유리화 동결/융해된 인간 배반포 및 생쥐의 생식 결과에 영향을 미치는 조건들에 관한 연구

박재균 차의과학대학교 일반대학원 2021 국내박사

Vitrification is a rapid cooling/warming method that produces solidification similar to glass-like state. The use of high concentrations of cryoprotectants (CPAs) during vitrification allows for the dehydration of the cells and avoids the formation of ice crystals. Thus, vitrification is currently the recommended technique to cryopreserve not only embryos but also oocytes. Vitrification is currently producing very satisfactory outcomes. However, continuous studies are still required to validate and improve the efficiency of vitrification of human oocytes and embryos. Thus, I have performed retrospectively analyzed clinical data of vitrified human blastocysts and a series of vitrification experiments using mouse oocytes in this study. In part I, studies on the various conditions that influence the reproductive outcomes of vitrified/warmed human blastocysts include three topics. In part I.1, the effects of cryo-storage duration on clinical outcomes after transfer of vitrified blastocysts stored in the open carrier system (OC) with slush liquid nitrogen (SLN2) were evaluated. A cohort data with 1632 autologous VW blastocyst transfer cycles was retrospectively analyzed. Cryo-storage duration was divided into four groups (Group I-IV: 0–6, 7–12, 13–24, and ≥25 months). There were no significant differences between groups in survival rate (SR), clinical pregnancy rate, live birth rate (LBR), and neonatal outcomes. Multivariate logistic regression analysis showed no risks of cryo-storage duration on LBR. Vitrification using SLN2 and long-term cryo-storage in the OC are safe and effective and does not significantly affect clinical outcomes after blastocyst transfer. In part I.2, the second cohort data was retrospectively analyzed to evaluate the effect of the culture duration of post-warmed blastocysts on clinical outcomes. Embryos were divided into two groups depending on their post-warming culture duration, long-term (20–24 hours) and short-term (2–4 hours). Embryo morphology was analyzed with a time-lapse monitoring device to estimate the appropriate timing and parameters for evaluating embryos with high implantation potency. No significant differences were observed in implantation rate (IR) and ongoing pregnancy rate (OPR) between both groups (long-term vs. short-term: 56.3 vs. 67.9% and 47.3 vs. 53.6%, respectively). After warming, there were more expanded and hatching/hatched blastocysts in the long-term group, but there was no significant difference in embryo grade. The time to complete blastocyst re-expansion after warming is significantly shorter in women who became pregnant than in those who did not in both culture groups (long-term: 2.2±0.6 vs. 4.1±0.8 hours; short-term: 1.2±0.3 vs. 1.9±0.8 hours, respectively). The IR and OPR outcomes of VW blastocyst transfer were not significantly different between the short-term and long-term cultures. In part I.3, the third cohort data with 329 single VW blastocyst transfer cycles was retrospectively analyzed to evaluate the pre-vitrified and post-warmed morphological factors of blastocysts for the embryo selection process. Logistic regression analysis was conducted to select independent morphological factor(s) associated with ongoing pregnancy. The degree of blastocoel expansion [expanded blastocoel; adjusted odd ratio (aOD) 3.15, 95% CI 1.17-8.44, p = 0.02] in a pre-vitrified blastocyst and the grade of inner cell mass (ICM) (grade B; aOR 0.47, 95% CI 0.27-0.83, p = 0.01, grade C; aOR 0.22, 95% CI 0.09-0.56 p < 0.01) in post-warmed blastocysts significantly predicts the ongoing pregnancy rate (OPR). After fertilization, embryos developed to the blastocysts on day 5 showed a significantly higher OPR than that on day 6 (aOR 0.5, 95% CI 0.26-0.94, p = 0.03). ICM grade is more predictive than trophectoderm grade for the selection of blastocysts with higher OPR in single blastocyst transfers. In part II, The OC is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. The closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC was significantly lower than with OC. I aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to CPAs as key parameters for the CC effectively improved the SR and developmental competence of VW oocytes. When all the conditions that improved outcomes were applied to the conventional CC, hereafter named modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the OC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the total cell number of the blastocyst developed from VW oocytes. mVW-CC optimized for mouse oocytes could be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future. In summary, retrospective data analyses in part I suggested the clinical effectiveness of duration of cryo-storage, different post-warmed culture time, and blastocysts morphologic parameters on ongoing pregnancy and neonatal outcomes. The mVW-CC for vitrification of mouse oocytes in part II suggested an option for human oocytes without concerns on possible cross-contamination during vitrification in the future. Collectively, improvement of efficiency of vitrification process and understanding of key selection parameters to predict clinical outcomes of VW oocytes and embryos will help clinical embryologists further improve assisted reproductive technology procedures.

Comparative analyses of cellular and molecular features of oocytes from young and aged mice after vitrification

이주희 차의과학대학교 일반대학원 2020 국내석사

Advanced maternal age (AMA) is one of the major factors affecting the accomplishment of a normal pregnancy by assisted reproductive technology (ART), because of an age-related decrease in the quality and quantity of oocytes. The introduction of cryopreservation methods to preserve embryos in ART provides greater chances of having a successful pregnancy. Vitrification, the currently preferred cryopreservation method, utilizes an ultra-rapid freezing procedure using high concentrations of cryoprotectants to minimize cryo-damage. Embryo vitrification has become an efficient and safer technique for ART outcomes. However, the effectiveness of vitrification procedure on oocytes still needs to be optimized. Furthermore, oocytes from older women tend to have vulnerable cellular structures and are sensitive to various insults. Thus, extra caution(s) and/or modifications may be required for vitrification of oocytes from older women. To evaluate if AMA affects the developmental outcomes of oocytes after vitrification, cellular and molecular features of fresh and vitrified/warmed (VW) oocytes from young (5- to 7-week-old) and aged (10- to 12-month-old) mice were comparatively examined in this study. The number of oocytes retrieved from aged mice following superovulation was significantly lower than that of young C57BL/6 mice (28.3±1.85 vs 7.96±0.7; p<0.001). Similar results were also obtained in ICR and B6D2F1 mice. The proportion of oocytes with irregular morphology gradually increased in an age-dependent manner from 7.2 % in young mice to 35.9 % in aged mice. The survival rate of VW oocytes from aged mice was significantly lower than that of young mice (94.2 % vs 74.4 %; p<0.05). The fertilization rate of VW oocytes from aged mice tended to decline (81.4 % vs 71.3 %), whereas fresh oocytes from young and aged mice showed similar results (91.7 % and 89.9 %, respectively). Furthermore, the cleavage rate of the fertilized VW oocytes from aged mice significantly decreased (74.5 % vs 59.8 %; p<0.05) and the blastocyst formation rate tended to decline (52.4 % vs 43.7 %), as compared to those in the young mice. Molecular features of VW oocytes from young and aged mice were examined with immunofluorescence staining, live cell imaging, and/or transmission electron microscopy. Microtubules were found to be organized in a barrel-shape in metaphase spindle of both fresh and VW oocytes from young mice. However, the incidence of abnormal spindle structures, such as spindle pole decompaction and/or spindle fragmentation was significantly increased in both fresh and VW oocytes from aged mice. The mitochondria of fresh oocytes from young mice were evenly distributed throughout the ooplasm, whereas they were aggregated in the central region after vitrification. Aggregated patterns of the mitochondria were observed even in fresh oocytes, however, they became more severe in VW oocytes from aged mice. Transmission electron microscopy analyses demonstrated that the number and size of microvacuoles in fresh oocytes were similar between young and aged mice. However, vitrification increased the number of microvacuoles in oocytes from both young and aged mice. Furthermore, not only the number but also the size of microvacuoles was significantly increased in VW oocytes from aged mice. Collectively, this study provides evidence that vitrification could affect the cellular and molecular features of oocytes, and AMA further deteriorates the vulnerable conditions of VW oocytes. These results suggest that current vitrification protocols optimized for oocytes from young donors and pre-implantation embryos need to be modified to efficiently preserve the integrity of oocytes from women at AMA. Keywords: oocyte, vitrification, advanced maternal age, developmental potential, fertility preservation

