• 한국수정란이식학회지
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• 제32권3호
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• pp.65-71
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• 2017

혹서기에 있어 고온 다습한 환경은 동물의 생산성과 생리적 반응에 영향을 주어 HS를 유도하는것으로 알려져 있다. 본 연구에서 HS처리 효과가 도축장 유래 난소에서 채취한 난자의 체외 성숙율, 난할율 및 수정란의 발생능력에 미치는 영향을 비교 검토하였다. 대조군으로서 COCs를 $38.5^{\circ}C$에서 22시간 배양하였으며 실험군은 전배양을 동일하게 21, 18 및 12시간 배양 후 $40.5^{\circ}C$에서 각각 1, 4 및 12시간 동안 후배양하여 HS를 유도하였다. 22 시간 숙성시킨 후, COCs를 체외수정하여 mSOF 배지에서 8 일 동안 배양하였을 때 난자의 성숙율과 수정란의 발생 능력을 조사 하였다. 대조군과 1 및 4 시간동안 HS처리된 난자에서 성숙율과 난할율에는 차이가 없었으나(p > 0.05), 4 시간 HS처리군에서 배반포 형성율이 유의적으로 감소하였다(p < 0.05). 또한, 4시간 이상의 지속적인 HS에 대한 노출은 배반포 형성율과 세포사멸도에 영향을 주는 것으로 관찰되었다(p < 0.05). 이러한 결과는 HS가 난자의 성숙 과정에서 유도되면 수정란의 발생 능력에 부정적인 영향을 줄 수 있음을 시사하며 HS에 의한 소 배반포에서 세포사멸현상이 나타나고 있음을 보여주고 있다.

• 한국가축번식학회지
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• 제26권1호
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• pp.33-39
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• 2002

소 체세포 핵이식 시 융합액 내 $Ca^{2+}$ 농도에 따른 융합율, 핵형의 변화, 배 발달율을 검토한 결과는 다음과 같다. 1. 융합액 내 $Ca^{2+}$ 농도를 0.05, 0.1, 0.5 및 1.0 mM로 각기 다르게 처리한 결과, 0.5와 1.0 mM의 CaC$_2$에서 융합율이 80.5 와 84.3%로 나타나 0.05 mM CaCL$_2$에서의 융합율 56.6%에 비하여 유의적(P＜0.01)으로 높았다. 2. 융합액 내 $Ca^{2+}$ 농도에 따른 재구축배의 핵형을 검토한 결과, CaCL$_2$농도 0.05와 0.1 mM 에서는 88.4와 84.0%의 난자가 PCC이후 염색질괴를 형성한 반면, 0.5와 1.0 mM 에서는 54.5와 59.3%가 PCC 형태를 거치지 않고 직접 전핵을 형성하여, $Ca^{2+}$ 농도가 증가함에 따라 PCC 형태를 거치지 않고 직접 전핵을 형성하는 난자의 비율이 증가하였다. 3. 핵이식란의 체외 발육율을 검토한 결과, 1.0 mM CaCl$_2$에서는 배반포 발육율이 30.6%로 나타난 반면, 0.1 mM CaCl$_2$에서 는 20.0%로 나타나 유의적(P＜0.05)인 차이를 나타냈다. 본 연구의 결과는 융합액 내 $Ca^{2+}$농도의 증가가 소 체세포 핵이식란의 융합율 및 배반포 발육율을 향상시킬 수 있음을 시사한다.

• 한국수정란이식학회지
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• 제32권3호
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• pp.131-138
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• 2017

본 연구에서는 체외성숙 배양액 내 caffeine 첨가가 돼지 난자의 성숙과 단위발생 및 체세포 핵이식 후 배 발육에 미치는 영향을 조사하였다. 난세포질 및 난구세포 부착정도의 형태학적 특징에 따라 MGCOCs와 MPCOCs로 구분된 미성숙난자를 각각 무처리군(대조군)과 2.5 mM caffeine이 첨가된 배양액에서 체외성숙 22-42(20시간), 34-42(8시간), 38-42(4시간)동안 처리하는 군으로 나누어 체외성숙을 유도하였다. 또한 체외성숙 난자를 단위발생 및 체세포 핵이식에 공여하여 배아를 생산한 후 7일 동안 체외배양하여 체외성숙 동안 caffeine 처리가 분할률, 배반포 형성률 및 배반포의 세포수에 미치는 영향을 조사하였다. 연구 결과, 분할률 및 배반포 세포 수는 caffeine의 처리 시간에 따라 유의적인 영향을 받지 않았다. 그러나 MPCOCs 유래 난자에서 체외성숙 후기 4시간 동안 caffeine 처리는 체세포 핵이식 배아의 배반포 형성률을 유의적으로 증가시켰다. 이 결과는 caffeine 처리가 체외성숙 동안 난자의 MPF 수준의 감소를 억제시킴으로써 체세포 핵의 리모델링이나 리프로그래밍에 영향을 미쳐 핵이식 배아의 발육능에 영향을 미친 것으로 사료된다.

