and ET method and that the in vivo development rates of delayed embryos were decreased with longer culture duration in vitro. It means that more effective cryopreservation was obtained in day 4 early blastocysts than in day 5 expanding blastocysts.

The Cytoskeletal and Chromosomal Constitution in Vitrified Mouse Oocyte

이봉경,김은영,남화경,이금실,윤산현,박세필,임진호,Yi, B.K.,Kim, E.Y.,Nam, H.K.,Lee, K.S.,Yoon, S.H.,Park, S.P.,Lim, J.H. The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.3

본 연구는 동해방지제인 EFS35를 이용한 생쥐 미수정란의 초자화동결이 cytoskeleton과 염색체에 미치는 영향을 indirect immunocytochemistry와 염색체 분석으로 확인하고자 실시하였다. 생쥐 미수정란은 M2 배양액에 35% ethylene glycol, 18% ficoll, 0.3 M sucrose와 10% FBS가 함유된 EFS35를 이용하여 초자화동결되었다. 본 연구에서 얻어진 결과는 다음과 같다 초자화동결된 미수정란의 융해후 생존율은 89.3%로써 노출군 (97.7%)과 차이가 없었다. 초자화동결이 미수정란의 microtubule과 microfilament에 미치는 영향을 조사하였던 바, 동결군 (95.5, 100%)의 microtubule과 microfilament의 정상적인 형성율은 대조군 (97.5, 100%) 또는 노출군 (92.3, 100%)의 결과와 비교하였을때 차이가 나타나지 않았다. 또한, 체외수정 후 동결 군에서 정상적인 염색체수를 가진 난자의 비율도 73.5%로써, 대조군 (79.5%)과 노출군 (78.7%)의 결과와 차이가 없었던 바, 생쥐 미수정란을 EFS35에 노출하고 동결하는 것이 미수정란의 cytoskeleton과 염색체 성상변화에 영향을 미치지 않는다는 것을 알 수 있었다. 따라서, 본 연구에서 사용된 동해방지제인 EFS35를 이응한 초자화동결법은 생쥐 미수정란의 동결에 적합하다는 것을 시사한다고 하겠다. The objective of this study was to confirm whether the vitrification method using EFS35 has detrimental effect for cytoskeleton and chromosome constitution of the mouse oocytes by indirect immunocytochemistry and chromosome analysis. Mouse oocytes were vitrified using EFS35 which consisted of 35% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS in M2 medium. The results obtained in this experiment were summarized as follows: When the survival rates after being exposed or vitrified in EFS35 were examined, there were not different between two groups (97.7 and 89.3%). Also, when the microtubule morphology and microfilament distribution in vitrified oocytes were examined, normal percentage of two cytoskeleton in vitrified group (95.5 and 100%) was not different from that in control (97.5 and 100%) and exposed group (92.3 and 100%). In addition, the rate of oocytes containing a normal chromosome number in vitrified group (73.5%) after IVF was not different from that in control (79.5%) and exposed group (78.7%). These results indicated that the cytoskeletal morphology and chromosome constitution of mouse oocytes were not affected by cryoprotectant (EFS35) or freezing apd that vitrification methods using EFS35 was suitable for cryopreservation of mouse oocytes.

In Vitro/In Vivo Development of Mouse Oocytes Vitrified by EFS

김묘경,김은영,이봉경,윤산현,박세필,정길생,임진호,Kim, M.K.,Kim, E.Y.,Yi, B.K.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

