• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.93-100
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• 2001

This study was conducted to evaluate the nuclear development of preantral follicle oocytes in dog following collecting from different estrus ovaries and oocyte diameter. To do this, ovaries were collected from Sunchon livestock station by ovarioectomy and then transported to laboratory in 3$0^{\circ}C$ saline within 2 h. All of the ovaries were washed three times with saline supplemented with 100 IU Penicillin and 100 $\mu\textrm{g}$/$m\ell$ Streptomycin and then sliced with blade in 100 mm dish. All of the oocytes collected were classified the oocyte size such as over 110 ${\mu}{\textrm}{m}$ or under 110 ${\mu}{\textrm}{m}$ and the estrus status. To induce the nuclear development, oocytes were cultured in TCM199 $\alpha$-MEM supplemented with 10% FCS, 0.1 $\mu\textrm{g}$/$m\ell$ sodium pyruvate, 100 ng/$m\ell$ FSH, 100 ng/$m\ell$ EGF, 1% ITS, 100 IU/$m\ell$ penicillin, 100 $\mu\textrm{g}$/$m\ell$ streptomycin at 38.5$^{\circ}C$, 5% $CO_2$ incubator for 72 h. After culture, all of the oocytes were stained in 1% orcein following fixed in 45% Acetic acid for 48 h. The oocyte recovery rates of over 110 ${\mu}{\textrm}{m}$ from estrus ovary (63.6%) were significantly higher rather than that in anestrus status (51.5%). Oocyte recovery rate per ovary of over 110 ${\mu}{\textrm}{m}$ in estrus status ovary (22.6/63.8%) was also significantly higher rather than that in anestrus status ovary (8.2/51.6%). Nuclear development to MI of over 110 ${\mu}{\textrm}{m}$ in estrus ovary (24.3%) was significantly higher rather than those in under 110 ${\mu}{\textrm}{m}$ or over and under 110 ${\mu}{\textrm}{m}$ in anestrus (2.5, 6.8 and 0.0%), respectively (P ＜ 0.05). Nuclear development to AT and MII of over 110 ${\mu}{\textrm}{m}$ in estrus was more developed in other groups. Nuclear development to MI in TCM199 (21.8%) was significantly higher than in $\alpha$-MEM (10.0%). Altho $\mu\textrm{g}$h the development rate to AT was significantly different between TCM199 (7.3%) and $\alpha$-MEM (1.1%), but to MII was not different between TCM199 (0.9%) and $\alpha$-MEM (1.1%). The results indicated that over 110 ${\mu}{\textrm}{m}$ oocytes was could be recovery from estrus status ovary, bigger oocytes were more developed to MI, AT or MII in TCM199.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.71-76
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• 1998

This study was carried out to examine whether the developmental capacity of bovine immature oocytes frozen ultra-rapidly using electron microscope (EM) grids and EFS30 can be obtained. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. As criterior of oocyte viability, the rates of maturation, fertilization and embryonic development were determined. The results obtained in this experiment were summarized as follows: When ultra-rapidly frozen immature oocytes were thawed, 43.2% of them were survived. The rates of maturation (84.1%) and normal 2 pronuclei formation (57.5%) of frozen immature oocytes were not significantly different when compared to those of control (92.5, 65.0%). In addition, the rates of $\geq2$-cell (65.0%) and blastocyst formation (30.8%) of freezing group were not significantly different when compared to those of control (73.7, 35.7%). These results demonstrate that developmental capacity of frozen-thawed bovine immature oocytes can be successfully obtained when survived from the ultra-rapid freezing method using EM grid and EFS30.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.21-26
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• 2007

