• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.165-171
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• 2002

Follicular fluid (FF) contains an oocyte maturation inhibitor with unknown chemical properties. This study was carried out to chemically define the factor(s) inhibiting cumulus cell denudation (CD) and first polar body formation (PBF). Porcine FF (PFF) was extracted with methanol and the extract was serially separated using gel filtration on Superose 12 and Superdex columns. A Superdex fraction was derived with PITC and analyzed with an amino acid analysis column. The results obtained are as follows; PFF had an activity inhibiting both CD and PBF of porcine primary oocytes. Superdex fractions RV2.11 prepared from PFF exhibited an activity inhibiting CD and PBF. By amino acid analysis, the fraction RV2.11 appeared to be proline having the same activity inhibiting CD and PBF. In conclusion, PFF had oocyte maturation inhibitors, of which proline should inhibit CD and PBF.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.12-12
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• 2002

난포액은 난자의 성숙을 억제하는 성분을 함유하고 있으나, 억제성분의 화학적 성질은 연구자에 따라 다르게 보고되고 있다. 따라서 본 연구는 난자의 성축과 관련하여 제l극체의 형성을 억제하는 단백성 성분을 화학적으로 규명하기 위하여 실시하였다. 난포액 함유 난자성숙 억제 인자를 분리하기 위하여 난포액을 methanol로 추출한 다음 Superose 12 및 Superdex column 을 이 용하여 gel filtration 을 실시하였다. 회수한 Superdex 분절을 PITC로 처리한 다음, Amino Acid Analysis column을 이용, 억제 인자를 동정하였다. (중략)

• Applied Microscopy
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• v.33 no.2
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• pp.105-116
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• 2003

In Vitro fertilization of U. unicinctus eggs observed by immunofluorescence and electron microscopes revealed an overview of the meiotic pattern of the tide animals. The eggs have been fertilized early at germinal vesicle stage, followed by germinal vesicle break down (GVBD), but pre-mitotic aster like structure could not be resolved by the methods employed in this work. The meiotic features, such as rotation-shift movement of spindle fibers, behavior of spermatozoonmonaster in the egg cytoplasm and active spindle fiber of the 1st polar body, have been observed. The antitubulin-FITC fluorescence show the 2nd meiotic apparatus appeared firstly parallel to the tangential line of the oolemma, proceeding the meiosis, its bipolarity is rotated and shifted towards the oolemma. The polar bodysite of the oolemma was not amorphous, but active in a sense of anti-tubulin-FITC reactions during the extrusions of the polar bodies. The immunofluorescence reactions of the spermatozoon centriole appeared at a later stage of the 2nd meiosis. During the time periods, the fertilized spermatozoon resided in the egg cytoplasm. Activating the centrioles, spermatozoon approaches towards the chromosomal materials of the 2nd oocyte. This suggests that spermatozoon centrioles initiate and play a roll to fuse male and female pronuclei.

• Journal of Aquaculture
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• v.10 no.2
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• pp.123-131
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• 1997

Growth of triploid abalone, Haliotis discus hannai induced by cody (3$^{\circ}C$) shock and its feed efficiency were investigated from larva to adult for 51 months. After 51 months from triploidy induction, the triploid abalones have outgrown to diploid abalones in shell length and total weight. Triploid abalones with inhibition of extrusion of first polar body (3n-1pb) were outgrown to diploid abalones, however, triploid abalones with inhibition of extrusion of second polar body (3n-2pb) were not significantly different from diploid controls in shell length and total weight through the whole rearing period (P<0.05), because of their heterozygosity differences. Daily feeding rates and feed conversion rates decreased with the growth of abalones and both rates had no differnce between two experimental groups. After 51 months from inducing triploid, conditin index of triploid abalone (64.1%) was higher than that of diploid control (59.4%) (P<0.05). Survival rate was 63.0% in triploid group (3n-1pb 62.0%, 3n-2pb 64.0%) and 62.0% in diploid group during the experimental period.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.169-175
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• 2006