본 연구는 생쥐 미수정란을 초자화 동결하였을 때, 체내/외 발달율에 미치는 영향을 연구하고자 실시하였다. 생쥐 미수정란은 30, 35, 40% ethylene glycol, 18% ficoll과 0.5 M sucrose가 함유된 M2 배양액으로 구성 된 EFS30, 35, 40을 이 용하여 초자화 동결되었다. 노출 또는 초자화 동결-융해 후, 형태학적으로 정상적인 미수정란은 $1-2\times10^6/ml$ 농도의 정자로 체외수정되었고, 수정 율과 체내/외 발달율 그리고 배 반포의 세포수 (inner cell mass 와 tropectoderm cell)가 조사되었다. 본 실험에서 얻어진 결과는 다음과 같다. 35%의 ethylene glycol이 함유된 EFS35에서 EFS30과 EFS40보다 높은 분할율을 나타냈다. 초자화 동결-융해 후 체외수정된 미수정란의 2-세포기까지의 발달을 (51.1%)은 동결없이 초자화 동결액에 노출만된 군 (60.0%)과 대조군 (68.2%)에 비해 유의하게 감소하였다 (p<0.05). 그러나 이들 처리군에 있어서 난할된 난자로 부터의 배반포기 배까지의 발달율에는 유의한 차가 없었다. (75.0, 73.3 과 80.0%). 또한 초자화 동결된 군의 배반포기배 세포수 $(92.5{\pm}2.9)$도 노출군 $(98.5{\pm}5.3)$, 대조군 $(100.9{\pm}4.8)$과 유사하였다. 초자화 동결-융해 하여 얻어진 배반포기배를 가임신 생쥐에 이식하였을 때 체내발달율인 산자발달율 (50.7%)과 착상율 (80.0%)도, 대조군 (58.2, 78.2%)과 유사하였다. 이러한 결과로 보아, 생쥐 미수정란은 ethylene glycol를 기본으로 한 EFS35라는 초자화 동결액을 이용하여 동결보존될 수 있음을 알 수 있었다. This study was carried out to investigate in vitro/in vivo development of vitrified mouse oocytes. Mouse oocytes were vitrified using EFS30, 35 and 40 (30, 35 and 40% ethylene glycol, 18% ficoll and 0.5 M sucrose in M2 medium). After being exposed or vitrified-thawed, oocytes of normal morphology were inseminated in vitro by $1-2\times10^6/ml$ of epididymal sperm. The rates of fertilization, in vitro/in vivo development and cell number (inner cell mass and tropectoderm cell) of blastocysts in each treatment group were examined. The results obtained in these experiments were summarized as follows: The cleavage rates were obtained in EFS35 containing 35% ethylene glycol higher than in EFS30 and EFS40. The development rate of vitrified-thawed oocytes to two-cell stage after in vitro fertilization (51.1%) was significantly different compared to that of exposed to vitrification solution without cooling (60.0%) and control (68.2%) (p<0.05). However, there were no differences in the blastocyst formation from the cleaved embryos among groups (75.0, 73.3 and 80.0%). Also, the mean number of cells per blastocysts of vitrified group $(92.5{\pm}2.9)$ was similar to that of the exposed $(98.5{\pm}5.3)$ and control $(100.9{\pm}4.8)$. In vivo development of the blastocysts derived from vitrified-thawed oocytes resulted in fetal development (50.7%) and implantation rates (80.0%) which are very similar to those of control (58.2, 78.2%). These results suggest that mouse oocytes could be cryopreserved using vitrification solution (EFS35) based on ethylene glycol.

Sister Chromatid Exchange (SCE) Frequency and In Vitro Development of Mouse Zygote Cryopreserved by Vitrification

김묘경,백청순,엄상준,김은영,윤산현,박세필,정길생,임진호,Kim, M.K.,Baik, C.S.,Uhm, S.J.,Kim, E.Y.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.3

본 연구는 생쥐 1-세포기 수정란의 초자화 동결과 동결액 노출에 따른 수정란의 배발달율과 SCE 빈도를 조사하고자 실시하였다. 체외수정된 생쥐 1-세포기 수정란의 동결은 EFS40 (40% ethylene glycol, 30% Ficoll, 0.3 M sucrose)의 초자화동결법을 이용하였으며, $25^{\circ}C$에서 30 초동안 EFS40에 노출시킨 다음, 곧바로 액체질소에 침투시키거나, 동결액의 독성 검사를 위해 동결없이 배양하여 다음과 같은 결과를 보였다. 융해후, 2-세포기 까지 생존율은 EFS40에 노출 혹은 초자화동결시 각각 95.2, 98.5% 로서 대조군의 100%와 비교했을때 큰 차이가 없었다. 그러나 배반포와 탈출배반포까지의 배발달율에 있어서 초자화 동결된 군 66.7, 50.0%는 동결액에 노출만된 군 94.0 78.8%와 대조군 93.9, 81.8%에 비해 낮았다 (p<0.05). 동결액에 노출 혹은 초자화동결된 생쥐 1-세포기의 체외 발달에 따른 SCE 빈도를 조사한 결과, 동결액 노출 ($20.2{\pm}2.1$) 혹은 초자화동결시 ($21.4{\pm}3.2$) SCE 빈도가 대조군 ($16.8{\pm}1.5$)에 비해 증가하였다. 이러한 결과를 종합하여 고찰할 때, 초자화동결된 1-세포기 수정란의 배반포 또는 탈출 배반포까지의 낮은 배발달율은 동결액의 영향을 받지 않았으나, SCE 빈도는 동결액에 노출 혹은 초자화 동결시 증가 된다는 것을 알수 있다. This study was undertaken to investigate the sister chromatid exchange (SCE) frequency and embryonic development after exposure to cryoprotectants and vitrification of mouse zygotes. Mouse IVF zygotes were cryopreserved by vitrification using vitrification solution, EFS40 (40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). After mouse zygotes were exposed to EFS40 at $25^{\circ}C$ for 30 sec., they were immediately plunged into $LN_2$ or cultured for cryoprotectant toxicity test without freezing. The results obtained in these experiments were summarized as follows: After thawing, survival rates to the 2-cell stage of zygotes exposed to or vitrified in EFS 40 (98.5%, 95.2%) were not significantly difference compared with that of control (100%). However, the developmental rates upto blastocyst and hatching blastocyst in vitrified groups (66.7, 50.0%) were lower than those of control (93.9, 81.8%) or exposed group (94.0, 78.8%) (p<0.05). When the influence of vitrification and exposure to cryoprotectant on the in vitro development of mouse zygotes was assessed by the SCE frequency, the SCE frequency in exposed ($20.2{\pm}2.1$) to or vitrified embryos ($21.4{\pm}3.2$) was higher than that in control embryos ($16.8{\pm}1.5$). These results suggest that the frequency of SCE was increased after cryoprotectant exposure or Vitrification although developmental rates of zygotes upto blastocysts and /or hatching blastocysts were not afected by cryoprotectant.

