• Title/Summary/Keyword: developmental rate

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Neural and Cholinergic Differentiation of Mesenchymal Stem Cells Derived from the Human Umbilical Cord Blood (인간 제대혈액에서 유래된 중간엽 줄기세포의 신경 및 콜린성 분화)

  • Kam, Kyung-Yoon;Kang, Ji-Hye;Do, Byung-Rok;Kim, Hea-Kwon;Kang, Sung-Goo
    • Development and Reproduction
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    • v.11 no.3
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    • pp.235-243
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    • 2007
  • Human umbilical cord blood(HUCB) contains a rich source of hematopoietic stem cells, mesenchymal stem cells and endothelial cell precursors. Mesenchymal stem cells(MSCs) in HUCB are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. We studied on transdifferentiation-promoting conditions in neural cells and cholinergic neuron induction of HUCB-derived MSCs. Neural differentiation was induced by addingdimethyl sulphoxide(DMSO) and butylated hydroxyanisole(BHA) in Dulbeco's Modified Essential Medium(DMEM) and fetal bovine serum(FBS). Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor(bFGF), retinoic acid(RA) and sonic hedgehog(Shh). MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including $\beta$-tubulin III, GFAP and MBP, was markedly elevated during this acute differentiation. The differentiation rate was about $32.3{\pm}2.9%$ for $\beta$-tubulin III-positive cells, $11.0{\pm}0.9%$ for GFAP, and $9.4{\pm}1.0%$ for Gal-C. HUCB-MSCs treated combinatorially with bFGF, RA and Shh were differentiated into cholinergic neurons. After cholinergic neuronal differentiation, the $\beta$-tubulin III-positive cell population of total cells was $31.3{\pm}3.2%$ and of differentiated neuronal population, $70.0{\pm}7.8%$ was ChAT-positive showing 3 folds higher in cholinergic population than neural induction. Conclusively, HUCB-derived MSCs can be differentiated into neural and cholinergic neurons and these findings suggest that HUCB are alternative cell source of treatment for neurodegenerative diseases such as Alzheimer's disease.

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Retinol Supplemented to a Maturation Medium Stimulated In Vitro Development of Porcine Oocytes after In Vitro Fertilization (돼지 체외 수정란 생산 효율 제고를 위한 레티놀 첨가 효과)

  • Kim Y.S.;Song S.H.;Cho S.K.;Kwack D.O.;Kim C.W.;Park C.S.;Chung K.H.
    • Journal of Embryo Transfer
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    • v.21 no.2
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    • pp.101-107
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    • 2006
  • The objective of this study was to investigative the effects of retinol supplement to IVM and/or IVC medium on maturation, fertilization and development of pig oocytes. North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF) was used as base medium. Each 1 uM, 5 uM and 10 uM concentration of retinol was supplemented to IVM and /or IVC medium. When the retinol was supplemented to maturation medium, the maturation rates were not different (p>0.05) among treatment groups ($66.7{\pm}6.0{\sim}69.2{\pm}5.3%$), but the developmental rate to blastocyst stage was higher (p<0.05) in $5{\mu}M$ group ($20.4{\pm}2.6%$) than in 0 uM ($13.6{\pm}2.1%$) and 10 uM groups ($9.7{\pm}1.7%$). Moreover, total cell number was significantly greater (p<0.05) in the 5 uM group ($37.0{\pm}1.6$) than in the other groups ($29.8{\pm}1.0{\sim}33.2{\pm}1.0$). Retinol supplement to maturation medium did not significantly affect the rates of fertilization and polyspermy (p>0.05). When the retinol was supplemented to culture medium or both maturation and culture medium, the rates of cleavage, and develop to morula and blastocyst stage were not affected, while those of 10 uM group were significantly decreased (p<0.05). These results indicate that 5 uM retinol supplement in maturation medium significantly stimulates embryo development, also improves the total cell number of blastocyst stage in pig.