Effects of caffeine / MG132 on the vitrification of mouse mature oocyte and further embryonic development

백지이 차의과학대학교 대학원 2016 국내석사

Cryopreservation is the technique used to stop all biological activities by cooling the cells to low temperature (-196 ℃) for future use. This is important to remove the intracellular water of the oocyte by dehydration using short exposure to high concentrations of cryoprotectants (Fuller et al, 1996). Most importantly, it could reduce the damage to oocytes without the forming of ice crystals Cryopreservation has been developed to store surplus embryos after Assisted Reproductive Technology (ART). Recently, vitrification is replaced with slow freezing protocol, because of improved survival rates and clinical outcomes. Vitrification using a high concentration of CPAs and it makes the cell toxicity increased. Also risk of contamination increases due to plugged into liquid nitrogen directly. Ice crystal formation, bubble formation could happen during vitrification and warming process and may reduce the survival and developmental rates (Fiamigni et alL, 2004). Generally, Cyclin B1 interacts with Cdk1 to form a complex known as the MPF, which is plays an essential role in the cell cycle regulation and maintaining the meiotic arrest of oocytes (Pines, 1999; Labbe et al, 1989). Succu (2011) reported that, MPF activity has been observed to decrease in MII ovine oocytes after vitrification, which could compromise the oocyte development after in vitro fertilization. Likewise, MAPK also responsible for the beginning of germinal vesicle breakdown and is essential for the maintenance of the meiotic arrest of oocytes at MII-stage (Dupre et al, 2002; Araki et al, 1996). The present study were designed to investigate the effects of the Caffeine and MG132 of vitrified/warmed MII mouse oocytes on the activity of MPF and MAPK. We evaluated their survival, fertilization, cleavage and developmental rates. Ovulated MII oocytes were retrieved from 6 weeks old B6D2F1 female mice at 14 hr post hCG injections. Collected MII oocytes were vitrified/warmed into divided 4 groups (non-treated, 10 mM caffeine, 10 μM MG13, 10 mM caffeine + 10 μM MG132). After warming, MPF and MAPK activity was evaluated using ELISA and Western blotting. Also, verified the fertilization and blastocyst rates of mature oocytes after in vitro fertilization. Blastocyst formation was increased in 10 mM caffeine, 10 μM MG132 and 10 mM caffeine + 10 μM MG132. Also MPF activity were same tendency. However, statistically no difference was found all groups in MAPK activity and differential staining. These results suggest that during vitrification/warming process treatment of caffeine and MG132 can prolongs the MPF activity and stabilized the chromatin configuration, it helps these fertilization and blastocyst rates. In conclusion, caffeine and MG132 may reduce Cryoinjury during vitrification/warming process, and furthermore, these caffeine and MG132 could be useful for the development and supplementation of cryopreservation method.

Optimal vitirification protocol and transplantation condition for mouse ovarian tissue

염혜원 서울대학교 대학원 2014 국내박사

Introduction: In clinical medicine, ovarian tissue (OT) cryopreservation together with transplantation has been used to restore their fertility for the women suffering from infertility caused by cancer treatment. Lots of studies on transplantation of cryopreserved-thawed OTs have been performed, and now this is the promising alternatives to preserve fertility in cancer patients. Due to the limited availability of human OT in experiments, the mice have been used as an effective model for such purposes in many studies. Cryopreservation and transplantation of murine ovaries is a useful tool to assess the risk of malignant recurrence after re-transplantation of OT, to analyze the recovery of ovarian function, and to improve the related protocols for future clinical application. Optimization of cryopreservation and transplantion procedures is the most important steps to improve OT survival. The aim of this study was to determine the optimal cryopreservation especially vitrification protocols for mouse OT survival, assess the impact of 4 different OT grafting sites and evaluated the effect of angiopoietin-2 (ANG-2) as an angiogenic factor on the follicular pool and OT integrity after grafting. Methods: For the vitrification experiment, a total of 644 ovaries were collected from BDF1 mice. Of these, 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition, such as EDS, ES, ED, EPS, EF, EFS, E, and EP, respectively (E: EG, D: DMSO, P: PrOH, S: sucrose, and F: Ficoll). And the remained 73 ovaries were used for control group. After warming, each 8 group of ovaries was further randomly divided into 4 subgroups and in vitro cultured for 0, 0.5, 2, and 4 h, respectively. Ovaries of the best 2 groups among 8 groups were autotransplanted after IVC. To evaluate the quality of vitrified OTs, OT morphological histology, follicular apoptosis, proliferation and FSH level was observed. For the grafting site experiment, the B6D2F1 mice were randomly assigned to 4 groups. One group was used for the control (sharm). The ovaries from the other 4 groups were collected and autotranplanted directly to the different transplantation sites: back muscle (BM), fat pad (FP), kidney capsule (KC) and subcutaneous (SC). Assessment of the follicular density, integrity, classification, apoptosis, fibrosis, FSH level and the quantity and the quality of oocytes from the OT grafts was carried out on day (D) 2, 7, 21 and/or 42 after grafting. The Five-week-old B6D2F1 female mice were divided into 3 groups (a control and two ANG-2 groups) followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without ANG-2 injection (50 or 500ng/kg), and then killed at day(D)2, 7, 21 and 42 after transplantation. Total 2,437 follicles in OT grafts were assessed for the follicular density, integrity and classification by hematoxylin and eosin staining. Apoptosis and revascularization were evaluated by Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate Nick End Labeling assay and CD31 immunohistochemistry respectively. Serum FSH levels were measured by Enzyme-linked immunosorbent assay. Results: Morphologically intact (G1) and apoptotic follicles were compared after vitrification. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8% and 46.6%, respectively, whereas the EP group showed the worst results at 42.2% and 12.8%, respectively. Apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All the 8 groups showed significant decreases in G1 follicle ratio and increases in apoptotic follicle ratio as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 ratio (84.9%). For the proliferation ratio, only the ES 4 h group (80.6%) showed significant decrease. There was no significant difference in apoptotic ratio and FSH level between all the groups. In the grafting site experiment, the graft recovery rate was lowest in the FP group (83.9%), and the mean number of follicles only in the KC group (D7: 14.7 and D21: 15.9) was comparable to that of the control group (D7: 17.0 and D21: 18.8). The antral follicles appeared in BM (0.9%) and KC (2.2%) groups on D2 while no antral follicles in FP and SC groups yet, and were increased as the grafting duration increased showing the lowest percentage in the SC group (7.8%) on D21. For the intact (G1) follicles, all the grafting groups showed low ratios (31.5%-37.7%) compared to the sharm group (61.3%) on D2 followed by dramatic increase in FP (74.0%) and KC (71.9%) groups on D21 (sharm: 63.0%, BM: 59.5%, and SC: 70.9%). The ratios of apoptotic follicles were lowest in the KC group (8.2%) and highest in the SC group (24.5%) on D7, but no significant difference was observed on D21. Marked increase in tissue fibrosis was observed in the FP (8.3%) and SC (15.0%) groups on D2, and it was continued in the FP group (D7: 5.9% and D21 7.5%). The FSH levels were significantly increased in all grafting groups on D2, but only the SC group showed the high FSH level (5.5ng/ml) compared to the others (1.6-2.5ng/ml) on D7 followed by no sign of significant difference among the groups on D21. When collecting the oocytes from the experimental 4 sites, the mean oocyte numbers of 4 sites ovarian grafts from 4 graft sites were significantly highest in KC group and lowest in SC group (BM; 4.6±1.6, FP; 3.5±1.3, KC; 10.4±2.3 and SC; 2.1±0.9 respectively). However the ratio of MII oocyte and normal spindle were not different among the 4 experimental groups. For the ANG-2 experiment, all the ANG-2 groups (50ng and 500ng) showed remarkable increase of morphologically intact follicle ratio across all the grafting duration except D21 (no statistical difference). The numbers of CD31 positive vessels (the sum of 3 fields at x400 magnification) were significantly increased in both ANG-2 groups compared with the control group at all the grafting duration. Especially at D42, the 500ng ANG-2 group showed significantly more vessels than the 50ng ANG-2 group as well as the control group. However the mean follicle numbers of grafts, apoptosis ratio and serum FSH levels showed no significant difference among the groups. Conclusions: We compared 8 vitrification protocols according to CPA composition and IVC duration and found the EDS to be the optimal protocol among them. For the grafting site, we confirmed that the different grafting sites influenced the outcomes of the grafting. The KC site was the most optimal for OT transplantation in murine model. In contrast, the SC sites were not favorable to OT grafts. And we found that ANG-2 treatment to reduce the ischemic damage and improve angiogenic ability could preserve ovarian function via its beneficial effects on transplants.

