02 poster.hwp

Poster Presentation - 33 -

KSET-01 Production of Transgenic Miniature Pig Expressing Human Heme Oxygenase 1 Gene Ok Jae Koo, Jung Taek Kang, Hee Jung Park, Dae Kee Kwon, Sol Ji Park, Su Jin Kim, Ma. Ninia Gomez, Goo Jang, Byeong Chun Lee Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University Heme oxygenase 1 (HO1) is an ubiquitous enzyme in hemoglobin metabolism. Since HO1 has had anti inflammatory as well as anti apoptotic effect, it has been regarded as a candidate gene for transfection in xenotransplantation research. In this study, we produced transgenic miniature piglets expressing human HO1 gene using somatic cell nuclear transfer. The cdna fragments of human HO1 under the influence of CMV IE promoter were inserted into pcdna vector. The constructed vector was transfected into cultured ear fibroblast derived from miniature pig using lipofection. Cloned transgenic embryos were derived from enucleated in vitro matured porcine oocytes and a transfected ear fibroblast. A total of 305 reconstructed embryos was transferred to uterine tubes of four surrogate crossbreed farm pigs (60 to 100 for each surrogate). One out of four surrogates was diagnosed as pregnancy at 28 day after embryo transfer by ultrasonography, and delivered one live piglet and three abortuses by normal labour at 113 day of gestation. The alive piglet were deceased at 10 th day after born due to severe diarrhea suffered from bacterial infection. As a result of autopsy, there is no congenital disorders. For expression of the gene using RT PCR analysis, tissue samples from heart, lung, liver, pancrease, spleen, kidney, testis, and skin were isolated. The HO1 gene was well expressed in all sampled organs. In conclusion, present study demonstrate that an ear fibroblast with expression human HO1 gene was reprogrammed in enucleated porcine oocytes and consequently a cloned transgenic piglet was successfully born. This study was supported by Gyeonggido Veterinary Service, Korean MEST through BK21 program and Hanhwa L&C. Key words: xenotransplantation, heme oxygenase, transgenic pig, somatic cell nuclear transplantation - 35 -

KSET-02 Pig ADAM3 Disintegrin Domain has an Important Rolein the Sperm Egginteraction Ekyune Kim 1, Ji Su Kim 2, Dong chul Baek 1, Jae Woong Lee 1, Sang Rae Lee 1, Myeong Su Kim 1, Sang Hyun Kim 1, Chan Shick Kim 3, Deog Bon Koo 2, Kyu Tae Chang 1 1 National Primate Research Center, 2 Center for Regenerative Medicine, KRIBB 3 Faculty of Biotechnology, Cheju National University The A Disintegrin and Metalloprotease (ADAM) protein family has important roles in various biological processes, such as fertilization, neurogenesis, myogenesis, and inflammation. Most ADAM proteins have a unique organization, containing an N terminal signal sequence, followed by a pro domain, metalloprotease, disintegrin, Cys rich, epidermal growth factor (EGF) like, transmembrane, and cytoplasmic tail domains. The metalloprotease domain possesses shaddase activity toward the ectodomain of membranous precursor proteins, whereas the cytoplasmic domain is related to the interaction with Src family protein tyrosine kinases. It is known that the disintegrin domain is involved in cell to cell adhesion. Although the roles of several testis specific ADAMs have been identified in knockout (KO) mice, they were expressed in a species specific manner. Thus, the specific role of testis specific ADAM molecules in the sperm egg interaction remains to be determined. Cyritestin, known also as ADAM3, is a member of the ADAM family, and is expressed specifically in male germ cells. It is suggested that cyritestin is able to bind to the ZP through the disintegrin domain because the pretreatment of eggs with peptides of the ADAM3 disintegrin domain inhibited IVF in mouse. However, little is known about ADAM3 in other species, such as pigs. We have identified and characterized porcine ADAM3 in an attempt to elucidate its role(s) in the interaction with the ZP. In this study, we found two isoforms of porcine ADAM3, ADAM3a and ADAM3b, which are present on the surface of sperm, and are involved in the interaction with the ZP. Theses results indicate that both ADAM3a and ADAM3b could have important roles in fertilization. Key words: adam family, cyritestin, sperm egg interaction - 36 -