The objective of this study was carried out to examine the polar body extrusion of in vitro matured porcine follicular oocytes as a non-invasive marker of oocyte quality to know the developmental competence in advance. The porcine oocytes matured for 48 hours were examined the polar body extrusion and some parts were stained. The examined oocytes were matured for additional $16{\sim}18$ hours and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 hours for diploid formation. The treated oocytes were washed and cultured for 7 days. The polar body extrusion and degeneration rates were varied with $9.9{\sim}52.4%$ and $21.4{\sim}61.8%$ by repetition. The polar body extruded oocytes were shown the polar body chromosome and metaphase II plate by staining. However the non-extruded oocytes were shown expanded nucleus with 39.1%, premature chromosome condensation with 19.6%, metaphase I plate with 10.9 %, metaphase II with 13%, condensed chromatin with 6.5%, and absent nuclear material with 8.7%. The oocytes that were not examined for the polar body extrusion were cleaved 45.0%, and developed to blastocyst stage with 11.3%. In examined oocytes for polar body extrusion,. the polar body extruded oocytes were cleaved 94.2% and developed with 42.5%. This result suggests that discarding of the degenerating oocytes and oocytes that not extruded polar body will be effective for experiments of culturing effect in porcine embryos and embryo biotechnology.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.187-190
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• 2004

Protein kinase C exists as a family of serine/threonine kinases which are broadly classified into three groups as cPKC nPKC and aPKC depending on their cofactor requirements. Previous studies have shown that the role of PKC in the process of mouse oocyte maturation. For example, phorbol 12-myristate 13-acetate which is known as an activator of cPKC and nPKC inhibits germinal vesicle break down and 1st polar body extrusion in maturing oocytes. In this study, the effect of thymeleatoxin, a specific activator of cPKC not nPKC, was tested comparing with PMA to address the roles of cPKC and nPKC during mouse oocyte maturation. Cumulus-oocyte complex were cultured in M16 medium for 6 or 12 hr with each of these PKC activators to investigate the effect of germinal vesicle breakdown (GVBD) or the extrusion of 1st polar body. IC/sup 50/ of GVBD were at concentrations of 50nM in PMA and 400nM in thymeleatoxin and of 1st polar body extrusion were 20nM in PMA and 200nM in thy- meleatoxin. The results suggest that activation of nPKC is more closely related to the inhibition of GVBD and 1st polar body extrusion than activation of cPKC. Additionally, we found that the oocytes inhibited 1st polar body extrusion with PMA or thymeleatoxin were arrested in metaphase I of first meiosis.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.229-237
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• 2004

The main objective of this study was to examine the effects of serum and gonadotropins supplement during in vitro maturation(IVM) of bovine oocytes on nuclear maturation and embryo development, and we also examine the cell number. 1 . The first polar body(PB) extrusion rates of Korean native cow(KNC) oocytes matured in medium with FBS or gonadotropins were similar among treatment groups. The development rate to the blastocyst stage was significantly higher in the group of both supplement FBS and gonadotropins(26.0％) than in the group of non-supplement(9.9％) and gonadotropins (12.0％). The numbers of inner cell mass (ICM) and trophectoderm (TE) cells and total cell numbers of blastocysts were highest in the group of both supplement FBS and gonadotropins, and the number of ICM cells was increased by FBS supplementation (p<0.05). 2. The PB extrusion rates of KNC oocytes matured in medium with FBS in the different duration of IVM was significantly higher in the 0-18hr(63.1％) and in the 9-18hr(63.4％) group than in the 0-9hr.(37.4％) group (p<0.05). The embryo development rates did not differ among treatment groups. The numbers of TE cells and total cell numbers of blastocysts were similar among treatment groups, but the number of ICM cells of the 0-18h. group were significantly higher than the other treatment groups (p<0.05). The results indicate that although TCM199 alone can support bovine oocyte maturation and development to the blastocyst stage, a high quality of blastocysts can be produced from oocytes matured in medium containing serum and gonadotropins.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.219-226
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• 1998