(A) study of fertility preservation options using mouse ovarian tissue

이재왕 서울대학교 대학원 2016 국내박사

Introduction: As the diagnosis and treatment of cancer have been dramatically improved, fertility preservation in female cancer patients has been up in light to improve their quality of life. Oocyte banking and embryo cryopreservation commonly used as fertility preservation options, however, they cannot be applied to pre-pubertal girls and unmarried women. Fertility preservation with ovarian tissue (OT) can provide alternative instead of oocyte and embryo while there are major hurdles to enhance the efficiency of the procedures. First, cryoinjury is usually occurred during cryopreservation process. Second, ischemic injury is also spontaneously happened until re-vascularization, such as 2 days and 5 days are needed to initiate the re-vascularization. Finally, OT has a risk to re-implant the malignant cells after transplantation. This study was aimed to 1) compare the deleterious effects of cryoinjury and ischemic injury on the quality of OT after cryopreservation and transplantation process 2) decrease the cryoinjury after vitrification-warming process using several types of antifreeze proteins 3) diminish the ischemic injury via enhancement of re-vascularization after auto-transplantation 4) optimize the two-dimensional follicle culture system using mouse model. Methods: For the comparison of cryoinjury and ischemic injury (Exp I), a total of 160 ovaries were harvested from 6-week-old female B6D2F1 mice. Ovaries were randomly divided into eight different groups consisting of two control groups (fresh and vitri-con) and six experimental groups according to the presence or absence of vitrification and transplantation. (fresh OT [FrOT]-day [D] 2, FrOT-D7, FrOT-D21, vitrified OT [VtOT]-D2, VtOT-D7, and VtOT-D21). In the fresh control group, OT was fixed immediately after ovariectomy, and in the vitri-con group, OT was fixed after the vitrification-warming procedure. All six experimental groups were auto-transplanted with fresh or vitrified-warmed OT, and then the mice were sacrificed by cervical dislocation at 2, 7, or 21 days after grafting. To investigate the detrimental impacts of these injuries, histology, cell-death, blood vessel distribution in OT and ELISA for FSH level For decrease cryoinjury during vitrification-warming process (Exp II.), a total of 140 mice were sacrificed to collect sexually mature ovaries from 6-week-old aged female B6D2F1 mice. In Exp II-I, a total of 240 whole ovaries were randomly distributed to one of three groups: the fresh control group, the vitrification control group, or the AFP-treated group. The AFP-treated group was further divided into nine subgroups according to AFP type (e.g., FfIBP, LeIBP, and type III AFP) and dose (0.1, 1.0, and 10 mg/mL). After two-step vitrification and four-step warming process, the quality of ovary was assessed. Then, auto-transplantation of ovary was carried out to determine whether the cryoprotective effects of LeIBP could also be seen in OT after transplantation (Exp II-II). A total of 20 B6D2F1 mice were randomly divided into two groups: one group that received 10mg/ml of LeIBP-treated ovaries and another that received the vitrification control ovaries. We used only 10 mg/ml of LeIBP-treated group for this experiment because this group showed the best results in Experiment II-I. The quality was evaluated by histology, TUNEL, immunohistochemistry and serum FSH level on each evaluation days (Day 2, 7 and 21 days after transplantation). Next, a combination of simvastatin and/or methylprednisolone was treated to diminish of ischemic injury during avascular period after transplantation (Exp III). Following comparison study, the mice were treated with 5 mg/kg of simvastatin and/or methylprednisolone 2 h before ovareictomy and then the ovaries were cryopreserved by two-step vitrification process as previously described in Exp I. One week later, vitrified OTs were warmed by four-step warming process and then auto-transplanted under bilateral kidney capsules. Similar to the Exp II-II, the mice were sacrificed by cervical dislocation on 2nd, 7th or 21st of the transplantation period to assess the quality of OT. Macroscopic and microscopic examination, immunohistochemistry for blood vessel, flow cytometry for CD45, serum AMH ELISA were carried out on each evaluation days. Moreover, oocyte retrieval from graft and further in vitro fertilization were also performed to evaluate the drug safety on gametogenesis and embryogenesis. Finally, Pre-antral follicles were mechanically isolated from 2-week-old BDF-1 mice and randomly assigned into two groups according to the culture methods (with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layer with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layerwith or without oil layerwith or without oil layer with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layer , Exp IV). In vivo matured oocytes were collected using superovulation to compare the growth, cytoplasmic normality, gene expression and embryonic development. Ovarian follicles were in vitro cultured for 10 days and cumulus-oocyte complexes were harvested at 16-18 hours after hCG and EGF treatment. Mature oocytes were assessed their maturational ability and developmental competence in vitro. Results: In Exp I, The vitrification-warming procedure decreased the intact (grade 1, G1) follicle ratio in the vitri-con and FrOT-D2 groups compared with that in the fresh control, and this ratio was reduced more by ischemic injury after transplantation (fresh: 64.2%, vitri-con: 50.3%, and FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovarian tissue than in the fresh control, and it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0%, and FrOT-D2: 26.8%). The mean number of follicles per section and CD31-positive area was significantly reduced after vitrification and transplantation. (the number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, and FrOT-D2: 17.9 ± 2.1; CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9%, and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1%, and FrOT-D21: 70.7%; CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6%, and FrOT-D21: 7.5 ± 0.8%). In Exp II-I, the percentage of grade 1 total follicles was significantly higher in only the 10 mg/mL LeIBP group than in the vitrification control while all of AFP-treated groups had significantly improved grade 1 primordial follicle ratio compared with the vitrification control. The apoptotic (TUNEL-positive) follicle ratio was significantly decreased in the 1 and 10 mg/ml of LeIBP treated groups. The proportion of τH2AX positive follicles was significantly reduced in all AFP-treated groups while the Rad51-positive follicle ratio was significantly decreased in only FfIBP and LeIBP treated groups. In ExpII-II, after auto-transplantation of OT vitrified with 10 mg/ml of LeIBP, the percentage of total Grade 1 follicles and primordial Grade 1 follicles, the extent of the CD31-positive area were significantly increased. Moreover, the level of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated group than in the control group. In Exp III, The group that received simvastatin and methylprednisolone showed a significantly improved intact (G1) follicle ratio (D2: p<0.001, D7: p<0.05 and D21: p<0.001), apoptotic follicle ratio (D21: p<0.05), CD31-positive area (D7: p<0.05 and D21: p<0.05), and serum AMH level (D7: p<0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rate, the number of total and apoptotic blastomere per blastocyst and ICN/TE ratio among the four transplantation groups. In Exp IV, With respect to the follicular growth, the diameter of follicles in oil layer culture was significantly higher than that of without oil layer. In addition, maturational criteria including survival in oil layer, pseudo-antral like cavity formation, ovulation and oocyte maturation, also significantly increased compared with the without oil layer. Late stage of culture, estradiol on D10 and progesterone on D11 in spent medium of oil layer was statistically significant different between without oil layer culture condition. When comparing the mRNA expression in matured oocytes, no significant difference was observed between in vivo and in vitro derived oocytes. On the other hand, in vitro grown and matured oocytes increased the level of reactive oxygen species and decreased the mitochondrial activity when compared with the in vivo mature oocytes with statistical significance. Moreover, cortical granules of both in vitro derived oocytes seemed to be more clumped and unevenly distributed than in vivo control. However, no significant difference was noted in actin filament configuration and spindle normality between in vitro and in vivo derived oocytes. Conclusions: Cryoinjury and ischemic injury are main cause of follicular depletion during fertility preservation process using ovarian tissue. Inevitable post-transplantation ischemia seems to be more deleterious than cryoinjury during cryopreservation process. Then, cryoinjury and ischemic injury could be decreased by use of AFPs and a combination of simvastatin and methylprednisolone. It can improve the quality of OT via promotion of vessel integrity in OT after cryopreservation and transplantation process. Therefore, minimizing cryoinjury and ischemic injury by enhancing vascularization is needed to improve the ovarian function after fertility preservation. Finally, optimization of two-dimensional in vitro follicle culture method was also attempted to avoid the re-implantation of residual malignancy cells in OT after transplantation and found the cause of reduction in embryonic development competence from in vitro derived oocytes.