The Development and Life Table Parameters of Tetranychus kanzawai (Acarina: Tetranychidae) on Leaves of 'shiranuhi' and Japanese Violet in the Laboratory (한라봉과 왜제비꽃 잎에서 차응애의 발육과 생명표 통계량)

  • Hyun, Heejeong;Kim, Subin;Kim, Dong-Soon
    • Korean journal of applied entomology
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    • v.59 no.2
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    • pp.153-163
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    • 2020
  • This study was conducted to evaluate the fitness of Tetranychus kanzawai Kishida on different host plants: young and old leaves of the mandarin orange 'shiranuhi' ((Citrus unshiu × C. sinensis) × C. reticulata), Japanese violet (Viola japonica Langsd.) and kidney bean (Phaseolus vulgaris L.). The development and oviposition experiments were conducted at constant temperatures (20, 25 and 30℃) and a life table parameters were estimated. T. kanzawai could complete it's development on 'shiranuhi' young leaves, japanese violet and kidney bean, while all died during the immature period on 'shiranuhi' old leaves. The total developmental period of T. kanzawai feeding on 'shiranuhi' young leaves was 17.4, 13.4 and 10.2 days at each temperature, respectively, which was longer than 16.1, 9.5 and 7.0 days of kidney bean. The female longevity of T. kanzawai on young leaves of 'shiranuhi' were 19.1, 15.0 and 12.3 days at each temperature, respectively, and there was no significant difference from 22.1, 14.1 and 10.9 days investigated from kidney bean. The fecundity was 18.1, 23.9 and 17.8 eggs per female, which was less than them of japanese violet and kidney bean at each temperature, respectively. As a result of estimating the life table parameters based on the experimental data, intrinsic rate of increase (rm) were significantly different from each other, and appeared in the following order: kidney (0.1542, 0.2563 and 0.3251), japanese violet (0.1087, 0.2007 and 0.2673) and 'shiranuhi' young leaves (0.0868, 0.1002 and 0.1217) at each temperature, respectively. Finally, the management strategy against T. kanzawai in citrus orchards was discussed based on the results.

Toxicological Effects of Some Insecticides against Welsh Onion Beet Armyworm (Spodoptera exigua) (Metaflumizone을 포함한 몇 가지 약제의 파밤나방(Spodoptera exigua)에 대한 독성검정)

  • Kang, E.J.;Kang, M.G.;Seo, M.J.;Park, S.N.;Kim, C.U.;Yu, Y.M.;Youn, Y.N.
    • Korean journal of applied entomology
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    • v.47 no.2
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    • pp.155-162
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    • 2008
  • The Welsh onion beet armyworm, Spodoptera exigua (Hubner), has attacked Welsh onion and is now the most important pest of Welsh onion in southwestern Korea. The beet armyworm has a wide host range, occurring as a serious pest of vegetable and fields. The relatively high abundance of beet armyworm has stimulated frequent application of insecticides to foliage. Insecticide resistance is a major problem in management of this insect. Accordingly, pesticide application for the control of beet armyworm was tried in both the open field and in laboratory, using 4 synthetic compounds such as metaflumizone and chlorfenapyr, indoxacarb, flufenoxuron, emamectin benzoate. In the laboratory, each developmental stages from eggs, larva to pupa was tested against 4 insecticides. Against the eggs of welsh onion beet armyworm, there was no significantly different with each other. These tested chemicals no killing effect to eggs. However, the population of 1st larva hatched from eggs were reduced because they eaten the egg shell with residual insecticides. The tested insecticides were taken very high mortalities to 1st to 3rd larva of Welsh onion beet armyworm. Otherwise, there were decreased the death rate from 4th to 6th larva. On the other hand, their value of control effects were relatively good against Welsh onion beet armyworms in the field between 87.2 and 90.5% on 10 days after insecticide application.