Developmental Capacity of Bovine Follicular Oocytes after Ultra-Rapid Freezing by Electron Microscope Grid - I. Cryopreservation of Bovine Immature Oocytes

김은영,김남형,이봉경,윤산현,박세필,정길생,임진호,Kim, E.Y.,Kim, N.H.,Yi, B.K.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30. 본 연구는 소 미성숙난자를 electron microscope (EM) grid와 동해제인 EFS30을 이용하여 초급속 동결하였을 때 정상적인 배 발달의 유도가능성 여부를 조사하고자 실시하였다. 동해제는 30% ethylene glycol, 18% ficoll, 0.5 M sucrose와 10% FBS 등이 PBS에 첨가되어 제작된 EFS30을 사용하였다. 난자 생존의 평가기준으로는 성숙, 수정 및 배발달을 조사하였다. 본 연구에서 얻어진 결과는 다음과 같다. 초급속동결-융해 후, 소 미성숙란의 생존율은 43.2%을 나타내었다. 동결-융해군의 체외 성숙 (84.1%)과 정상 자웅전핵 형성율 (57.5%)은 대조군의 결과 (92.5, 65.0%)와 비교하여 볼 때 유의한 차이는 없었다. 또한, 동결군의 체외수정 이후의 $\geq2$-세포기 형성 (65.0%)과 배반포형성율 (30.8%)도 대조군(73.7, 35.7%)의 결과와 유의한 차이를 나타내지 않았다. 따라서 소 미성숙난자는 EM grid와 EFS30 동결액을 이용한 초급속 동결방법에 의해 정상적인 배발달이 유도될 수 있다는 것을 알 수 있었다.

Study on Epidermal Growth Factor (EGF) and Expression of EGF-Receptor (EGF-R) in Mouse IVF/IVC Embryo;I. Additive Effect of EGF and Expression of EGF-R on Mouse IVF Embryo Development

김은영,엄상준,김묘경,윤산현,박세필,정길생,임진호,Kim, E.Y.,Uhm, S.J.,Kim, M.K.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

본 연구는 EGF가 체외수정된 생쥐배의 착상전 발달 및 inner cell mass (ICM) 과 trophectoderm (TE) 세포수에 미치는 영향을 조사하고, 그와 더불어 간접 면역형광방법을 이용하여 배 발달 단계에 따른 EGF-receptor (EGF-R) 단백의 발현유무를 조사하기 위해 실시하였다. 그 결과를 요약하면 다음과 같다. 2-세포기 배의 group (5/25 ${\mu}l$)배양은 단독 배양보다 양호한 배 발달을 유도 할 수 있었으며, 단독 배양에서의 저조한 배 발달은 EGF를 첨가함으로서 개선시킬 수 있었다. 특히, 10 ng/ml의 EGF가 첨가된 단독배양군 (62.4%)은 단독대조군 (47.9%)에 비하여 유의하게 높은 배 발달을 나타냈다. 또한, ICM과 TE세포수 공히 EGF첨가에 의해 증가되는 것을 differential labelling기법으로 확인 할 수 있었다. 발달단계에 미치는 EGF의 효과를 검토하였던 바 4-세포기 이후의 배 발달에 유의하게 영향을 미치는 것을 알 수 있었고, 특히 배반포기배의 ICM도 동시에 증가되는 것을 알 수 있었다. 한편, 간접면역형광에 의한 EGF-R의 발현유무를 조사한 결과, 4-세포기 이후에 EGF-R가 발현되는 것을 확인할 수 있었다. 따라서, EGF는 착상전 생쥐배의 4-세포기 이후에 발현되는 EGF-R에 반응하여 배 발달을 유기하며, 또한 배반포기배의 ICM과 TE세포수의 증가에 영향을 미치는 것을 알 수 있었다. The objective of this study was to determine the effect of EGF on the preimplantation development of mouse IVF embryos and their ICM and TE cell number. And also, we examined the expression of EGF-R protein on embryonic development by indirect immunofluorescence. The results obtained in these experiments were summarized as follows; Group culture (5 embryos/ 25 ${\mu}l$) showed more improved development rate to blastocyst than singly culture. This inferior development of singly cultured 2-cell embryos improved by the addition of EGF. Especially, 2-cell embryos cultured singly in 10 ng/ml of EGF (62.4%) indicated significant difference in development to blastocyst compared with control group (47.9%). Also, cell number of ICM and TE by differential labelling showed the increased pattern in the EGF treatment group. The stimulating effect of EGF with the development level was significantly increased after 4-cell stage (p<0.05). ICM proportion also showed the increased pattern with the developmental level in the EGF treatment group. In addition, expression of EGF-R by indirect immunofluorescence detected after 4-cell stage. Therefore, EGF could stimulate preimplantation mouse embryo development by binding with expressed EGE-R after 4-cell stage and produce the more increased ICM and TE cell number of blastocyst.