Identification of new membrane-associated markers in assessing vitrification efficiency of oocytes from aged mice

엄다은 건국대학교 대학원 2019 국내석사

유리화 동결은 여성의 생식능력을 유지할 수 있는 효과적인 생식 보조술이다. 35세 이전의 여성에서 채란 된 난자의 유리화 동결 이후 수정 및 배아 발달율은 신선 난자와 비교했을 때 임상적으로 유의적 차이를 보이지 않는다. 최근 생식능력을 유지하고자 하는 고령의 여성에서 유리화 동결의 수요가 증가하고 있는 반면, 유리화 동결의 기본 프로토콜은 젊고 어린 여성의 난자에 맞춰져 있다. 많은 연구결과에 따르면, 여성의 나이가 생식능력 뿐 아니라 동결효율에 영향을 미치는 주요한 인자이다. 유리화 동결 과정은 난자에 삼투 및 동결 손상을 유발하며 이는 동결 난자의 해동 후 생존에 여부에 영향을 미친다. 고령의 여성의 난자는 특히 유리화 동결에 낮은 효율을 보이며, 이는 세포막 약화와 관련이 있을 것으로 보인다. 세포막은 난자의 가장 자리에서 세포 내부와 외부를 구분 지으며, 외부의 물리적 화학적 충격에서 세포를 보호한다. 본 연구에서는 고령 난자의 유리화 동결 취약의 원인으로 세포막에 초점을 맞춰 진행되었다. 가장 먼저, 나이 든 생쥐는 어린 생쥐보다 적은 수의 난자를 배란 하는 것을 확인했으며, 또한 유리화 동결 생존율이 크게 떨어졌다. 또한 유리화 동결 과정을 거친 고령 생쥐의 난자의 세포막을 염색해 보았을 때 세포막의 비연속적으로 염색됨을 관찰하였고, 세포 내부의 지질류의 감소가 확인되었다. 또한 유리화 동결 과정을 거친 고령 생쥐의 난자는 SYTOX Green 염색약에 DNA가 염색됨을 확인하였다. 이러한 결과는 고령 난자의 세포막이 유리화 동결 과정을 거치며 손상 받았음을 의미한다. 또한 본 연구에서는 이러한 세포막 약화현상이 Necroptosis와의 연관성을 살펴보았다. 마지막으로 유리화 동결 과정에 PEG8000을 첨가함으로써 고령 생쥐의 난자의 동결 효율이 개선됨을 확인하였다. 이것은 약화된 고령 난자의 세포막을 보강하여 고령 여성의 난자의 유리화 동결 효율의 향상에 도움이 될 수 있음을 시사한다. 종합적으로 본 연구에서는 유리화 동결에 취약한 고령 생쥐 난자의 세포막 약화현상의 분자적 세포학적 근거를 제시한다. Vitrification is an effective method to preserve the fecundity. In humans, vitrification of oocytes and their survival after warming does not affect fertilization and developmental potential in women under 35 years of age. However, while the demand for vitrification in aged women has been increasing in recent years, the standard vitrification protocol is focused on healthy and fertile oocytes from young women. Accumulating evidence suggests that maternal age is one of the most important factors of female reproductive ability as well as vitrification efficiency. The vitrification procedure accompanies osmotic damage and cryoinjury to oocytes which lead to low survival rates of vitrified-warmed oocytes. Oocytes from aged women with poor vitrification efficiency may have membrane-associated weaknesses. In this study, I focused on the plasma membrane of oocytes as the main factor of low efficiency of vitrification. The plasma membrane is the outermost layer, protecting oocytes against various physical and chemical stresses. I first confirmed that ovulation and survival rates after vitrification significantly decreases in oocytes from aged mice. Besides, the oocytes from aged mice showed membrane discontinuity after vitrification and warming procedure and the cytosolic lipid contents were reduced. The majority of these oocytes were stained positively for SYTOX Green dye after vitrification and warming. I show that the membrane weakening phenomenon in oocytes from aged mice were associated with necroptotic cell death. PEG8000 was partially effective in improving survival rates after vitrification and warming of oocytes from aged mice, suggesting a possibility that membrane fortifying method may help improve survival of vitrified-warmed oocytes from aged mice. Collectively, this study provides the molecular and cellular evidence underlying the weakened plasma membrane of oocytes in aged mice.

宿根 안개초(Gypsophila paniculata L.)의 組織培養에 있어서 Vitrification의 發生原因과 防止에 關한 硏究

최영윤 全南大學校 1990 국내석사

器內培養方法을 利用한 宿根 안개초의 大量增殖 過程에 있어서 問題가 되고 있는 vitrification의 發生原因과 防止를 위한 實驗을 實施한 바 몇가지 基礎資料를 얻었기에 報告하는 바이다. 1. MS 培地에 BA量을 增加시킬수록 vitrification率은 增加하였으나, BA의 濃度가 높더라도 agar의 濃度를 높여 줌으로써 苗의 透明化率을 減少시킬 수 있었으며, agar의 濃度를 1.6%까지 增加시켜 줌으로써 苗의 vitrification율을 33%까지 減少시킬 수 있었다. 2. NH₄NO₃의 量이 많은 MS基本培地 보다는 NH₄NO₃量을 반으로 줄인 MS-1/2NH₄NO₃ 培地가 苗의 透明化率이 낮았다. 3. 日長이 8時間 以下일 境遇에는 苗의 透明化率을 增加시켰으나, 그 以上의 日長處理에 있어서는 차이가 거의 없었다. 4. 光度處理에 있어서는 2000lux를 基準으로, 너무낮을 境遇를 除外하고는 苗의 透明化率에 큰 影響을 미치지 못했다. 5. 培養用器의 크기와 苗의 vitrification과의 관계는 거의 없는 것으로 나타났다. 6. 培地內에 ABA를 0.3ppm 添加하므로써 苗의 透明化率을 36.4%까지 減少시킬 수 있었다. The vitrification in Gypsophila paniculata L. is a serious problem when they are mass-propagated in vitro. the present study was, therefore, performed in order to examine its cause and prevention. The results obtained were summarized as follows: 1. There was a tendency that the rate of vitrification was increased when the higher concentration of BA was used in the MS medium. However, the rate of plantlet's vitrification could be reduced by using the higher agar concentration in the medium even though BA concentration was high. If the concentration of agar was raised up to 1.6%, the rate of vitrification was reduced down to 33%. 2. The amount of nitrogen in the medium also affected the rate of vitrification. When the half the amount of NH₄NO₃recommended in MS medium was used, the vitrification rate was decreased. 3. The day length below 8 hours increased the rate of plantlet's vitrification, while the longer photoperiod did not show any differences. 4. The light intensity less than 2000 lux increased the rate of vitrification. However, higher light intensity did not affect the vitrification rate. 5. There was no relationship between the size of container and the rate of vitrification. 6. By the addition of ABA in the medium (0.3ppm), the rate of vitrification was reduced up to 36.4%.

