• 한국가축번식학회지
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• 제23권3호
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• pp.221-227
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• 1999

미성숙한 소 난자 동결보존 기술의 개발은 체외수정, 복제동물 및 형질전환동물 생산에 필요로 하는 난자를 시간과 공간의 제약 없이 공급이 가능해지기 때문에 그 효용가치가 많으나 아직까지 성공 보고례가 없다. 본 연구에서는 전자현미경용 grid를 이용한 초급속 동결 방법에 의해 미성숙한 소 난자를 동결 보존한 후, 이 난자를 융해하여 체외성숙, 체외수정 및 배발달을 유도하였다. 동해제는 PBS에 40% ethylene glycol, 0.5M sucrose, 18%과 10% fetal bovine serum이 들어 있는 EFS40을 사용하였다 . 동결ㆍ융해 후의 난자의 생존율은 48% 정도로 대조군에 비해 현저히 낮았으나 metaphase-II까지의 성숙율은 78%로, 정상 자웅전핵 형성율 75%로 대조군에 비해 차이가 없었다. 또한 배반포까지의 배발달율은 대조군23%와 동결군은 5%로 동결 융해한 것이 낮았으며 수정후 108~120시간 째 배반포를 염색하여 세포수률 알아본 결과는 각각 128$\pm$5와 98$\pm$8이었다. 항동해제내 $Na^{＋}$ 이온의 농도에 따른 미성숙된 난자의 생존율, 성숙, 수정 및 배발달율을 조사하였으나 유의차가 없었다. 이상의 결과는 동결 보존된 미성숙 소 난자가 융해후 체외성숙 및 수정에 의해 배반포까지의 발달이 가능하다는 것을 시사하고 있다.

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.23-28
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• 2003

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into $30^{\circ}C$ water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p<0.05). The effect of room temperature ($25^{\circ}C$ for 10 min) and cold temperature ($4^{\circ}C$ for 10 min) on COCs were determined in this study. After IVF, the cleavage and blastocysts rate of oocytes exposed to room temperature and cold temperature in VS+5% PVP was significantly different (2 cell: 63.20% vs 37.97%, blastocysts: 18.40% vs 2.53%). The cleavage rates of frozen-thawed oocytes were 20.53% with PVP and 22.13% without PVP (p>0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation.

• 한국가축번식학회지
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• 제19권2호
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• pp.129-134
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• 1995

This study was carried out to investigate on the survival rate and in vitro developmental rate of bisected porcine embryos and immature oocytes by manipulator and micropipette. Bisected embryos and immature oocytes cultured for 1∼5 days in TCM 199 medium with 20% FCS. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos and oocytes were 26.1%, 22.7%, respectively. The survival rate of bisected embryos and oocytes was significantly lower than that of non-bisection embryos(62.5%). 2. The survival rate of bisected porcine embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 26.9%, 19.2%, 19.2% and 11.5%, respectively. 3. The in-vitro developmental rate with and without zona pellucida of bisected porcine embryos by micromanipulator were 30.8%, 25.0%, respectively.

• Clinical and Experimental Reproductive Medicine
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• 제45권4호
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• pp.189-194
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• 2018

Objective: The aim of this study was to compare the rate of maturation, fertilization, and embryo development of in vitro-matured human oocytes derived from pregnant and non-pregnant women. Methods: Immature oocytes were obtained by needle aspiration from 49 pregnant women (group 1) who underwent a cesarean section at term and 77 non-pregnant women (group 2) who underwent a gynecological operation during the same period (8 months). Healthy immature oocytes (530 in group 1 and 539 in group 2) were cultured and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. Results: The percentage of degenerated oocytes was significantly higher (12.1% vs. 6.3%; p<0.001) in group 1 than in group 2. There was no significant difference in the maturation rate (66.8% vs. 68.1%; p=0.698), fertilization rate (66.7% vs. 67.6%; p=0.857), or the rate of formation of good-quality blastocysts (46.2% vs. 47.2%; p=0.898) in oocytes obtained from pregnant and non-pregnant women. Conclusion: The developmental competence of immature oocytes did not differ between pregnant and non-pregnant women.