Human Blastocysts;The Correlation Between Embryo Microscopical Assessments and Their Cell Number

김은영,엄상준,김묘경,윤산현,박세필,정길생,임진호,Kim, E.Y.,Uhm, S.J.,Kim, M.K.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.3

본 연구는 인간의 체외수정 program으로 부터 생산된 여분의 배반포기 배를 이용하여 현미경에 의한 형태학적 판정과 differential labelling 기법을 이용한 세포 수의 상관관계를 조사하고자 실시하였다. 공시된 인간 배반포기 배는 체외수정 후 5일째에 36명의 환자로부터 76개를 얻어 배반포강의 확대와 투명대 두께의 감소를 기준으로 early (ErB), early expanding (BEB), middle expanding (MEB) 및 expanded blastocyst (EdB)로 구분하였다. 분류된 배반포기 배의 크기와 투명대의 두께를 micrometer로 측정하였을 때, 그 크기는 각각 $148.8-217.6{\mu}m$, $1.2-14.4{\mu}m$로 나타나 같은 배양조건에서 생산된 배아라도 그 차이는 크게 나타났다. Hoechst 염색을 이용하여 배반포기 배의 총 세포수를 조사하였을 때, 체외수정 후 5일째 생산된 배반포기 배는 ErB $(39.1{\pm}3.6)$ 에서 EdB $(89.6{\pm}3.3)$ 로 진행되는 동안 두배에서 세배정도 증가되는 양상을 나타내었다. 또한, differential labelling기법 을 이용한 배반포기 배의 inner cell mass (ICM) 와 trophectoderm (TE) 세포수 조사 결과, 각각 $11.9{\pm}1.8-22.2{\pm}4.3$, $24.5{\pm}3.6-70.0{\pm}7.7$을 나타내어 발달이 진행될수록 ICM과 TE세포수도 증가되는 것을 알 수 있었다. 특히, 형태학적으로 약한 ICM으로 판정된 EdB의 경우 differential labelling 후, 역시 적은 ICM 세포수를 나타내어 형태학적 판정과 세포수간에 밀접한 상관관계가 있음을 알 수 있었다. 따라서, ICM과 TE를 differential labelling하는 기법은 인간 배반포기 배의 quality를 평가하는데 매우 유용한 기법으로 형태학적인 구분과 병용된다면 인간 배반포기 배 이식 program의 임신율 증진을 위한 배아 선별의 중요한 자료로서 이용될 수 있다는 것을 시사한다. The objective of this study was to investigate correlation between the morphology by microscopic assessments of surplus blastocysts produced in human IVF program and their cell number obtained by differential labelling method. For these experiments, 76 surplus human blastocysts were obtained from 36 patients on day 5 after IVF, the embryos were classified to early (ErB), early expanding (EEB), middle expanding (MEB), expanded blastocyst (EdB) according to their blastocoel expansion and zona thickness. When the ovum size and zona thickness of the classified blastocysts were measured using micrometer, although the embryos were produced in the same culture condition, there were significant variances in ovum size ($148.8 217.6{\mu}m$) and zona thickness ($1.2-14.4{\mu}m$). Total blastomere cell number counted after hoechst staining was increased by two to three fold during the transition period from ErB ($39.1{\pm}3.6$) to EdB ($(89.6{\pm}3.3)$) stage on day 5 after IVF. ICM ($11.9{\pm}1.8-22.2{\pm}4.3$) and TE ($24.5{\pm}3.6-70.0{\pm}7.7$) cell numbers using differential labelling were also showed the increased pattern according to the developmental level. Especially, EdB which showed poor ICM morphologically also indicated the low ICM cell number after differential labelling. This demonstrated that there is good correlation between the morphological assessment and the cell number. The count of ICM and TE nuclei using differential labelling can be used as an important criterion, if it is accompanied with morphological assessments, in selecting the better embryos for improving the pregnancy rates in human blastocyst transfer program.