Effect of antifreeze protein and necrostatin on ovarian cryopreservation and transplantation

이정렬 서울대학교 대학원 2013 국내박사

INTRODUCTION: Ovarian cryopreservation and transplantation is one of the most promising options of fertility preservation for women have survived malignant disease. However, the survival rate of cryopreserved ovarian tissue after transplantation is still not sufficient for the application of this procedure in routine clinical practice. Possible causes of ovarian damage after cryopreservation and transplantation are cryodamage during cryopreservation and ischemic damage after transplantation. Cryoprotective and anti-necrotic agents are expected to reduce this damage. Antifreeze proteins (AFPs) are a class of polypeptides produced by animals such as antarctic fish; these proteins allow their survival in subzero environments. Several recent studies on the protective effect of AFP for cryopreservation of animal cells and organs have been reported. However there has been no study on AFP for ovarian tissue cryopreservation. Necrostatin-1 (Nec-1) is a receptor-interacting protein 1 kinase (RIP1) inhibitor and known to have inhibitory effect for necroptosis via RIP1. The purpose of this study was to investigate the effects of AFP and Nec-1 supplementation during ovarian tissue vitrification and transplantation. METHODS: Ovaries from 4-week-old ICR mice were used for vitrification. Ovaries were vitrified using a two-step procedure involving exposure to equilibrium and vitrification solutions. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. All solutions were based on Dulbecco’s phosphate buffered saline (DPBS) containing 20% fetal bovine serum (FBS). Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. To investigate the effect of AFP, 0, 5, or 20mg/mL of AFP III was added into the vitrification solution and to investigate the effect of Nec-1, 0, 25, or 100 μM of Nec-1 was added into the vitrification solution. After warming, follicular morphology and apoptosis were assessed by histological analysis and the TUNEL assay. A part of ovaries vitrified in each group were warmed and autotransplanted. In the experiment assessing Nec-1, 0, 25, or 100 μM of Nec-1 was added to each warming solution. In addition, the same concentrations of Nec-1 were injected intraperitoneally to a final volume of 0.3 mL. After 2 weeks, follicular morphology and apoptosis of transplanted ovaries were assessed. Immunostaining with Ki-67 and vascular endothelial growth factor (VEGF) antibodies was performed for analyses of proliferative and angiogenic activity. RESULTS: Morphological analysis after vitrification and warming showed a significantly higher intact follicle ratio in the AFP treated groups (control, 28.9%; 5 mg/mL AFP treated group, 42.3%; and 20 mg/mL AFP treated group, 44.7%). The rate of apoptotic follicles (TUNEL positive) was significantly lower in the AFP treated groups (control, 26.6%; 5 mg/mL AFP treated group, 18.7%; and 20 mg/mL treated group, 12.6%). After transplantation of the vitrified-warmed ovaries, morphological analysis showed a significantly higher intact follicle ratio in the 20 mg/mL AFP treated group compared with control and 5 mg/mL AFP treated groups. The rate of apoptotic follicles was similar among the groups. Ki-67 positive follicle ratio increased significantly as the AFP dose increased (control, 3.3%; 5 mg/mL AFP treated group, 31.3%; and 20 mg/mL AFP treated group, 49.0%). Mean immunohistochemical intensity score of VEGF staining was significantly higher in both AFP treated groups in follicles and in the 20 mg/mL AFP treated group in the stromal cells. Morphological analysis after vitrification and warming showed a significantly higher intact follicle ratio in the Nec-1 treated groups (control, 45.1%; 25 μM Nec-1 treated group, 51.7%; and 100 μM Nec-1 treated group, 57.9%). The rate of apoptotic follicles was lower in the Nec-1 treated groups (control, 11.2%; 25 μM Nec-1 treated group, 8.5%; and 100 μM Nec-1, 7.2%). After transplantation of the vitrified-warmed ovaries, morphological analysis showed a significantly higher intact follicle ratio in the Nec-1 treated groups (control, 43.1%; 25 μM Nec-1 treated group, 60.6%; and 100 μM Nec-1 treated group, 70.7%). Nec-1 treated groups showed a lower rate of apoptotic follicles (control, 5.3%; 25 μM Nec-1 treated group, 2.5%; and 100 μM Nec-1 treated group, 2.0%). The Ki-67 positive follicle ratio was not different according to Nec-1 treatment. The mean intensity score of VEGF staining of the stromal cells was significantly higher in the 100 μM Nec-1 treated group. CONCLUSIONS: The results of the present study suggest that supplementing AFP in the vitrification solution and Nec-1 during vitrification, warming, and transplantation has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation. 서론: 난소동결보존과 이식은 여성 암 생존자에서 가임력보존의 가장 이상적인 방법으로 기대된다. 그러나 동결보존된 난소의 생존율이 낮아 아직 임상에서 널리 사용되기에는 부족한 점이 많다. 난소 동결보존과 이식 시 난소 손상은 주로 동결 시 발생하는 동결손상과 이식 시 발생하는 허혈성 손상에 기인한다. 동결방지제와 항괴사제는 이러한 동결손상과 허혈성괴사를 감소시킬 것으로 기대된다. 항동결단백 (antifreeze protein, AFP)은 극지생물의 체내에서 생성되는 폴리펩티드의 한 종류로 이들이 극지에서 살아가는 것을 가능하게 하는 물질이다. 최근 들어 항동결단백이 동물세포와 기관의 동결보존 시 보호효과를 보인다는 연구가 보고되었으나 항동결단백의 난소동결에서의 효과에 대해서는 연구가 이루어진 바가 없다. Necrostatin-1 (Nec-1)은 receptor-interacting protein 1 kinase (RIP1)의 억제제로 이를 통한 necroptosis를 억제하는 역할을 하는 것으로 알려진 물질이다. 본 연구의 목적은 항동결단백과 Nec-1의 첨가가 난소조직 동결보존에 미치는 효과를 탐색하는 것이다. 방법: 4주령 ICR마우스를 사용하여 유리화 동결 실험을 진행하였으며 유리화 동결 과정은 평형용액과 유리화동결용액에 노출시키는 2단계 방법으로 진행되었다. 평형용액은 20% ethylene glycol (EG), 유리화동결용액은 40% EG, 18%Ficoll, 0.3M sucrose로 구성되었으며 두 용액 모두 20% fetal bovine serum (FBS)을 포함한 Dulbecco’s phosphate buffered saline (DPBS)을 기본용액으로 하여 제작되었다. 적출된 난소를 1mL의 평형용액에 10 분간 처리한 후 0.5mL의 유리화동결용액에 5분간 처리하였다. 항동결단백 첨가의 효과를 탐색하기 위해서는 각각 0, 5, 20mg/mL의 항동결단백을 유리화동결용액에 첨가하였으며, Nec-1첨가의 효과를 탐색하기 위해서는 각각 0, 25, 100 μM의 Nec-1을 유리화동결용액에 첨가하였다. 동결과 해동 후 난포형태와 세포자연사 여부를 조직학적 검사와 TUNEL assay를 이용하여 평가하였다. 유리화동결된 난소 중 일부는 해동하여 자가이식을 시행하였다. Nec-1 효과를 보기 위한 실험에서는 각각 0, 25, 100 μM의 Nec-1을 해동용액에 첨가함과 아울러 각각 0, 25, 100 μM의 Nec-1용액 0.3mL를 이식 30분 전에 복강내 주사하였다. 이식 2주 후 이식된 난소의 난포형태 분석과 세포자연사 여부 평가를 시행하였다. Ki-67과 혈관내피성장인자 (vascular endothelial growth factor, VEGF)항체의 면역조직화학 염색을 시행하여 이식된 난소의 증식과 혈관생성을 평가하였다. 결과: 유리화동결과 해동 후 형태학적 분석 결과 항동결단백 처리군에서 유의하게 높은 정상난포 비율을 보였다 (대조군: 28.9%, 항동결단백 5mg/mL투여군: 42.3%, 항동결단백 20mg/mL 투여군: 44.7%). 세포자연사가 진행된 난포 (TUNEL 양성 난포)의 비율은 항동결단백 처리군에서 유의하게 낮았다 (대조군: 26.6%, 항동결단백 5mg/mL투여군: 18.7%, 항동결단백 20mg/mL 투여군: 12.6%). 해동-이식 후 형태학적 분석 결과 항동결단백 20mg/mL처리군에서 유의하게 높은 정상난포 비율을 보였다. 세포자연사가 진행된 난포의 비율은 각 군에서 유의한 차이가 없었다. Ki-67 양성 난포의 비율은 항동결단백의 투여용량에 따라 유의하게 증가하는 양상을 보였다 (대조군: 3.3%, 항동결단백 5mg/mL투여군: 31.3%, 항동결단백 20mg/mL 투여군: 49.0%). VEGF의 immunohistochemical intensity score는 난포에서는 항동결단백 투여군 모두에서, 기질세포에서는 항동결단백 20mg/mL투여군에서 유의하게 높았다. Nec-1투여후 유리화동결과 해동 시 형태학적 분석 결과 Nec-1 처리군에서 유의하게 높은 정상난포 비율을 보였다 (대조군: 45.1%, Nec-1 25 μM 투여군: 51.7%, Nec-1 100 μM 투여군: 57.9%). 세포자연사가 진행된 난포의 비율은 Nec-1 처리군에서 낮은 양상을 보였다 (대조군: 11.2%, Nec-1 25 μM 투여군: 8.5%, Nec-1 100 μM 투여군: 7.2%). 해동-이식 후 형태학적 분석 결과 Nec-1 처리군에서 유의하게 높은 정상난포 비율을 보였다 (대조군: 43.1%, Nec-1 25 μM 투여군: 60.6%, Nec-1 100 μM 투여군: 70.7%). 세포자연사가 진행된 난포의 비율은 Nec-1 처리군에서 낮은 양상을 보였다 (대조군: 5.3%, Nec-1 25 μM 투여군: 2.5%, Nec-1 100 μM 투여군: 2.0%). Ki-67 양성 난포비율은 각 군에서 차이를 보이지 않았고, 기질세포에서의VEGF의 immunohistochemical intensity score는 Nec-1 100 μM 투여군에서 유의하게 높은 결과를 보였다. 결론: 본 연구의 결과 난소조직의 유리화 동결 시 항동결단백의 첨가와 유리화동결, 해동 및 이식 시 Nec-1의 첨가가 난소조직의 동결보존과 이식 시 유익한 효과를 나타낼 수 있다는 점을 확인할 수 있다.

