• Clinical and Experimental Reproductive Medicine
• /
• 제30권3호
• /
• pp.241-248
• /
• 2003

Objective: The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method. Methods: Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass. Results: The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively. Conclusion: This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.

• 한국가축번식학회지
• /
• 제23권4호
• /
• pp.313-321
• /
• 1999

본 연구는 체외생산된 배반포기배의 vitrification 을 위한 용기로서 glass micropipette(GMP)을 이용할 수 있는지, GMP 와 OPS 로 동결융해 후 생존율의 비교 및 GMP vitrification 후 hatching 율의 향상을 위하여 실시하였다. GMP vessel은 열전도율과 수정란을 포함하는 적은 질량 때문에 OPS 보다 동결 및 융해속도를 높일 수 있다. 3개의 체외수정란을 vitrification 용액에 노출시키고 OPS 또는 GMP vessel에 loading 시킨 후 액체질소에 침적하는데까지 20~25초 이내에 실시하였다. 동결ㆍ융해한 배반포기배는 0.25와 15 M sucrose solution 및 TCM 1999에 각각 5분씩 차례로 희석한 후 10% FCS가 첨가된 TCM 199에 24시간동안 배양하였다. OPS(75.9%)와 GMP(80.0%) 방법간의 re-expanding 율은 유의적 (P＜0.05)인 차이가 없었다. OPS(34.1%)와 GMP(37.5%) 방법에서 hatching 율은 intact group(54.3%) 보다도 유의적 (P＜0.05)으로 낮았다. 비록 GMP straw 당 3개 이하의 blastocysts 를 loading 하였더라도 narrow portion(83.3%) 보다도 wide portion(S6.7%)에서 vitrified 되었다면 re-expanding 율이 유의적 (P＜0.05)으로 낮았다. 비록 30초 처리군과 무처리군 간에는 유의적인 차이가 없었지만 0.05% pronase 용액에 30, 60 및 90초간 처리군 (45.9, 54.7 및 57.5%)의 hatching 율은 무처리구 (35.0%) 보다 유의적 (P＜0.05)으로 높았다. 이러한 결과들은 OPS와 GMP vitrification vessel은 체외생산된 배반포기배의 높은 생존율을 얻을 수 있다. 그러나 GMP vessel은 L$N_2$침적 후 vessel의 floating을 방지하기 위한 또 다른 cap 이 필요하지 않다는 유리한 점을 가지고 있다. 수정란의 loading 위치, 즉 narrow 또는 wide portion에 따라 소 체외 생산된 배반포기배의 생존력에 제한적인 요인으로 고려된다. 0.5% pronase 용액에 60 또는 90초간의 노출은 융해후 hatching 율을 향상시킬 수 있었다.

• 한국가축번식학회지
• /
• 제26권4호
• /
• pp.311-320
• /
• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• 대한생식의학회:학술대회논문집
• /
• 대한불임학회 2005년도 제49차 추계학술대회
• /
• pp.131.2-132
• /
• 2005