KSET-03 체외배양액의종류에따른돼지단위발생란의발육능비교 김진영, 이주형, 유진영, 박준홍 1, 이은송 강원대학교수의학부대학, 1 서울대학교농업생명과학대학 최근국내외에서돼지체외성숙난자를이용한체외수정및체세포핵이식란생산에관한연구가활발히진행되고있다. 그러나현재까지체외수정란과핵이식수정란의배반포발육능은체내유래수정란에비해낮은실정이다. 본연구에서는돼지체외성숙난자를이용하여작성된단위발생난자의체외배양에서돼지수정란의체외배양에광범위하게사용되고있는 North Carolina State University(NCSU) 3 배양액과 porcine zygote medium(pzm) 3 배양액이배반포로의발육에미치는영향을검토하였다. 도축된돼지난소로부터미성숙난자를채취하여 ecg/hcg가포함된 TCM 199배양액에서 22시간배양한후호르몬이첨가되지않은배양액에서 25시간추가배양하여체외성숙을유도하였다. 체외성숙 47시간째에극체를배출한체외성숙난자를선별하여전기자극 (2 DC pulses, 1.2 kv/cm, 60 μs) 으로난자의활성화를유도하였다. 활성화난자를 7.5 μg/ml cytochalasin B가첨가된 NCSU 23aa 배양액에서 4시간동안배양한후실험설계에따라각각의배양액으로옮겨 39, 5% CO 2, 5% O 2, 90% NO 2 의조건에서 7일간배양하였다. 본실험에서는 (1) 2% 필수아미노산과 1% 비필수아미노산용액이첨가된 NCSU 3 배양액 (NCSU 3aa), (2) NCSU 3aa + 2.77 mm myo inositol, 0.34mM trisodium citrate, 10 μm β mercaptoethanol(mncsu 3aa), 그리고 (3) PZM 3 + 2.77 mm myo inositol, 0.34mM trisodium citrate, 10 μm β mercaptoethanol (mpzm 3) 의세가지배양액을이용하여단위발생난자를체외배양한후이들배양액의배발육효과를비교, 검토하였다. 체외배양 48시간에분할율을, 7일째에배반포로의발육및배반포의평균세포수를조사하였다. 실험결과분할율은 NCSU 3aa, mncsu 3aa 및 mpzm 3에서각각 91%, 88% 및 92% 로유의적인차이가나타나지않았으나각각 33%, 37% 및 45% 의배반포로의발육률을보여 mpzm 3에서배발육촉진효과가가장높은것으로관찰되었다. 배반포의평균세포수는 31~36개로처리군간에유의적인차이가관찰되지않았다. 이상의결과에서 mpzm 3는 NCSU 3에비해돼지단위발생난자의발육능을유의적으로개선하는것으로밝혀졌다. 향후체외수정란과체세포핵이식란의배발육효과에대한추가적인연구를통하여이들수정란의발육능향상에기여할수있을것으로생각된다. This work was supported by a grant (# 20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea. Key words: 단위발생, 체외배양, PZM 3, NCSU 23, 돼지 - 37 -

KSET-04 흑염소의발정동기화처리기간단축에따른임신율 김현종, 최창용, 조상래, 손동수, 최선호, 최순호, 김성재, 장용석 1, 임경순 2 농촌진흥청축산과학원가축유전자원시험장, 1 유정목장, 2 서울대학교동물자원과학과 농가에서적용가능한흑염소인공수정기술을확보를위해발정동기화처리기간을단축하는연구를실시하였다. 흑염소는자연발정을관찰하기가어려워서인공수정을위해서는발정동기화가필요하다. 흑염소의발정동기화는일반적으로프로게스테론제제를질내에 11~19일정도삽입하였다가제거하여난포의발육과배란을유기하는방법을사용한다. 본연구에서는프로게스테론제제인 CIDR G(Pharmacia & Upjohn, New Zealand) 를 11일간삽입하거나, 2일단축하여 9일간삽입하였을때임신율의차이를조사하였다. 1~3세사이의재래흑염소암컷의질내에 CIDR G를삽입하여 9일째에 PMSG(Intervet, Holland) 400IU와 PGF 2α (Pharmacia, Belgium) 7.5 mg을근육주사하여황체를퇴행시키고난포의발육을유도하였으며, CIDR G 를삽입후 11일째에제거하여발정을유도하여 CIDR G 제거후 24시간, 32시간째에 2회인공수정하였다. 한편, 기간을단축하기위한방법으로 CIDR G 삽입후 7일째에호르몬을주사하고, 9일째에 CIDR G를제거한후 24시간, 32시간째에인공수정하였다. 발정동기화처리기간단축에따른수태율을확인하기위하여자궁경관입구에정자수 1억개인 0.5 ml 스트로로정액을주입하여수정을유도하여수정후 35 일경에임신진단을실시하고, 분만여부로분만율을구하였다. 9일간 CIDR G 삽입개체들은 25% (1/4) 의임신율을보였으며, 7일간 CIDR G를삽입한개체들은 50% (3/6) 의임신율을보였다. 특히 7일간삽입암염소들 6두중 3두는액상정액, 나머지 3두는동결정액으로인공수정을실시하여액상정액인공수정개체는 1두, 동결정액인공수정개체는 2두가임신하여액상정액보다동결정액에서더높은임신율을보였다. 이상의결과로처리두수가적어발정동기화기간단축이더욱유리하다고제시하기는어려우나, 기간단축이수태율을떨어뜨리지는않는것으로생각된다. 10두에서 4두의임신으로농가활용가능성을확인하였으며, 임신된 4두중 3두가동결정액으로임신이이루어져동결정액이염소인공수정에이용될수있음을확인하였다. Key words: 흑염소, 발정동기화, 인공수정, CIDR G, 기간단축 - 38 -