A study on the role of antifreeze protein for improvement of effectiveness in ovarian tissue cryopreservation

공현선 서울대학교 대학원 2018 국내박사

연구 목적: 난소조직 동결보존 시 발생할 수 있는 얼음 결정의 성장은 조직에 동결 손상을 주게 된다. 항동결단백질은 얼음 결정의 성장을 막아준다고 알려져 있다. 따라서 본 연구에서는 항동결단백질을 마우스 혹은 소 난소조직 동결보존 시 처리하여 동결손상 극복을 도모하고자 ① 각기 다른 특성을 갖는 항동결단백질을 마우스 난소 유리화동결에 사용하여 각 단백질이 난소 동결보존에 어떤 영향을 미쳤는지 알아보고자 하였고 ② 마우스 난소 유리화동결-해동 시 항동결단백질 처리 단계가 다른 두 가지 프로토콜을 비교하여 최적의 프로토콜을 확립하고자 하였으며 ③ 항동결단백질을 유리화동결 보존한 소 난소 조직 해동 시 처리하였을 때 동결손상 극복에 효과를 보였는지, 또한 해당 조직 중 일부는 이종 이식하여 이식 후 어떤 결과를 보이는지 알아보고자 하였다. 방법론: 첫 번째 연구에서는 항동결단백질 중Notched-fin eelpout (nfe)이라는 물고기 유래의 항동결단백질 isoform들 중 nfeAFP6, nfeAFP8, nfeAFP11을 선정하였다. 더불어 좀 더 다양한 특성의 항동결단백질을 비교 분석 하고자 세 가지 isoform의 변이형을 만들어 각각 nfeAFP6_tri, nfeAFP8_tri, nfeAFP11_tri로 명명하였다. 마우스 난소 동결보존 실험에 앞서, 총 6개의 nfeAFP들에 대한 특성을 분석하고자 각 항동결단백질의 온도 이격 (thermal hysteresis; TH)과 얼음 재결정 억제 (ice recrystallization inhibition; IRI) 활성을 측정하였다. 측정을 마친 nfeAFP들은 마우스 난소 유리화동결 및 해동 과정에 10 mg/mL 의 농도로 처리하여 사용하였다. 총 139개의 5주령 B6D2F1 마우스 난소를 무작위로 동결대조군이나 6개의 nfeAFP 처리군으로 (nfeAFP6, nfeAFP8, nfeAFP11, nfeAFP6_tri, nfeAFP8_tri, nfeAFP11_tri) 분배하였다. 각 군의 난소는 해동 후 H&E 염색을 통한 난포형태 분석이나 TUNEL assay를 통한 난포 세포자멸사 분석, τH2AX/Rad51 면역 염색을 통한 난포의 이중가닥 DNA 손상/회복 정도를 분석에 이용하였다. 두 번째 연구에서는 항동결단백질의 한 종류인 LeIBP를 사용한 기존 LeIBP 처리 프로토콜과 (LeIBP를 유리화동결 및 해동 첫 단계에 처리) 새로운 LeIBP 처리 프로토콜을 (LeIBP를 해동 첫 단계에만 처리) 비교하고자 하였다. 본 실험에는 총 42개의 5주령 B6D2F1 마우스 난소를 사용하였고, 이는 무작위로 동결대조군이나 두 개의 실험군인 LeIBP-all 군 (기존 LeIBP 처리 프로토콜 실험군), LeIBP-w 군(새로운 LeIBP 처리 프로토콜 실험군)에 배정하였다. LeIBP는 각 군에 맞게 10 mg/mL의 농도로 유리화동결-해동 메디아에 처리하여 사용하였다. 샘플 분석으로는 H&E염색을 통한 난포형태 분석, TUNEL assay를 통한 난포 세포자멸사 정도 분석, τH2AX/Rad51 면역 염색을 통한 난포의 이중 가닥 DNA의 손상/회복 정도를 분석을 사용하였다. 세 번째 연구에서는 소 난소 조직 중 원시난포가 분포하고 있는 피질만 5 ⨯ 5 ⨯ 1 mm3로 분리하여 fresh대조군, 동결대조군, LeIBP-10군 (LeIBP 10 mg/mL을 해동 첫 단계 메디아에 처리), LeIBP-20 군 (LeIBP 20 mg/mL을 해동 첫 단계 메디아에 처리)에 배정하였다. 각 군의 난소조직 중 일부는 바로 H&E를 통한 형태학적 분석에 사용되었고, 일부 조직은 면역결핍쥐에 이종이식한 후 7일째에 회수하여 난포의 형태학적 분석 및 이식체의 혈관형성정도, 섬유화정도 분석에 사용하였다. 결과: nfeAFP isoform 특성 분석 결과, nfeAFP6은 낮은 TH 및 낮은 IRI를, nfeAFP8은 높은 TH 및 높은 IRI를, nfeAFP11은 낮은 TH 및 낮은 IRI를 나타냈다. Isoform 변이형의 경우, nfeAFP6_tri는 높은 TH 및 높은 IRI를, nfeAFP8_tri는 낮은 TH 및 낮은 IRI를, nfeAFP11_tri는 높은 TH 및 높은 IRI를 나타내었다. nfeAFP를 이용한 난소 동결보존 실험에서는 총 22,286개의 난포가 난포의 형태학적 분석에 사용되었다. 동결대조군 대비 nfeAFP 처리군들에서 정상 형태의 총 난포율이 유의하게 높은 것을 확인할 수 있었다 (동결대조군: 36.3%, nfeAFP6: 56.1%, nfeAFP8: 60.4%, nfeAFP11: 54.1%, nfeAFP6_tri: 61.2%, nfeAFP8_tri: 51.1%, nfeAFP11_tri: 60.7%). 원시 난포의 경우 동결대조군 대비로 nfeAFP8_tri군만 제외하고 모든 군에서 유의하게 증가한 정상 형태 원시난포율을 확인할 수 있었다 (동결대조군: 53.6, nfeAFP6: 61.4%, nfeAFP8: 69.0%, nfeAFP11: 62.3%, nfeAFP6_tri: 70.2%, nfeAFP8_tri: 58.0, nfeAFP11_tri: 66.9%). 실험군간 비교에서는 전반적으로 높은 TH와 높은 IRI특성을 보인 실험군이 다른 실험군에 비해 유의하게 높은 정상 형태 난포율을 보였다. 난포 내 세포자멸사 정도 비교 결과, 동결대조군 대비 모든 nfeAFP처리군에서 유의하게 낮은 결과를 확인할 수 있었다. (동결대조군: 33.2%, nfeAFP6: 22.7%, nfeAFP8: 17.0%, nfeAFP11: 27.5%, nfeAFP6_tri: 20.8%, nfeAFP8_tri: 27.5%, nfeAFP11_tri: 22.2%). 특히 실험군 중에서도 nfeAFP8 군이 가장 유의하게 낮은 난포 세포자멸사를 기록했다. τH2AX-positive 난포의 경우 nfeAFP8 (43.6%) 과 nfeAFP11_tri (48.9%)군에서 동결대조군 (55.2%) 대비 유의하게 낮은 결과를 보였다. 더불어 Rad51-positive 난포의 경우 nfeAFP8 (43.0%) 군만 동결대조군 (48.9%) 대비로 유의하게 낮아진 결과를 나타냈다. 두 번째 연구에서는 LeIBP 처리군 (LeIBP-all: 원시난포: 73.0%, 성장 난포: 60.3%, 총 난포: 66.1%; LeIBP-w: 원시난포: 74.4%, 성장 난포: 55.3%, 총 난포: 63.2%)이 동결대조군(원시난포: 66.6%, 성장 난포: 50.2%, 총 난포: 56.3%)과 비교하여 증진된 정상 난포율을 보였다. 난포 세포자멸사 정도는 두 LeIBP 처리군(LeIBP-all: 9.2%, LeIBP-w: 9.3%)이 대조군(17.5%)보다 유의하게 낮았고, 두 실험군간 비교에서는 통계적 유의차 없었다. 두 LeIBP 처리군 (LeIBP-all: τH2AX-positive 난포: 31.2%, Rad51-positive 난포: 31.6%; LeIBP-w: τH2AX-positive 난포: 29.6%, Rad51-positive 난포: 32.8%)은 동결대조군(τH2AX-positive 난포: 36.9%, Rad51-positive 난포: 38.4%)과 비교하여 유의하게 감소한 이중 가닥 DNA 손상 (τH2AX-positive)/회복 (Rad51-positive) 난포율을 보였고, 두 LeIBP처리군간 비교 시 유의차 없는 결과를 보였다. 세 번째 연구에서 동결대조군 (원시난포: 66.7%, 총 난포: 58.6%)이 fresh군 (원시난포: 80.1%, 총 난포: 74.5%)보다 유의하게 낮은 정상 원시난포율과 총 난포율을 보였다. 그러나 동결대조군과 대비 LeIBP-10 군 (원시난포: 78.7%, 총 난포: 72.7%)과 LeIBP-20군 (원시난포: 73.8%, 총 난포: 73.3%)에서정상 원시난포율과 총 난포율의 결과가 향상되는 것을 확인할 수 있었다. 이식 후 결과에서는 LeIBP-20군(원시 난포: 57.8%, 총 난포: 55.7%)의 형태학적 정상 원시난포 및 총 난포율이 동결대조군(원시난포: 48.9%, 총 난포: 44.2%)과 비교하여 증가함을 보였다. 이식체의 미세혈관형성 정도 및 섬유화 정도의 경우 군간 유의차 없었다. 결론: 높은 TH와 IRI 특성을 가지는 항동결단백질이 난소 동결보존 효율 증진에 효과적인 것을 확인할 수 있었다. 높은 TH와 IRI특성을 보이는 항동결단백질이라도 변이체보다는 원형태의 단백질의 동결보존 효율이 더 좋은 것 또한 확인할 수 있었다. LeIBP 처리 프로토콜 비교 연구에서는 기존 LeIBP처리 프로토콜 (유리화동결 및 해동 첫 단계에 모두 처리), 새 LeIBP 처리 프로토콜 (해동 첫 단계에만 처리) 모두 난포 형태 유지나 난포 세포자멸사, 이중 가닥 DNA 손상 극복 면에서 증진된 결과를 확인할 수 있었다. 따라서, 본 연구 결과를 통해 난소 조직 유리화 동결 해동 시 새 프로토콜을 사용할 경우 기존 프로토콜 대비 유사한 항동결 효과를 나타내면서, 사용하는 항동결단백질의 양은 최소화 할 수 있다는 결론을 도출하였다. 세 번째 연구결과 처음으로 소 난소동결 시 항동결단백질을 사용하여 동결보존효율 증진 효과를 확인할 수 있었다. LeIBP를 소 난소 해동 시 처리할 경우 농도에 상관없이 잘 보존된 난포의 형태를 확인할 수 있었고, 해당 조직을 이종이식 할 경우 고농도의 LeIBP 처리군에서 잘 보존된 난포의 형태를 볼 수 있었다. Objectives: Cryodamage caused by ice crystal growth is a major problem in ovarian tissue (OT) cryopreservation. Antifreeze proteins (AFPs) are known to have ice growth inhibition activity. To improve the outcome of OT cryopreservation, the role of AFPs were investigated in mouse and bovine OT cryopreservation. This study was aimed to ① investigate the effect of AFPs with different characteristics on mouse OT vitrification-warming (chapter I), ② compare different AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only (chapter II) and ③ investigate the AFP treatment effect on bovine OT vitrification-warming and xenotransplantation by treating AFPs only on the warming process (chapter III). Methods: Fish origin AFP isoforms, nfeAFP6, nfeAFP8 and nfeAFP11, were recombined for this study. Three mutant AFPs named nfeAFP6_tri, nfeAFP8_tri and nfeAFP11_tri were made from the each isoforms nfeAFP6, nfeAFP8 and nfeAFP11 to compare different AFPs with more variable antifreeze characteristics in chapter I study. For antifreeze characteristic evaluation, the six nfeAFPs were analyzed by thermal hysteresis (TH) and ice recrystallization inhibition (IRI) assays. After the antifreeze characteristics evaluations, the nfeAFPs were treated for the mouse OT vitrification-warming procedures. The mouse OTs (n=139) from five-week-old B6D2F1 mice were randomly divided into a vitrified-warmed control and the six different nfeAFP treated groups. In accordance with the nfeAFP treated groups, 10 mg/mL of nfeAFPs were added to the vitrification and warming solutions. After the vitrification-warming, the OTs were evaluated for follicle morphology, apoptosis and DNA double-strand break (DSB) damage/repairing by hematoxylin-eosin staining, TUNEL assay, and