Industrial restructuring and uneven regional development in the 1980s (산업구조조정과 지역불균등발전 : 1980년대)

  • ;Choi, Byung-Doo
    • Journal of the Korean Geographical Society
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    • v.29 no.2
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    • pp.137-165
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    • 1994
  • Structural adjustment of industry (or industrial restructuring) seems to be inherent in the process of capitalist economic development, which tends to be proceeded with shifts from one stage to another in order to overcome structural crises generated in each stage. The structural adjustment of industry is necessarily accompanied with regional restructuring, since it is not only projected on spece, but also mediated by space. Such a restructuring necessitates industrial and uneven regional devlopment through which capital can seek excessive profits over the rate of socio-spatial average. The industrial restructuring and uneven regional development in the 1980s in Korea can be seen as a process in which capital attempted with a strong support of the govenment to overcome the crises in the end of 1970s and hence to go on rapid economic growth. In this process, capital, especially monopoly capital concentrated into few conglomerates, pursued both extensive expansion and intensive development of industry simultaneously. In results, the Korean economy could eliminate some of peripheral characters and maturate the Fordist accumulation system. The extensive expansion of the Korean industry in the 1980s was stimulated mainly through the enlargement and adjustment of investment for equipment facilities which was planned to exclude or rationalize traditional light industries on some places, and to continue rapid growth of key heavy-chemical industries, especially of fabricated metal industry, on other places. In this process, keeping mainly the existing developmental axis which polarized the Seoul Metroplitan region and the Southeast region in Korea, the enhancing spatial mobiiity of capital and the further differentiating division of labour enforced a tendency of concentration of all types of industry in the Seoul Metropolitan region, and at the same time provoked the diffusion of some industries over Jeolla and Chungchong regions in a considerable extent. The intensive development of industriai structure in the 1980s was pursued through the strategic encouragement of subcontracting small firms mainly which produced assembling components, the technical enhancement and factory (semi-) automation, and the enrichment of service industries for estate management, finance, distribution and retailing which supported and complemented the production of goods. In this process, enabling capital to extend and elaborate its domination over space through the reorganization of regulating systems, the Fordist division of labour generated a socio-spatial hierarchy in the nation-wide scale that characterized: the Seoul Metropolitan region as an overmaturated (or overarching) Fordist region performing the conceptive functions of management, research and development, in which all types of industry (including service industries) tended to be reconcentrated; Kyungsang region as a maturated Fordist region with excutive branches of large conglomerates and with subcontracting firms around them which produced standardized products through the automized production processes in secialized Fordist industries or rationalized traditional industries; and Jeolla and Chungchong regions as newly devloping Fordist regions with newly migrated branches and some subcontracting small firms-in relatively older Fordist industries or partly rationalized traditional industries. From these analyses, it can be argued that the structural adjustment of the Korean industry in the 1980s, which had carried out both through the extensive expansion and the intensive deveiopment, strengthened further uneven regional development process, even though it appears to have reduced apparently the economic and regional disparity by balancing numerically large and small firms and by extending the Fordist industrial space nation-wideiy. And it seems more persuasive to see that the Korean industrial structure in the 1980s maturated the Fordist system of accumulation, but not yet transformed towards the post-Fordist (or the so-called flexible) accumulation system, even though the Korean economy in the 1990s seems to be under a pressure of restructuring towards the latter system.

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The Role of the Endometrium and Embryo in Human Implantation (인간 착상 과정에 자궁내막과 배아의 역할)