KSET-05 Effect of Fluoxetine on In Vitro Maturation and Fertilization of Bovine Oocytes and Embryonic Development Sun Young Park, Changyong Choe 1, Jae Ik Lee 2, Hye Jin Park, Jaehee Han, Dawon Kang Medical Research Center for Neural Dysfunction, Departments of Physiology and 1 Animal Genetic Resources Station, National Institute of Animal Science, RDA, Namwon 590 832, Korea 2 Animal Science, College of Medicine and Institute of Health Sciences, Gyeongsang National University, Jinju 660 751, Korea Fluoxetine, a antidepressant of the selective serotonin reuptake inhibitor class, regulates a variety of physiological processes, such as cell proliferation and apoptosis. The fluoxetine could induce apoptosis or cell proliferation in a dose or/ and time dependent manner. This study was undertaken to investigate the effect of fluoxetine on in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes and embryonic development. The fluoxetine treated for IVM, normally 24 hours, significantly decreased blastocyst formation rate compared to that of control, whereas the fluoxetine treated for 6 hours enhanced the blastocyst formation rate. Fluoxetine treated during IVF, normally 6 hours, increased the blastocyst formation rates by 20%. The fluoxetine treated for in vitro culture (IVC) 24 hours also enhanced the blastocyst formation rate by 20%. These results showed that fluoxetine treated during IVF and IVC increased blastocyst formation rate, but the fluoxetine treated during IVM decreased blastocyst formation rate in a time dependent manner. Fluoxetine also regulated mouse embryonic developmental rate in a time dependent manner but not in a dose dependent manner (10μM to 50 μm). All the 2 cell stage embryos exposed to fluoxetine for 2 days were arrested at the 2 cell or 4 cell stages. In contrast, the embryos exposed to fluoxetine for 1 day showed higher blastocyst formation rate and total cell numbers than control untreated with fluoxetine. fluoxetine induced blastocyst showed normal karyotype. These results suggest that fluoxetine could be good drug to enhance the embryonic developmental rate when bovine and mouse oocytes and embryos are cultured in vitro. Key words: fluoxetine, in vitro culture, mouse, bovine - 39 -

KSET-06 Effect of HAM 12 Treatment on Cellular Morphology of Bovine Ovary in In Vitro Sung Jai Park, Jun Kyu Son, Kwang Soo Baek, Soo Bong Park, Byeong Soon Jeon, Hyeon Shup Kim, Tai Young Her, Byoung Ho Park, Seok Jin Kang, Joong Hui Lee, Wang Shik Lee, You Lim Choi National Institute of Animal Science, Republic of Korea This study investigated the effect of Ham 12 injection using micro tubing system (5% O 2, 5% CO 2 ) on cellular morphology of bovine ovary in in vitro. Holsteinovaries were procured from slaughter house and shipped to laboratory within 1 hour. Ovaries were treated with HAM 12 (for 6 h at media flow rate 1 ml/ 1 minute) using circulating machine (ISMATEC, MCP Motor, Switzerland) in an incubator. Then the Ham treated ovaries were transferred in a 10% formalin solution for 24 h. Ham untreated ovaries were directly transferred to formalin without any treatment. A 4 μm cross section was taken from each of the Ham treated or untreated ovary. The cross sections were stained with hematoxylin, eosin, and TUNEL. These cross sections were analyzed using microscope (imaging analyses system) at 400X to estimate intercellular distance, oocyte diameter, follicular membrane, and living cell. Inter cumulus cell distance and oocyte diameters were higher in Ham treated ovary compared to untreated ovary. Follicularmembrane was normal and living cell number was reduced in Ham treated ovary. It may be conclude that Ham 12 treatment is not sufficient to enhance the bovine ovarian cell life. Further research is needed to understand cellular and molecular mechanisms of ovarian cells to better succeed in their in vitro cultures. Key words: bovine ovary, in vitro culture, follicular membrane, oocyte diameter - 40 -

