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The Cytoskeletal and Chromosomal Constitution of Vitrified Immature Mouse Oocytes
박세필,이봉경,김은영,남화경,이금실,윤산현,정길생,임진호,Park, Se-Pill,Yi, Bong-Kyung,Kim, Eun-Young,Nam, Hwa-Kyung,Lee, Keum-Sil,Yoon, San-Hyun,Chun, Kil-Saeng,Lim, Jin-Ho The Korean Society for Reproductive Medicine 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.3
연구목적: 본 연구는 동해방지제인 EFS40을 이용한 초자화동결이 생쥐 미성숙란의 cytoskeleton과 염색체의 성상에 미치는 영향을 indirect immunocytochemistry방법과 염색체 분석법으로 알아보고자 실시하였다. 연구재료 및 방법: 본 실험은 생쥐 미성숙란을 EFS40 (40% ethylene glycol, 18% ficou과 0.5 M sucrose가 들어있는 M2배양액)으로 초자화 동결하여 융해한 후 16시간동안 체외 성숙을 유도하여, 제 1극체가 나타난 성숙된 난자를 기준으로 동해제노출군 또는 대조군과 비교 조사하였다. 결과: 초자화동결된 미성숙란의 응해 후 생존율과 체외성숙율은 90.3%과 64.7%로써, 동해제노출군 (86.7%, 69.2%)과 대조군 (100%, 58.3%)에 유사하였다. 초자화동결이 미성숙란의 microtubule과 microfilament에 미치는 영향을 조사하였던 바, 동결군의 microtubule과 micro-filament의 정상적인 형성율 (93.9%, 100.0%)은 동해 제노출군 (94.4%, 100.0%)과 대조군 (100.0%, 100.0%)의 성적과 유사하게 나타났다. 또한, 초자화동결군에서 정상적인 염색체수를 가진 난자의 비율도 65.8%로써, 대조군(79.6%)과 노출군 (69.0%)의 결과와 유의한 차이가 없었다. 결론: 생쥐 미성숙란을 EFS40에 노출하고 동결하는 것이 미성숙란의 cytoskeleton과 염색체성상에 영향을 미치지 않으며, 본 연구에서 사용된 EFS40을 이용한 초자화동결법은 생쥐 미성숙란 동결에 적합하다는 것을 알 수 있었다. This study was to confirm whether the vitrification method using EFS40 freezing solution has detrimental effect on the cytoskeleton and chromosome constitution of the immature mouse oocytes by indirect immunocytochemistry and chromosome analysis. Immature mouse oocytes were vitrified using EFS40 (40% EG, 18% ficoll, 0.5 M sucrose diluted in M2 medium), thawed and then survived oocytes were in vitro matured for 16 hr. When the microtubule morphology and micro filament distribution in vitrified-thawed immature mouse oocytes were examined, normal percentage of two cytoskeleton in vitrified group (93.9 and 100.0%) was not significantly different from that in control (100.0 and 100.0%) and exposed group (94.4 and 100.0%). The rate of oocytes containing a normal chromosome number in vitrified group was 65.8%, this result was not significantly different from that in control (79.6%) and exposed group (69.0%). These results indicated that exposure to cryoprotectant or freezing has not effect on the alteration of cytoskeleton morphology and the chromosome constitution of mouse oocytes and that our vitrification methods using EFS40 freezing solution was suitable for the cryopreservation of immature mouse oocytes.
탈핵 후 동결한 MII 난자의 활성화 시기가 체세포 핵치환 이후 소 난자의 체외발달에 미치는 영향
박세필,김은영,김선균,이영재,길광수,박세영,윤지연,이창현,정길생 한국동물번식학회 2002 Reproductive & developmental biology Vol.26 No.3
This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 ${\mu}{\textrm}{m}$ ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into L$N_2$. Thawing was taken by 4-step procedures at 37$^{\circ}C$. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide and 2.5 $\mu\textrm{g}$/$m\ell$ of cytochalasin D added CRlaa medium for 1 h, and then in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.
체외수정 유래 생쥐 배아줄기세포와 유사한 특성을 보유한 단위발생 유래 생쥐 배아줄기세포
박세필,김은영,이금실,이영재,신현아,민현정,이훈택,정길생,임진호,Park, Se-Pill,Kim, Eun-Young,Lee, Keum-Si,Lee, Young-Jae,Shin, Hyun-Ah,Min, Hyun-Jung,Lee, Hoon-Taek,Chung, Kil-Saeng,Lim, Jin-Ho 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.2
Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.
우 난포란의 체외성숙에 관한 연구 III. 체외성숙 우난포란의 체외수정과 발달
박세필,박태균,윤산현,고대환,정길생 한국동물번식학회 1989 Reproductive & developmental biology Vol.13 No.2
These experiments were carried out to obtain the basic information for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2-6mm of diameter. Bovine oocytes were matured in vitro for 24-26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM199 supplemented with hormones, pyruvate, FBS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2-3 hours in BO solution containing bovine serum albumin(5mg/ml) and caffein(2.5mM). Insemination was made by introducing about 10-15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after inseminatin the eggs were transferred to TCM 199 supplemented with FBS(10%) for in vitro development. The results obtained in these experiments were summarized as follows : 1. The maturation rate of oocytes following incubation for 24-26 hours was 78.4%(228/291). 2. Of total 250 oocytes, 172 embryos extruded 2nd polar body following in vitro culture with spermatozoa for 20 hours, and the rates of embryos developed to 2-, 4-, 8-, 16-cells and morula or early blastocyst were 64.0, 39.2, 22.0, 15.2 and 11.2%, respectively. 3. The time needed for development to 2-, 4-. 8-, 16-cell stage and morula was 42.5$\pm$5.4, 58.0$\pm$9.2, 74.4$\pm$11.5, 96.1$\pm$13.4 and 119.0$\pm$18.2 hours, respectively.