본 연구는 mSOF(modified synthetic oviduct fluid medium) 배양액을 이용하여 $100{\mu}1$와 $10{\mu}1$ 배양 소적에서 일본 흑우의 수정란 생산 효율을 개선하기 위하여 수행하였다. 난구세포가 부착된 미성숙 난자는 각각 단독 배양조건($S;\;10{\mu}1$ 소적) 및 그룹 배양 조건 ($G;\;100{\mu}1$ 소적)에서 실시하였고 배양액은 TCM-199의 기본 배지에 10% FCS, 0.02IU/ml FSH와 $1{\mu}g/ml$ $estradiol-17{\beta}$를 첨가하여 사용하였다. 배반포 단계로 발육한 수정란은 1.5M ethylene glycol로 직접 이식법에 의한 동결 방법으로 동결을 실시하였고, 세포수는 융해 후 생존 수정란에 대해 조사하였다. 체외 배양 시간이 $16{\sim}17$시간 배양 조건에서 난자의 성숙율은 그룹 배양 조건$(27.1{\pm}16.8%)$보다는 단독 배양조건$(57.1{\pm}15.0%)$에서 성숙율이 높았다(p<0.05). 그러나 체외 배양 시간이 $18{\sim}19$ 시간과 $20{\sim}21$시간 배양시는 유사한 성숙율을 보였다. 난자의 체외 배양율은 체외 배양 시간의 증가에 의해 성숙도가 $86.3{\pm}9.9%$로 증가하였다. 접합체(zygote)의 분할율은 단독이나 그룹 배양 조건에서도 유사한 결과를 얻었다. 배반포 발달율은 배양 $7{\sim}8$일째에 조사한 결과 단독 배양 방법보다는 그룹 배양 방법에서 발달율이 높았으나, 분할된 접합체를 기준으로 한 경우 배반포 발달율$(S;\; 21.4{\pm}10.6%,\;G;\;39.0{\pm}13.1%)$에서는 유의적인 차이가 없었다. 단독 배양과 그룹 배양에서 $6.5{\pm}8$일 사이에 배반포로 발달된 수정란의 세포수 조사에서는 유사한 결과를 얻었다. 동결 융해 후 24시간 배양 후 배반포 생존율(5; 24.2%, G; 30.2%), 부화율(S: 20.9%, G: 12.7%) 및 생존 수정란수(S; 45.2%, G: 42.8%)에서도 배양 조건에 따른 유의적인 차는 없었다. 결론적으로 mSOF배양액을 이용하는 경우 미성숙 난자의 체외 성숙 유도 배양 시 단독이나 그룹 배양 시 배반포 발달율에서 그룹간에 유의적인 차가 인정되었다(p<0.01).

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.269-274
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• 2003

This experiment was carried out to investigate the effects of different IVM-IVC culture media factors, such as development rates according to the maturation media, collecting times from slaughter to initiation of incubation and with cumulus cells, on in vitro maturation of oocytes collected from 3∼5mm diameter follicles of the swine abattoir The development rates significantly(p<0.05) higher when the oocytes were matured TCM-199 media than NCSU-23 media. In comparing with TCM199 medium in presence of Earle's salts and Hank's salt, there were no significantly differences between each salt balance in cleaved rate and in number of morulae plus blastocyst. Among 1,455 immature oocytes, 999(68.6％) of oocytes were cleaved. The number of development to the morulae and blastocysts were 617(61.8％) include 62 balstocysts(6.2％).

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.363-368
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• 1999

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.

• Korean Journal of Animal Reproduction
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• v.19 no.4
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• pp.323-329
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• 1996

In vitro cultrue systems for the growth of sma-II oocytes and for meiotic maturation are expected to provide a new source of a large population of oocytes as well as assistance in basic physiological studies of oogenesis. Mouse oocytes mid-growth phase can complete grovvth and acquire full developmental capacity in vitro. On the other hand, growing pig oocytes need some other factors. FSH at a low concentration maintains the viability of both oocytes and granulosa cells, and hypoxanthine promotes the meiotic competence of the oocytes during culture period. Considerable improvement in the culture systems for growth of pig oocytes, suggested from mouse studies, and for oocyte maturation could help to develop this technology in larger species.