KSET-07 Effects of the Addition of β Lactoglobulin and Bsa on the Development of Porcine Embryos Yong Soo Park 1, Hum Dae Park 2 1 Gyoengbuk Livestock Research Institute 2 Division of Life Food and Biotech, Daegu Universityu This study was performed to elucidate the effects of addition of β lactoglobulin and bovine serum albumin (BSA) in vitro maturation (IVM) and in vitro culture (IVC) medium on porcine embryo production. The development rate to the 2 cell (71.4~75.6%) and blastocyst stages (6.8~13.3%) with different BSA concentrations in IVM medium were similar among treatment groups. Blastocyst hatching rate was significantly higher in the control group (0.0 mg/ml) than in the group of 1.0 mg/ml supplement (20.0% vs. 0.0%; p<0.05). The development rate to the 2 cell (62.0~70.6%) and blastocyst stages (15.4%~38.5%) with different β lactoglobulin concentrations in IVM medium was similar among treatment groups. The development rate to the blastocyst was significantly higher in the group of 1.0 mg/ml (15.3%) than in the group of 0.5 mg/ml supplement (7.6%, p<0.05). The development rate to the 2 cell and blastocyst stages following the first addition of β lactoglobulin in IVM medium was significantly higher in the control group (77.0% and 18.9%) and was 0~44 hr(77.2% and 16.9%) greater than that observed in other treatment groups (p<0.05). The development rate to the 2 cell stage (68.1~ 74.8%) and blastocyst stages (9.2~12.7%) with different BSA concentrations in IVC medium was similar among treatment groups. However, blastocyst hatching rate was significantly higher in the group of 3.0mg/ml supplement (30.0%) than in the control group (0.0%; p<0.05). The development rate to the 2 cell stage (72.9~ 78.0%), blastocyst (7.1~14.2%) and hatching stages (33.3~38.1%) were not different. The development rate to the 2 cell stage (63.6~72.5%), blastocyst (8.4~16.1%) and hatching stages (18.2~37.5%) at the different culture periods were similar among treatment groups. This study suggested that if the addition level and periods of β lactoglobulin addition are adjusted, it is possible to replace BSA in the in vitro porcine embryo production. Key words: porcine, bsa, β lactoglobulin, in vitro culture - 41 -

KSET-08 Reproductive and Metabolic Endocrinology in Rams Selected for High or Low Plasma IGF I Concentrations Eun Kuk Park 1,2, Yun Sik Lee 1, Won Hyung Chio 1, T. J. Parkinson 2, J. F. Cockrem 2, K. S. Han 3, H. T. Blair 2, Jong Taek Yoon 4, Jong Phil Chu 1 1 Department of Medical Zoology, College of Medicine, KyungHee University, South Korea 2 Institute of Veterinary, Animal & Biomedical Sciences 3 Riddet Centre, Massey University, Palmerston North, New Zealand 4 Genetic Engineering Institute, Hankyong National University, South Korea The objectives of this study were to define reproductive and metabolic endocrinology in Romney rams selected at the time of weaning for high or low peripheral IGF I concentrations with particular relevance to the annual changes in the relationships between GH, IGF I and insulin and the relationship between that system and the activity of the reproductive endocrine axis. Parameters examined in detail included the response of testosterone to hcg, and expression of IGF I and its binding proteins in the testis. Blood samples were collected before and 40 mins after i/v administration of 1,000 IU hcg. Seasonal differences from July to March were evident in concentrations of testosterone, IGF I and insulin. IGF I line differences were also found in IGF I and insulin concentrations. Basal testosterone concentrations did not differ between lines, but the response to hcg was greater in high than low IGF lines. When IGF I concentrations were taken into consideration, basal testosterone concentrations also differed between lines. Samples of liver and testis tissue were collected from four animals for expression of mrna for IGF I, Type I IGF receptor and IGF binding proteins. It was found that genes for IGF I, Types I IGF receptor and IGF binding proteins 2, 3, 4, 5 and 6 were expressed in the testis. Expression of Type I IGF receptor and IGFBP 3 in the testis and of IGF I in the liver was greater in high than low IGF line rams. These studies suggested that circulating concentrations IGF I are associated with gonadotropin stimulated steroidogenesis that affects scrotal circumference, but differences in IGF I status between lines are modulated by the different expression of mrna for IGF IR and IGFBPs and by negative feedback between lines. Key words: ram, Igf I, Igfbindingproteins, insulin, testosterone - 42 -