• 한국수정란이식학회지
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• 제15권3호
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• pp.247-253
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• 2000

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 $\mu\textrm{g}$/mL), and higher rate of encleation was obtained at 7.5 and 15 $\mu\textrm{g}$/mL than at $\mu\textrm{g}$/mL. In Experiment 2, oocytes enucleated in 7.5 $\mu\textrm{g}$/mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 $mutextrm{s}$) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 $mutextrm{s}$ or 90 $mutextrm{s}$, but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

• 한국가축번식학회지
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• 제24권4호
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• pp.429-437
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• 2000

본 연구는 retrovirus vector system 에 의해서 EPO와 EGFP 유전자가 전이된 소 태아세포를 이용하여 핵치환된 소 난자에서의 이들 유전자의 성공적인 도입을 조사하였다. Non-starved 소 태아세포는 탈핵된 소 난자의 위란강내로 주입되었다. 소 태아 세포와 난자는 세포간 전기자극에 의해 융합시켰으며, 이후 calcium ionophore와 6-dimethylaminopurine를 이용하여 난활성을 유도하였다. 핵치환에 의해 재구성된 난자는 8일 동안 CRlaa 배양액에서 소 난관상피세포와 함께 공배양하였다. 핵치환에 의해 재구성된 187개와 210(EPO, EGFP)개의 소 난자 중에서, 149개와 158(EPO : 80.0%, EGFP : 75.2%)개의 난자가 분할되었고, 이들 분할된 난자 중 36개와 35(EPO : 24.2%, EGFP : 22.2%)개의 난자가 배반포까지 발달하였다. 이들 배반포에서, EPO 유전자는 PCR에 의해 36개의 모든 난자에서 삽입을 확인하였고, EGFP 유전자의 발현은 형광현미경 하에서 35개의 모든 난자에서 확인하였다. 이 결과는 외래유전자가 삽입된 소 태아 세포를 이용하여 핵치환된 난자는 배반포까지 성공적으로 발달할 수 있다는 것을 나타낸다. 더우기, 이러한 방법은 효율적인 형칠전환 소를 생산하는데 이용될 수 있으리라 사료된다.

• 한국수정란이식학회지
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• 제23권3호
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• pp.183-187
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• 2008

This study was conducted to analyze the expression pattern of inhibitor of DNA binding proteins (Id)1 and Id2 mRNA on folliculogenesis in rat ovary. The ovaries were obtained from 27 days old Sprague-Dawley rat, fixed, dehydrated, and paraffin embedded. For in situ hybridization, anti-sense and sense Idl and Id2 cRNA probes were prepared and applied to the ovarian section. The ovarian sections were coated with NTB-2 emulsion. After that, the slides were developed and counterstained with hematoxylin and eosin staining. In oocytes, the hybridizational signals of Id1 mRNA were strong in primordial and primary follicles, however, there were no signals in that of atretic or preovulatory follicles. The Id2 mRNA signals were also strong in the oocytes of primordial, primary and secondary follicles. Interestingly, the Id2 mRNA was expressed specifically granulosa cells, but nor in oocyte or theca cells in dominant and preovulatory follicles. Based on these results, Id1 and Id2 mRNA was expressed specifically at follicle stages and follicular tissue and might be closely related with follicle development.

