• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.9-9
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• 2000

The mammalian ovary has a large number of primordial and preantral follicles, which are a potential source of oocytes for the in vitro mass production of embryos. Several in vitro culture systems have been developed to support the growth and development of oocytes from mouse preantral follicles. Under the appropriate condition, meiotically incompetent oocytes from preantral follicles can grow to final size and complete nuclear maturation in vitro. Furthermore, the successful production of live young from in vitro grown and matured oocytes demonstrates that oocytes from preantral follicles are able to acquire full developmental capacity in vitro. However, the efficiency of in vitro production of embryos from mouse preantral follicles is still low. In farm animals as well as human, the growth of oocyte from preantral follicle to the meiotic competence stage has yet to be demonstrate. Therefore, further studies to improve the culture condition or to develope new culture system should be needed in the future. In addition, the visible progress in the establishment of the in vitro culture system for preantral follicles of farm animals and human could help to enlarge the populations of valuable agricultural, phamaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that jeopardize oocytes.

• 대한의생명과학회지
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• 제13권1호
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• pp.61-69
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• 2007

The cycle of the seminiferous epithelium and the development of spermatids of Apodemus agrarius coreae were observed using a light microscope. The cycle of the seminiferous epithelium was divided into 10 stages, and developing spermatids were subdivided into 10 steps. The Golgi phases occurs the first two steps ($St_1,\;St_2$), and the cap phases had the next two consecutive steps ($St_3$ and $St_4$). The acrosomal phases consisted of steps $5{\sim}8$ ($St_5-St_8$), and the remaining two steps consisted the maturation ($St_9$) and spermiation ($St_{10}$) phases, respectively. Type Ad spetmatogonia are appeared in all stages (I-X). Type Ap spermatogonia appeared from stage I and II, In spermatogonia from stage III, IV and V, and B spermatogonia from stages VI. The leptotene spermatocytes appeared from stage VII, zygotene from stages I, II, VIII, IX and X, pachytene from stage III to VIII, diplotene in stage IX, and meiotic figures and secondary spermatocytes in stage X. These data are considered in relation to interspecific differences in sperm morphology.

• Clinical and Experimental Reproductive Medicine
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• 제1권1호
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• pp.49-54
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• 1974

In the present studies, effect of progesterone on the germinal vesicle break-down of the mouse oocytes cultured in the micro tube was investigated. The results obtained are as follows: As dose of progesterone in the medium rose, accordingly the break-down of the germinal vesicle was suppressed. It was found that $ED_{50}$ was 15.7 ${\mu}g$/ml, and $ED_{90}$ 60.7 ${\mu}g$/ml of progesterone. The dose suppressing the oocyte maturation was apparently higher than that on the rabbit or on the mouse embryonal development. The inhibiting effect of progesterone on the GVBD was reversible. The germinal vesicle of the oocytes were broken down immediately in the medium upon removal of the hormone. Progesterone stops meiosis at any stage upon administration, while dbe AMP or theophylline supresses only the break-down of the nuclear membrane. Recovering of the meiotic division of the oocytes once exposed to progesterone was delayed a little. The inhibiting action of progesterone was not altered by adding more pyruvate or in the presence of higher concentration of the mineral ions in the culture medium.

• Asian-Australasian Journal of Animal Sciences
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• 제27권5호
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• pp.635-647
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• 2014

Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; $44h+10{\mu}M$ rapamycin/24 h, $47.52{\pm}5.68$) or control oocytes (44 h IVM; $42.14{\pm}4.40$) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, $22.04{\pm}5.68$) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

• 한국수정란이식학회지
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• 제19권2호
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• pp.185-189
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• 2004

본 연구는 호랑이 미성숙 난자의 체외 성숙의 가능성을 검토하였다. 1. 호랑이 난자의 직경 (176.5${\pm}6.1{\mu}m$)은 소 난자의 직경 (150.7${\pm}4.9{\mu}m$)보다 유의적으로 컷으며, 세포질 직경 (122.1${\pm}9.7{\mu}m$) 은 소 난자의 직경 (118.7${\pm}7.5{\mu}m$) 과 유의차가 없었다. 또한, 호랑이 난자의 투명대 두께 (20.4${\pm}2.9{\mu}m$) 는 소 난자의 두께 (12${\pm}2.6{\mu}m$) 보다 유의적으로 두꺼웠다(p<0.05). 2. 체외성숙 48시간째 제1극체의 출현율은 62.5%이며, 핵성숙은 GV 단계(12.5%)와 MII 단계(50.0%)의 핵 성숙율을 보였다. 이상의 연구결과로 보아 호랑이 난자도 체외성숙이 가능하리라 판단되며 보다 더 적합한 체외성숙조건의 검토가 요구된다.

• 한국가축번식학회지
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• 제17권2호
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• pp.85-92
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• 1993

In the previous study, we observed that Purine has a time dependent effect in maintaining the oocytes in meiotic arrest, and human fetal cord serum(HFCS) and human mature follicular fluid(HMFF) reverse the GVBD suppressed by purines. And it was reported that purine has a harmful effect on the development of oocytes or embryos, when they were cultured for a long time, in vitro. Therefore this study was performed to investigate the effects of purine on extrusion rates of 1st pb and viability of oocytes cultured for a long time, in vitro. Immature oocytes(GV stage) were collected from ovaries of 25~28 day old ICR mice at 48 hrs after PMSG injection. Cumulus-enclosed and denuded oocytes collected were assigned randomly to one of several culture conditions. Some of the oocytes were cultured in 4mM hypoxanthine for 24hr, and the extrusion rates of 1st pb and viability of the oocytes were assessed at every 12 hrs. In the viability, the oocytes showed granulation, pigmentation of cytoplasm or lysis of 1st pb extruded were regarded as degenerating oocytes. Also some of the oocytes were cultured in hypoxanthine for 12 hrs then the resulting oocytes were transferred to hypoxanthine-free medium and cultured for 12 hrs to determine whether the inhibitory effect of hypoxanthine on the 1st pb extrusion was reversible. The rest of the oocytes were cultured in medium containing hypoxanthine and adenosine for 18 hrs to compare the 1st pb extrusion be attendant upon hte concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominently. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine from the cultrue medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominetly. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine fromthe culture medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented.

• Reproductive and Developmental Biology
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• 제36권4호
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• pp.261-267
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• 2012

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII) oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• 한국발생생물학회지:발생과생식
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• 제16권1호
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• pp.59-67
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• 2012

Previously, we found that $Zap70$ (Zeta-chain-associated protein kinase) expressed in the mouse oocytes and played significant role in completion of meiosis specifically at MI-MII (metaphase I-II) transition. Microinjection of $Zap70$ dsRNA into the cytoplasm of germinal vesicle oocyte resulted in MI arrest, and exhibited abnormalities in their spindles and chromosome configurations. The purpose of this study was to determine the mechanisms of action of $Zap70$ in oocyte maturation by evaluating downstream signal networking after $Zap70$ RNAi (RNA interference). The probe hybridization and data analysis were used by Affymetrix Gene Chip Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Total 1,152 genes were up (n=366) and down (n=786) regulated after $Zap70$ RNAi. Among those genes changed, we confirmed the expressional changes of the genes involved in the regulation of actin cytoskeleton and MAPK (mitogen-activated protein kinase) signaling pathway, since the phenotypes of $Zap70$ RNAi in oocytes were found in the changes in the chromosome separation and spindle structures. We confirmed the changes in gene expression in the actin skeletal system as well as in the MAPK signaling pathway, and concluded that these changes are main cause of the aberrant chromosome arrangement and abnormal spindles after $Zap70$ RNAi.