  • Jee, Byung-Chul
    • Development and Reproduction
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    • v.13 no.1
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    • pp.1-11
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    • 2009
  • Implantation itself is governed by an array of endocrine, paracrine and autocrine modulators, of embryonic and maternal origin. Window of implantation is the unique temporal and spatial expression of factors allows the embryo to implant via signaling, appositioning, attachment, and invasion in a specific time frame of $2{\sim}4$ days. When the embryo has arrived in the uterine cavity, a preprogrammed sequence of events occurs, which involves the production and secretion of a multitude of biochemical factors such as cytokines, growth factors, and adhesion molecules by the endometrium and the embryo, thus leading to the formation of a receptive endometrium. Cytokines such as LIF, CSF-1, and IL-1 have all been shown to play important roles in the cascade of events that leads to implantation. Integrin, L-selectin ligands, glycodelin, mucin-1, HB-EGF and pinopodes are involved in appositioning and attachment. The embryo also produces cytokines and growth factors (ILs, VEGF) and receptors for endometrial signals such as LIF, CSF-1, IGF and HB-EGF. The immune system and angiogenesis play an important role. The usefulness of these factors to assess endometrial receptivity and to estimate the prognosis for pregnancy in natural and artificial cycles remains to be proven. Integrins, pinopodes, glycodelin and LIF (from biopsies) are promising candidates; from uterine flushings, glycodelin and LIF are also candidates. The ideal serum marker is not available, but VEGF, glycodelin and CSF have some clinical implications. Further evaluation that includes larger groups of infertile women and fertile controls are needed to elucidate whether their presence in plasma, flushing fluid, or endometrial samples can be used as some kind of a screening tool to assess endometrial function and prognosis for pregnancy before and after artificial reproductive therapy. A better understanding of their function in human implantation may lead to therapeutic intervention, thereby improving the success rate in reproduction treatment. New molecular techniques are becoming available for measuring both embryonic and endometrial changes prior to and during implantation. The use of predictive sets of markers may prove to be more reliable than a single marker. Ultimately, the aim is to use these tools to increase implantation in artificial cycles and consequently improve live-birth rates.

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Cryopreservation of Bovine IVM/IVF/IVC Hatched Blastocysts (체외생산된 소 완전탈출 배반포기배의 동결보존)

  • Lee, K.S.;Kim, E.Y.;Yi, B.K.;Nam, H.K.;Yoon, S.H.;Park, S.P.;Lim, J.H.
    • Korean Journal of Animal Reproduction
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    • v.23 no.2
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    • pp.141-148
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    • 1999
  • This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø>300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p<0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p<0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p<0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.

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The embryological studies on the interspecific hybrid of ginseng plant (Panax ginseng x P. Quiuquefolium) with special references to the seed abortion (인삼의 종간잡종 Panax ginseng x P Quinquefoilium의 발생학적 연구 특히 결실불능의 원인에 관하여)