• 한국수정란이식학회지
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• 제27권2호
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• pp.121-126
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• 2012

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and $10{\mu}M$ ${\beta}$-mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

• Asian-Australasian Journal of Animal Sciences
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• 제13권6호
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• pp.856-860
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• 2000

Cloning technology continues to capture widespread attention by the international news media and biomedical and agricultural industries. The future uses of this technology could potentially contribute to major advances in biomedical and agricultural sciences. Cloned transgenic dairy cattle possessing milk promoters directing transgenes will produce pharmaceutical proteins in their milk faster, more efficiently and less expensively than transgenic cattle created using microinjection techniques. Additionally, cloned transgenic fetuses and animals may become a source of cells, tissue and organs for xenotransplantation. Lastly, but maybe most importantly, enhanced production traits and disease resistance may be realized in animal agriculture by utilizing these new technologies. The recent advances in the cattle cloning technology are important but there are still major obstacles preventing widespread commercial use of this technology. The type of donor nucleus, recipient cytoplasm, and cloning procedures used will impact the potential number of clones produced and the uses of the technology. In addition, the new advances in cloning methodology have not improved the relatively low pregnancy rates or reduced the incidence of health problems observed in cloned offspring. These problems may require novel techniques to decipher their cause and new methods of preventing and/or diagnosing them in the preimplantation embryo. The commercial potential is enormous for cloning technology; however, little has been done to improve the efficiencies of the procedure. Improving procedural efficiencies is a critical developmental milestone especially for potential uses of cloning technology in animal agriculture.

• 한국수정란이식학회지
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• 제19권2호
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• pp.147-153
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• 2004

본 실험에서는 수술적 방법을 이용하여 난관 절제술을 실시한 성숙 난자의 회수율과 카데터를 이용한 난자 회수율 그리고 새롭게 고안된 개 난자 회수용 니들을 이용한 회수율을 비교하였으며, 각각의 회수 방법이 성숙난자의 외형에 미치는 영향을 조사한 결과는 12두에서 난관절제술로 채취한 성숙난자의 회수율은 89.7% 이었다. 수술적 회수방법에서는 본 연구실에서 개발한 난자 회수용 니들을 난관내에 삽입-결찰한 후 난관-자궁 접합부에서 난자 회수용 배지를 관류하는 방법으로 평균 83.0%의 회수율을 얻었다. 이 같은 결과는 TomCat 카테터를 이용한 회수율 (68.9%)과 난자 회수용 니들을 결찰하지 않고 관류한 방법 (73.5%) 보다 유의적으로 높은 회수율을 나타내었다 (p<0.05). 또한 난관 절제술과 각각의 수술적 방법으로 회수한 난자의 형태학적 차이는 관찰할 수 없었으나 난관 절제술과 난자회수용 니들을 결찰하여 회수한 난자의 형태와 난질이 Tom Cat 카테터나 결찰하지 않은 니들을 이용하였을 때보다 영향을 덜 받는 경향을 나타내었다 (각각 72.0%, 73.8%와 62.8%, 69.6%).

• 한국수정란이식학회지
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• 제5권1호
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• pp.1-20
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• 1990

Follicular atresia is a universal and characteristic phenomenon of both non-mammalian and mammalian vertebrates. Generally it is estimated that greater than 99% of follicles become atretic in higher domestic animals and human. The number of selected follicles developing to the preovulatory stage are thus fewer. Follicles can become atretic at any stage of development. The previous studies emphasized on descriptive and retrospect aspects of a limited population of the fully grown preovulatory follicle. The main efforts in ovarian physilogical researches are focused on follicular development culminating in ovulation but recent advances have resulted in a better understanding of atresia. Nowadays, recent studies are concentrated on the induction of atresia in a selected population of follicles and of the associated cellular, endocrine, biochemical and molecular changes. The factors initiating atresia and follicle selections are worthy of investigations. Another intriguing question is whether one can predict when a follicle will become atretic, i.e., what biochemical markers indicate that a follicle is destined for atresia. It is generally agreed that atretic process may vary even in antral follicles at different stages of their differentiations and among species. The dicisive factors are follicular responsiveness and the hormonal milieu. Some generalizations can be made on the basis of experimental induction of atresia. Alteration of the pattern of follicular steroid production is associated with the initiation stage of atretic process. Atresia appears to be a process unfolding gradually and affecting progressively in increasing number of functions and components of the follicle. The oocyte may be the latest to be afflicted in the atretic process. The high steroidogenic activity of atretic follicles lends support to the notion that atresia is not necessarily a degenerative process and that atretic follicles may play an essential role in ovarian physiology. The simultaneous occurence of growth and atretic processes may render the search for regulatory mechanisms involved in atresia difficult extremely. The questions such as how follicles are selected to undergo ovulation rather than atresia or what the mechanism of atresia is remain unanswered. However, the factors regulating or modifying ovarian hormonal milieu for the initiation of follicular growth and maturation or of atresia are being elucidated.