τH2AX/Rad51 immunostaining, respectively. For the chapter II study, 10 mg/mL of recombinant LeIBP (a type of AFP) was used to compare two different LeIBP treatment protocols: a conventional protocol with LeIBP treatment during vitrification and first-step warming, and a new protocol with LeIBP treatment during the first-step warming only. Five-week-old B6D2F1 mouse OTs (n=42) were randomly divided into a vitrified-warmed control and two experimental groups; one is the conventional LeIBP protocol treated group (LeIBP-all) and the other is the new LeIBP protocol treated group (LeIBP-w). For OT sample analysis, ratios of ovarian follicle integrity, apoptosis, and DNA DSB damage/repairing were evaluated by hematoxylin-eosin staining, TUNEL assay, and τH2AX/Rad51 immunostaining, respectively. Bovine OT cortex was separated and cut into 5 ⨯ 5 ⨯ 1 mm3 pieces for chapter III experiment. The OTs were randomly divided into four groups: a fresh control, a vitrified-warmed control (without LeIBP treatment), a LeIBP-10 group (10 mg/mL of LeIBP was treated during the warming process of vitrified OTs) and a LeIBP-20 group (20 mg/mL of LeIBP was during the warming process of vitrified OTs). Some of the OTs from each group was used for either OT morphology evaluation or xenotransplantation for OT grafts morphology, microvessel and fibrosis evaluations. For xenotransplantation, two pieces of OTs were xenografted to an ovariectomized nude mouse for a week. The OT morphology evaluation was performed by hematoxylin-eosin staining. Microvessel and fibrotic area of OTs were analyzed by CD31 immunostaining and Masson’s trichrome staining, respectively. Results: In terms of the outcomes of the nfeAFP isoforms antifreeze characteristics evaluations, nfeAFP6, nfeAFP8, and nfeAFP11 showed low TH with low IRI, high TH with high IRI and low TH with low IRI, respectively. With regards to the mutant nfeAFPs, nfeAFP6_tri, nfeAFP8_tri, and nfeAFP11_tri showed high TH with high IRI, low TH with low IRI and high TH with high IRI characteristics, respectively. A total of 22,286 follicles were evaluated for their morphological integrity in chapter I study. Compare to the vitrified-warmed control, the nfeAFP treated groups showed significantly higher intact total follicle ratios (vitrified-warmed: 36.3%, nfeAFP6: 56.1%, nfeAFP8: 60.4%, nfeAFP11: 54.1%, nfeAFP6_tri: 61.2%, nfeAFP8_tri: 51.1% and nfeAFP11_tri: 60.7%). Compared to the vitrified-warmed control, significantly increased intact primordial follicle ratios were observed in the nfeAFP treated groups, except the nfeAFP8_tri group (vitrified-warmed: 53.6%, nfeAFP6: 61.4%, nfeAFP8: 69.0%, nfeAFP11: 62.3%, nfeAFP6_tri: 70.2%, nfeAFP8_tri: 58.0 and nfeAFP11_tri: 66.9%). Among the experimental groups, the groups with high TH and IRI showed significantly higher intact total and primordial follicle ratios than the other groups. The apoptotic follicle ratios were significantly decreased in all nfeAFP treated groups compared to the vitrified-warmed control (vitrified-warmed: 33.2%, nfeAFP6: 22.7%, nfeAFP8: 17.0%, nfeAFP11: 27.5%, nfeAFP6_tri: 20.8%, nfeAFP8_tri: 27.5% and nfeAFP11_tri: 22.2%). Among the nfeAFP treated groups, the nfeAFP8 group showed the lowest apoptotic follicle ratio with a significant difference. The proportions of τH2AX-positive follicles were significantly decreased in the nfeAFP8 (43.6%) and the nfeAFP11_tri (48.9%) groups compared to the vitrified-warmed control (55.2%). With regards to the Rad51-positive follicle ratio, the nfeAFP8 (43.0%) showed significantly decreased results than the vitrified-warmed control (48.9%). In chapter II, the LeIBP-treated groups showed significantly higher intact follicle ratios (LeIBP-all: primordial follicle: 73.0%, growing follicle: 60.3% and total follicle: 66.1%; LeIBP-w: primordial follicle: 74.4%, growing follicle: 55.3%, total follicle: 63.2%) than the vitrified-warmed control (primordial follicle: 66.6%, growing follicle: 50.2% and total follicle: 56.3%). Apoptotic follicle ratios were significantly decreased in both LeIBP-treated groups (LeIBP-all: 9.2% and LeIBP-w: 9.3%) than the vitrified-warmed control (17.5%); however between the LeIBP-treated groups, the results were not significantly different. With regard to the DNA DSB damage (τH2AX-positive)/repairing (Rad51-positive) follicle ratios, significantly reduced results were shown in both LeIBP-treated groups (LeIBP-all: τH2AX-positive follicle: 31.2%, Rad51-positive follicle: 31.6%; LeIBP-w: τH2AX-positive follicle: 29.6%, Rad51-positive follicle: 32.8%) compared to the vitrified-warmed control (τH2AX-positive follicle: 36.9%, Rad51-positive follicle: 38.4%), and the results were similar between the LeIBP-treated groups. In terms of bovine OT (non-grafted OT) morphology evaluation, significantly decreased intact primordial and total follicle ratios were observed in the vitrified-warmed control (primordial follicle: 66.7%, total follicle: 58.6%) than the fresh control (primordial follicle: 80.1%, total follicle: 74.5%). However, the intact primordial and total follicle ratios were increased in LeIBP-10 group (primordial follicle: 78.7%, total follicle: 72.7%) and LeIBP-20 group (primordial follicle: 73.8%, total follicle: 73.3%) when compared with the vitrified-warmed control. Among the xenografted OT morphology evaluation results, the LeIBP-20 group showed the highest intact primordial and total follicle ratios. Additionally, the total intact follicle ratio of LeIBP-20 group was significantly higher than the ratio of vitrified-warmed control (vitrified-warmed: 44.2%, LeIBP-20: 55.7%). There was no significant difference with regards to the graft microvessel and fibrotic area measurement outcomes. Conclusions: The nfeAFPs treatment with high TH and high IRI showed better outcomes after the mouse OT vitrification-warming and the best OT quality was observed in the nfeAFP8 group. For the LeIBP treatment protocol comparison study, both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DNA DSB damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse OT vitrification-warming procedure using AFP, while minimizing the treatment steps. In chapter III study, the LeIBP treated groups (LeIBP-10 and LeIBP-20) showed a cryoprotective effect during the vitrified bovine OT warming procedure in terms of preserving follicle morphology. Between the two LeIBP treated groups, only the LeIBP-20 group beneficially affects the preserving follicle morphology after the bovine OT xenografting.