• The Korean Journal of Zoology
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• v.31 no.4
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• pp.265-272
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• 1988

세포융합방법을 사용하여 성장증인 포유동물의 난자에 들어있는 성숙억제요인(maturation inhibiting activity, 1연Al에 대해 조사하였다. 성장중인 생쥐난자와 성장한 미성숙난자를 1:1로 융합하여 배양했을 서 (14-17시간)에는 거의 모두 핵붕괴를 일으키었으나(90oyo), 2:1로 융합했을 때는 대부분(약 64%) 3개의 핵을 모두 간직하고 있었다. 돼지난자의 경우는 성장중인 것깎 성장한 것을 1:1로 융합하여 배양했을 때에도 융합체들은 모두 핵을 간직하고 있었으며 돼지의 성장중인 난자와 생쥐의 성장한 난자를 융합했을 때에도 모두 핵을 보존하고 있었다. 이에 반하여 돼지와 생쥐 모두에서 성장한 난자끼리 융합했을 때에는 예외없이 핵붕괴가 일어났다. 이러한 결과는 성장중인 생쥐나 돼지의 난자에 각IA가 존재한다는 열과 이종간에도 효과가 있다는 것을 보여주고 있다. 또한 이는 MIA와 성숙촉진요인(maturation promoting factor, MPH의 상대적인 양의 변화가 난자의 성숙조절에 증요한 9f할을 한다는 것을 시사해주고 있다.In an attempt to elucidate the nature of maturation inhibiting activity (MIA) in growing mamma-lian oocvtes, growing mouse and pig oocytes incompetent to resume meiosis were fused with fully grown immature oocvtes in various combinations and cultured for 14-17 hours. In slant cells composed of two mouse growing ooh임es and one large immature oocyte (2:기, their GVs remained well conserved (about 64%) after culture, but not in the ceils composed of one by one pairs. In giant cells of pig composed of one growing and onto large immature oocytes, both GVs remained conserved. In the cells composed of one pig growing and one mouse large oocytes, both GVs were also conserved. In contrast to this, pairs of large mouse oocvtes or those of large pig oocvtes had no CVs after culture. Thus, we could acertain the existEnce of MIA and none-pecificty of it in the mouse and pig growing oocvtes. The results also suggest that the relative amount of substances showlns MfA or MPF activity may be important in the regulation of oocyte amount of substances showing MIA or MPF activity may be important in the regulation of oocyte maturation.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.99-106
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• 1999

핵 이식에 공여되는 수핵난자의 효과적인 동결보존을 위하여 한우 성숙난자를 1.0 M dimethylsulfoxide(DMSO) 또는 1.0 M glycerol이 함유된 동결보호제를 이용하여 처리하거나, 동결보호제 처리 후 완만동결법을 이용한 동결융핼르 시행하여 상기 실험처리로 야기되는 투명대 경화현상을 관찰하였다. 도축장 유래의 난소에서 미성숙난자를 채취한 후 10% 소 태아혈청을 함유한 TCM-199을 이용하여 22∼24 시간 동안 체외성숙배양을 이해하였다. 배양후 작출된 성숙난자를 각각의 동결보호제로 처리, 혹은 처리 후 동격융해한 후 protease를 이용하여 투명 대의 경화현상 발생의 빈도를 조사하였다. 또한 동격란을 동결정액을 이용한 체외수정에 공여한 후 정자 침입농도능을 조사하였다. 동결보호제로 처리한 난자에 있어서 보호제의 종류와 관계없이 투명대 경화현상이 유의적 (P<0.05) 으로 증가하였으나 이후의 동결융해 처리에 의한 추가적인 경화현상의 발생은 증가하지는 않았다. 또한 투명대 경화현상의 발생양상을 동결보호제 처리 후 10분 간격으로 측정한 결과 DMSO의 경우 처리후 10분, glycerol의 경우 처리 후 20분 후부터 유의적으로 차를 발견할 수 없었으며, 수정율 및 난자 1개당 침입한 정자의 수는 동결란에서 유의적으로 증가하지 않았다. 본 연구의 결과 동결난자의 투명대 경화현상은 동결보호제 처리과정에서 이미 일어나지만, 이러한 투명대 경화현상이 난자의 동결보존 후 수정능에는 현자한 영향을 미치지 않는다는 사실이 규명되었다.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.10a
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• pp.65-65
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• 2001

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2001.05a
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• pp.38-39
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• 2001