Enucleation of oocytes is an important limiting step for embryo cloning. We propose an enucleation technique based on the removal of chromatin after oocyte activation by aspirating the second polar body containing complemented chromatin. In a preliminary experiment to determine an optimal age of oocytes enucleation in rabbits, oocytes were enucleated at 15~20 hours post hCG. Recently ovulated oocytes were enucleated at a higher rate than aged oocytes. Microsurgical removal of the complemented chromatin in the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes(96.8% versus 70.4%; P〈0.05). Moreover, compared with a nuclear transplantation protocol based on enucleation of metaphase-arrested oocytes and preactivated oocytes followed by treatment with 5 $\mu$M ionomycin for 5 min and 2 mM DMAP for 1 hr, there was no significant difference in the rate of blastocyst development. The ease with which modified technique can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

• The Korean Journal of Zoology
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• v.30 no.1
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• pp.89-98
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• 1987

The research of fused oocytes was conducted to investigate the in vitro mejotic maturation of immature oocytes (GV oocytes) fused with oocytes in germinal vesicle breakdown (GVBD oocytes) in the presence of dbcAMP which is known as one of the strong inhibitors to GVBD. The immature oocytes fused together as well as those fused with GVBD oocytes proceeded to GVBD in 3 hr culture in plain medium. But in the medium containing dbcAMP (100$\mu$g/ml), the immature oocytes fused together did not show any GVBD and thus the fusion itself could not affect the inhibitory activity of dbcAMP. However, all of the immature oocytes fused with GVBD oocytes underwent GVBD in 3 hr culture despite of the presence of dbcAMP. When the culture was extended to 20 hr, nearly all of the immature oocytes fused together were still arrested at the GV stage in the presence of dbcAMP. But most of the fused oocytes which had shown GVBD during 3 hr culture developed to metaphase II stage extruding one or two polar bodies regardless of the presence of dbcAMP. In this experiment, it was found that two sets of the metaphase chromosomes were somewhat concomitant with a pair of the polar bodies in the fused egg. Upon the results of the present studies, it is assumed that there may be a maturation promoting factor(s) in the cytoplasm of the GVBD occytes, and this factor(s) possibly nullifies the function of dbcAMP.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.175-180
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• 2005

This study was conducted to examine the effects of demecolcine-assisted enucleation and recipient cell cycle stage on the development of bovine somatic cell nuclear transfer (NT) embryos. In vitro cultured oocytes for $16\~20$ h were classified by first polar body (1st PB) extrusion and cell cycle stage (MI and MII) and treated $0.4\;{\mu}L/mL$ demecolcine for 40 min before enucleation. Enucleated oocytes were fused electrically with bovine ear skin cells, activated by Ca-ionophore+DMAP, and cultured in vitro. Most of eggs ($86.2\%$) treated with demecolcine protruded a chromosome mass and enucleated efficiently ($98.8\%$, (P<0.05). Demecolcine did not have a deteriorative effect on the development of NT embryos. Developmental rate of NT embryos reconstituted with oocytes extruded 1st PB significantly higher than that of NT embryos produced by oocytes without 1st PB ($18.2\%\;vs.\;4.6\%\cdot$, P<0.05). Cleavage and blastocyst formation rate of embryos reconstituted with MI oocytes ($69.4\%\;and\;5.9\%$, respectively) were significantly lower than those of embryos reconstituted with MII oocytes ($96.7\%\;and\;23.9\%$, respectively, P<0.05). From the present result, it is suggested that domecolcine is useful for the enucleation of recipient oocytes in bovine NT procedures, and MII oocytes rather than MI oocytes are more appropriate for recipient cytoplasm. Although, the potential to develop into blastocysts of NT embryos produced by 1st PB-nonextruded and MI oocytes was very low, these oorytes could be used for NT.