소 배반포 Droplet Vitrification 동결시 미세조작이 생존율에 미치는 영향

이영호 順天大學校 大學院 2005 국내석사

본 연구는 2004년 1월 동년 4월까지 3개월 동안 체외 생산된 배반포를 이용하여 미세조작이 VS1에서 배반포에 미치는 영향과 oplet동결·융해 하였을 때 미세조작이 소 배반포 생존율과 탈출율에 미치는 영향을 규명하고자 실시하여 얻어진 결과는 다음과 같다. 1. VS1에서 1분 동안 노출 시켰을 때 미세조작을 한 배반포가 미세조작을 실시하지 않은 배반포 보다 각각 103.3±10.3와 131.4±3.5 ㎛으로 수축율에서 유의적 차이를 보였다 (P<0.05). 2. 드롭동결 융해 후 24시간째 생존율에서 미세 조작한 그룹이 91.2%와 33.7%이고 탈출율 또한 미세조작한 처리군에서 62.9%와 25.0%로 유의적 차이를 보였다 (P<0.05). 이상의 결과를 볼 때 동결 시 수축율이 높은 것이 배반포 동결·융해율에 영향을 미치는 것으로 사료되며, 이로써 미세조작이 배반포 동결에 유효한 방법임을 증명하였다. 이는 체외수정란의 다량의 생산과 저장에 실용성에서의 한 걸음 더 다가섰다고 할 수 있다. The purpose of this study was to investigate the puncture manipulation on the post-thaw survival rate of bovine blastocyst by droplet vitrification. To in vitro embryo production (IVP), the oocytes were collected from ovarian follicles of bovine. The oocytes collected were cultured in TCM-199 medium containing 10% FBS, 1 ㎍/ml estradiol-17β, 10 ㎍/ml FSH, 0.2 mM sodium pyruvate and 0.6 mM cystein at 5% CO_(2) in air at 39℃ for 20 hrs and then inseminated with 1 × 10^(6) sperms/ml cultured for 24 hrs. The presumed bovine zygotes were cultured in vitro in CR1aa medium supplemented with 0.3% fatty acid free bovine serum albumin for 3 days and subsequently in CR1aa medium supplemented with 10% FBS for 4 or 5 days. To vitrification procedure, the vitrification solution was consisted of VSI {10% ethylene glycol (EG), 10% DMSO in holding medium (HM: TCM-199 supplemented with 20% FBS)} and VS2 {20% EG, 20% DMSO in HM}. The blastocysts collected were vitrified using ethylene glycol/dirnethyl sulfoxide method as reported previously (Vajta et al., 1998). Post-thaw blastocysts were serially washed in 0.3 and 0.15 M sucrose in a HM and then in TCM-199 for 5 min in both cases. They was then cultured in CR1aa supplemented with 10% FBS for 24 hrs. The re-expansion and hatching rats in punctured blastocysts were signifincanthly higher than those in non-puncture group (91.2 vs 33.7%; 62.9 vs 25.0%, p<0.05) These results indicated that the puncture manipulation can be increased the surviviability of droplets vitrification of bovine IVP blastocyst.