• Clinical and Experimental Reproductive Medicine
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• 제32권4호
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• pp.331-336
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• 2005

Objective: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. Method: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was $31.8{\pm}3.1years$. Ovulation was triggered by urinary or recombinant hCG. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/mL), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female (< 34 or ${\geq}34years$). Results: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). Conclusion: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes.

• 한국수정란이식학회지
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• 제33권4호
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• pp.281-286
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• 2018

Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes ($79.60{\pm}0.77{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found ($79.51{\pm}2.36{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured ($1.48{\pm}0.42{\mu}m$) than in vitro matured for 72 and immature oocytes ($1.10{\pm}0.16$ and $0.43{\pm}0.12{\mu}m$, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.

• Clinical and Experimental Reproductive Medicine
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• 제20권2호
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• pp.157-160
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• 1993

The human follicular f1uids(F.F.) may be considered to contribute the maturation of the oocytes on the in vitro culture. To investigate the effects of epidermal growth factor (E.G.F.), which is present in mature and immature follicular fluids, we had experiments of mouse oocytes maturation in vitro. The endpoints assayed were rated as percentage of oocytes undergoing germinal vesicle breakdown(G.V.B.D.) and polar body(P.B.) formation at 12 hours after in vitro culture. The rates of G.B.B.D. were 87% in mature F.F. 68% in immature F.F. and 78% in Ham's F-10 medium respectively. And overall the mature F.F. seem to stimulate on in vitro oocyte maturation compared with either immature F.F. or Ham's F-10 medium. As the concentration of addition of E.G.F. in immature F.F., the rates of G.V.B.D. and P.B. formation were 82 %, 23% in addition with 2 ng/ml while 84%, 32% in addition with 4 ng/ml respectivly. And at the concentration of addition of E.G.F. in Ham's F-10 media as well, the rates of G.V.B.D. and P.B. formation were 84%, 40% and 82%, 44% in addition with each 2ng, 4ng. AccordinglY there was no influence on the oocytes maturation at the addition of E.G.F. to each immature F.F. and Ham's F-10 medium. In conclusion, the E.G.F. is not able to induce oocyte maturation independent of it's effects in immature F.F. and Ham's F-10 media.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.234-237
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• 2011

Objective: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. Methods: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. Results: Percentages of immature oocytes were 30.8% and 32.9% ($p$=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% ($p$=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, $p$=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of $in$ $vivo$ matured oocytes (56.4% vs. 72.0%, $p$=0.005) and lower pregnancy rate (19.0% vs. 27.1%, $p$=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. Conclusion: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.

• Clinical and Experimental Reproductive Medicine
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• 제26권3호
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• pp.363-368
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• 1999

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.

• 한국가축번식학회지
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• 제21권3호
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• pp.211-217
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• 1997

This experiment was carried out to determine the possibility of using FDA on the selection of viable bovine immature oocytes. The results obtained in these experiments were summarized as follows; 1. The rates of nuclear maturation (metaphase II) of bovine oocytes according to FDA concentration were 92.5% (37/40) in the control and those rates in the dilution on FDA to 1:400,000, 1:800,000 and 1:1,600,000 were 74.4% (67/90), 80.3% (38/46) and 71.1% (32/45), respectively. 2. The fertilization rate ( 2-cell) in the control was 72.2% (39/54) and those rates in the dilution of FDA to 1:400,000, 1:800,000 and 1:1,600,000 were 28.8% (23/80), 66.7% (32/48) and 62.2% (28/45), respectively. In these results, a, pp.opriate concentration of FDA to bovine immature oocytes was 1:800,000. 3. Effect of FDA treatment to the blastocyst development of bovine oocytes was indicated that control was 22.2% (18/81) and FDA treatment groups which were classified to strong, partial and weak were 21.4% (36/168), 14.5% (9/62) and 0% (0/15), respectively. This result suggested that in vitro development to the blastocyst was severely reduced except strong group according to the FDA fluorescent level (P<0.05) and that using FDA is possible to select of bovine oocytes.