  • Jong-Kyu Hwang
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.5 no.1
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    • pp.69-86
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    • 1969
  • On the growing of the interspecific hybrid ginseng plant, the phenomena of hybrid vigoures are observed in the root, stem, and leaf, but it can not produce seeds favorably since the ovary is abortive in most cases in interspecific hybrid plants. The present investigation was undertaken in an attempt to elucidate the embryological dses of the seed failure in the interspecific hybrid of ginseng (Panax Ginseng ${\times}$ P. Quinque folium). And the results obtained may be summarized as follows. 1). The vegetative growth of the interspecific hybrid ginseng plant is normal or rather vigorous, but the generative growth is extremely obstructed. 2). Even though the generative growth is interrupted the normal development of ovary tissue of flower can be shown until the stage prior to meiosis. 3). The division of the male gameto-genetic cell and the female gameto-genetic cell are exceedingly irregular and some of them are constricted prior to meiosis. 4). At meiosis in the microspore mother cell of the interspecific hybrid, abnormal division is observed in that the univalent chromosome and chromosome bridge occure. And in most cases, metaphasic configuration is principally presented as 23 II+2I, though rarely 22II+4I is also found. 5). Through the process of microspore and pollen formation of F1, the various developmental phases occur even in an anther loclus. 6). Macro, micro and empty pollen grains occur and the functional pollen is very rare. 7). After the megaspore mother cell stage, the rate of ovule development is, on the whole, delayed but the ovary wall enlargement is nearly normal. 8). Degenerating phenomena of ovules occur from the megaspore mother cell stage to 8-nucleate embryo sac stage, and their beginning time of constricting shape is variously different. 9). The megaspore arrangement in the parent is principally of the linear type, though rarely the intermediate type is also observed, whereas various types, viz, linear, intermediate, Tshape, and I shape can be observed in hybrid. 10). After meiosis, three or five megaspore are some times counted. 11). Charazal end megaspore is generally functional in the parents, whereas, in F1, very rarely one of the center megaspores (the second of the third megaspore) grows as an embryo sac mother cell. 12). In accordance with the extent of irregularity or abnormality in meiosis, division of embryo sac nuclei and embryo sac formation cause more nucellus tissue to remain within th, embryo sac. 13). Even if one reached the stage of embryo sac formation, the embryo sac nuclei are always precarious and they can not be disposed to theil proper, respective position. 14). Within the embryo sac, which is lacking the endospermcell, the 4-celled proembryo, linear arrangement, is observed. 15). Through the above respects, the cause of sterile or seed failure of interspecific hybrid would be presumably as follows, By interspecific crossing gene reassortments takes place and the gene system influences the metabolism by the interference of certain enzyme as media. In the F1 plant, the quantity and quality of chemicals produced by the enzyme system and reaction system are entirely different from the case of the parents. Generally, in order to grow, form, and develop naw parts it is necessary to change the materials and energy with reasonable balance, whereas in the F1 plant the metabolic process becomes abnormal or irregular because of the breakdown of the balancing. Thus the changing of the gene-reaction system causes the alteration of the environmental condition of the gameto-genetic cells in the anther and ovule; the produced chemicals cause changes of oxidatio-reduction potential, PH value, protein denaturation and the polarity, etc. Then, the abnormal tissue growing in the ovule and emdryo sac, inhibition of normal development and storage of some chemicals, especially inhibitor, finally lead to sterility or seed failure. Inconclusion, we may presume that the first cause of sterile or seed abortion in interspecific hybrids is the gene reassortment, and the second is the irregularity of the metabolic system, storage of chemicals, especially inhibitor, the growth of abnormal tissue and the change of the polarity etc, and they finally lead to sexual defect, sterility and seed failure.

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Effect of Early Pregnant Heifer as Donor on the Ovum Pick-Up Derived Oocyte Aspiration and Embryo Production (초기 임신우의 공란우 활용이 초음파 유도 난자 채취 및 수정란 생산에 미치는 영향)

  • Jin, Jong-In;Kwon, Tae-Hyeon;Choi, Byeong-Hyun;Kim, Sung-Soo;Jo, Hyun-Tea;Bang, Jae-Il;Kim, Sam-Chul;Cho, Kyu-Woan;Lee, Jung-Gyu;Kong, Il-Keun
    • Journal of Embryo Transfer
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    • v.26 no.1
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    • pp.19-25
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    • 2011
  • This study was carried out to evaluate the effect of early pregnant cow as donor for Ovum Pick-Up (OPU) derived oocyte aspiration and embryo production in Holstein heifers. Four non-pregnant and 2 pregnant Holstein heifers were used as donor and then carried out total 17 OPU session for 10 weeks (2 times per week). Recovered cumulus-oocyte-complexes (COCs) were classified into 4 grade by oocyte cytoplasm and cumulus cells and matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol in 5% $CO_2$ and over 99% humidity for 24 h. After 24 h co-incubation with post-thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3 days and then changed CR1aa medium with 10% of FBS for another 3~4 days. The Mean number of aspirated follicles and collected oocytes in the early stage pregnant and non-pregnant heifers were $13.0{\pm}4.3$ and $10.6{\pm}3.9$, $5.4{\pm}3.4$ and $7.7{\pm}3.6$ per session, respectively. Rate of collected oocyte from aspirated follicles were 59.2% and 50.5%, respectively. The average number of good quality oocytes (Grade I and II) in the early stage pregnant and non-pregnant heifers was $3.7{\pm}2.7$ and $4.9{\pm}2.6$ (Mean${\pm}$SD). Cleavage and blastocyst developmental rates in Grade I and II were 22.2% and 25.5%, and then $1.7{\pm}0.9$ and $1.4{\pm}1.1$ blastocyst per session, respectively. In conclusion, OPU technology can be used in early stage pregnant and non-pregnant heifers without any problem and so applied OPU derived embryo production to maximize the ability of genetically valuable females.