• 한국가축번식학회지
• /
• 제23권1호
• /
• pp.85-92
• /
• 1999

본 연구는 동결동결보호제의 종류와 배발달단계가 생쥐의 OPP vitrification 동결방법에 미치는 영향을 알아보고자 실시하였다. 동결속도, 동결보호제 및 배발달단계는 vitrification 방법에 따른 수정란의 생존성에 영향을 미칠 수 있다. 본 연구에 사용된 동결보존액은 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose와 5% FCS가 첨가된 D-PBS (EFS) 및 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, 0.5 M sucrose 와 5% D-PBS (EDS)을 이용하였다. 배반포기배는 hCG 처리후 90시간째에 자궁으로부터 채취하여 실험 1에 이용하였고, 실험 2와 3에서는 zygote 를 hCG 처리후 18시간에 난관에서 채취하여 mHTF 배양액에 5% $CO_2$, 37$^{\circ}C$ 조건하에 배양하면서 2-, 4-, 8-cell, compacted morula, 또는 blastocyst를 이용하였다. 실험 1에서 배반포기배의 적당한 동결보존액을 결정하기 위하여 EFS 또는 EDS로 OPP vitrification 을 실시하였다. 재확장배반포기에 의한 생존율은 대조군과 EDS 처리군 (100, 100%) 이 EFS 군 (95.0%) 보다 유의적 (P＜0.05)으로 높게 나타났으나, 부화배반포기에서는 EFS 군 (90.0%) 이 대조군 (100%) 및 EDS 군 (95.0%) 보다 유의적으로 낮은 발달율을 보였다. 실험 2에서는 zygote, 2-, 4-, 8-cell, 상실배 빛 배반포기 등의 초기배에서도 OPP vitrification 동결방법이 적당한지를 판단하기 위하여 실시하였다. Zygote (70.0%) 는 동결융해 후 배발달율이 2, 4, 8, 상실배 및 배반포기배에 비하여 유의적으로 낮은 발달율을 보였다 (89.7, 90.0, 92.8, 97.6 및 97.5%) (P＜0.05). 또한 동결융해란의 할구수에서는 대조군 및 배반포기배 (35.7$\pm$2.98 및 39.6$\pm$2.81)에서 zygote, 2-, 4- 8-cell, 상실배 (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 및 30.8$\pm$2.93) 보다 유의적으로 높게 나타났다 (P＜0.05) 실험 3에서는 zygote의 VS1 에 노출시간에 따른 생존율을 조사한 결과 융해후 2-cell (91.6, 88.5 및 88.9%) 및 배반포기 (83.3, 74.3 및 69.4%) 까지 배발달율은 1,2 및 3분간의 노출시간에 따른 유의적인 차이를 보이지 않았다. 또한 융해후 노출시간에 따른 할구수에서도 유의적인 차이를 보이지 않았다 (36.4$\pm$4.76, 32.4$\pm$4.67 및 27.6$\pm$4.52). 이상의 결과에서 OPP vitrification 방법은 EFS 또는 EDS 동결보존액에 따른 유의적인 차이 없이 이용될 수 있는 것으로 판단된다. 배발달단계에 따른 생존율은 zygote 의 초기배는 2, 4, 8, 상실배 및 배반포기보다 유의적으로 저조한 생존율을 보였다. Zygote의 VS1에 노출시간에 따른 생존율도 1 분간의 노풀시간에서 높은 배발달율을 보였다. OPP vitrification 동결보존방법으로 생쥐수정란의 동결보존에 유용하게 이용가능한 것으로 판단된다.

• 한국수정란이식학회지
• /
• 제24권3호
• /
• pp.199-205
• /
• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• 한국수정란이식학회지
• /
• 제29권3호
• /
• pp.235-240
• /
• 2014

생쥐 2세포기, 4세포기, 8세포기를 각 발생 단계에서 채취하여 동결 보호제를 첨가한 서로 다른 배양액에서 배양하고, 배아의 동결 보존 및 융해 시 급속 처리와 저속 처리 단계로 비교하여 이들 조건이 배아의 생존과 발현에 미치는 영향을 조사하여 다음의 결과를 얻었다. 동결 보호제로 처리하여 배양액을 달리한 경우, 급속 단계에서는 모든 배양액에서 비슷한 발생율을 보였고, 저속단계의 4세포기와 8세포기는 D-PBS에서 높은 발생율을 보였다(P<0.05, P<0.01). 배아의 발생 시기에 따른 동결 보존 후, 발생율은 2, 4, 8세포기로 넘어갈수록 발생율의 증가를 보여 8세포기에서 발생율이 가장 높았다(P<0.01). 동결 보호제의 처리단계에 따른 발생율은 2세포기의 급속 단계에서는 유사하였으나, 4세포기와 8세포기는 저속단계에서 높은 발생율을 보였으며(P<0.05), 특히 8세포기에서 가장 높았다(P<0.01).

• Clinical and Experimental Reproductive Medicine
• /
• 제25권3호
• /
• pp.287-292
• /
• 1998

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.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2002년도 국제심포지엄
• /
• pp.78-78
• /
• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• Clinical and Experimental Reproductive Medicine
• /
• 제48권2호
• /
• pp.111-123
• /
• 2021

Objective: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality. Methods: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. Results: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2% ±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls. Conclusion: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.