The Developmental Effects of Radiation on ICR Mouse Embryos in Preimplantation Stage (착상전기(着床前期)에 있어서 ICR Mouse의 태아(胎兒)에 대한 방사선(放射線) 개체(個體) Level 영향(影響)의 연구(硏究))

  • Gu, Yeun-Hwa
    • Journal of Radiation Protection and Research
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    • v.21 no.4
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    • pp.273-284
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    • 1996
  • Embryos and fetuses are more sensitive to various environmental agents than are adults or children. The biological effects such as intrauterine death and malformation are closely connected with prenatal exposure very various agents. The sensitivity of these embryonic/fetal effects depends on the stage of pregnancy. From the viewpoint of fetal development, embryonic and fetal stages can be divided into three stages : Preimplantation, organogenetic and fetal. Each stage corresponds to 0 to 4.5days, 4.5 to 13.5days, and 13.5days of gestation in mice, respectively. Many studies on the biologcal effects of mice irradiated by ${\gamma}-rays$ at various stages during organogenesis and fetal period have been performed. Based on these results, the dose-effect and dose-response relationships in malformations, intrauterine death, or retardation of the physical growth have been practically modeled by the ICRP(International Commission on Radiological Protection) and other international bodies for radiation protection. Many experimental studies on mice have made it clear that mice embryos in the preimplantation period have a higher sensitivity to radiation for lethal effects than the embryos/fetuses on other prenatal periods. However, no eratogenic effects of radiation at preimplantation stages of mice have been described in many textbooks. It has been believed that 'all or none action results' for radiation of mice during the preimplantation period were applied. The teratogenic and lethal effects during the preimplantation stage are one of the most important problems from the viewpoint of radiological protection, since the preimplantation stage is the period when the pregnancy itself is not noticed by a pregnant woman. There are many physical or chemical agents which affect embryos/fetuses in the environment. It is assumed that each agents indirectly effects a human. Then, a safety criterion on each agent is determined independently. The pregnant ICR mice on 2, 48, 72 or 96 hours post-conception (hpc), at which are preimplantation stage of embryos, were irradiated whole body Cesium-gamma radiation at doses of 0.1, 0.25, 0.5, 1.5, and 2.5 Gy with dose rate of 0.2 Gy/min. In the embryos from the fetuses from the mice irradiated at various period in preimplantation, embryonic/fetal mortalities, incidence of external gross malformation, fetal body weight and sex ratio were observed at day 18 of gestation. The sensitivity of embryonic mortalities in the mice irradiated at the stage of preimplantation were higher than those in the mice irradiated at the stage of organogenesis. And the more sensitive periods of preimplantation stage for embryonic death were 2 and 48 hpc, at which embryos were one cell and 4 to 7 cell stage, respectively. Many types of the external gross malformations such as exencephaly, cleft palate and anophthalmia were observed in the fetuses from the mice irradiated at 2, 72 and 96 hpc. However, no malformations were observed in the mice irradiated at 48 hpc, at which stage the embryos were about 6 cell stage precompacted embryos. So far, it is believed that the embryos on preimplantation stage are not susceptible to teratogens such as radiation and chemical agents. In this study, the sensitivity for external malformations in the fetuses from the mice irradiated at preimplantation were higher than those in the fetuses on stage of organogenesis.

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