KSET-09 Effects of Selection for High and Low Serum Insulin like Growth Factor I on Reproductive Performance in Rams Eun Kuk Park 1,2, Yun Sik Lee 1, Yujing Mi 1, John. F. Cockrem 2, Hugh T. Blair 2, Paul R. Kenyon 2, Tim. J. Parkinson 2, Jong Taek Yoon 3, Jong Phil Chu 1 1 Dept of Medical Zoology, College of Medicine, KyungHee University, South Korea 2 Institute of Veterinary, Animal & Biomedical Sciences, Massey University, New Zealand 3 Genetic Engineering institute, Hankyong National University, South Korea This study examined effects of selection for high and low serum insulin like growth factor I (IGF I) on reproductive performance in rams. We examined the effect of genetic selection of rams for high or low circulating concentrations IGF I at the time of weaning upon seasonal differences and reproductive parameters. For preliminary experiment, animals were sampled in December 2005 and March 2006. Fifty seven Romney rams (17 from low IGF I line, 21 rams from high IGF I line and 19 from unselected group) were used in this study. For the main experiment, animals were examined and samples collected in July, September and November 2006 and March 2007 with two selection lines (13 high and 19 low). Scrotal circumference in the inguinal skin was recorded. Semen was collected by electroejaculation on 6 occasions over a 16 month period. Semen was evaluated according to standard procedures (volume, motility, density and morphology). Samples were collected from four animals for expression of mrna for IGF I and IGF type 1 receptor (IGF 1R) from testis and IGF I, IGF 1R and insulin receptor (IR) from liver. Blood samples were collected via jugular venipuncture for the measurement of IGF I, insulin and testosterone. The incidence of morphologically abnormal sperm cells, the scrotal circumference and sperm motility were higher in breeding season than in non breeding season. Seasonal changes were found in the percentage of abnormal sperm, scrotal circumference, sperm motility and sperm density, but there were no differences between lines in any reproductive parameters. High expression of IGF I and insulin receptor from liver was found in the high line, with differential expression of IGF I in the testis. Testosterone concentrations did not differ between lines, although the concentrations of IGF I and - 43 -

insulin were higher in the high line. Results suggested that IGF I may be locally produced in the liver and testis and selection for high IGF I may not be associated with improved reproduction, but seasonal variation does influence the male reproduction. Key words: ram, Igf I, reproductive parameter, semen seasonal variation - 44 -

KSET-10 Effect of Follicle Isolation Methods and Culture Conditions on Preantral Follicle Development in Pigs 1 Jun Hong Park 1, Joohyeong Lee, Jinyoung You, Jinyoung Kim, Eunsong Lee 1 College of Agriculture & Life Science, Seoul National University School of Veterinary Medicine, Kangwon National University Domestic pigs are an important food source and, because of the anatomical and physiological similarities to human, are also used extensively for biomedical research. Establishment of a culture system that can support in vitro growth and maturation of pig oocytes from preantral follicles (PF) may offer a great advantage for both biotechnological and fundamental research. Follicle maturation system has been developed and resulted in live birth in mice but has not been fully established in pigs. This study was conducted to examine the effects of PF isolation methods, addition of serum or pig follicular fluid (pff) to maturation medium, and type of culture media on PF development in vitro. Porcine PF isolated mechanically or enzymatically were cultured for 4 days in North Carolina State University (NCSU) 23 medium, minimum essential medium (MEM) or medium 199 (M199) containing 7.5% FBS and/or 5~10% pff. Recovered cumulusoocyte complexes (COCs) maturated in vitro for 2 days in medium M199. Parameters included follicular growth rate, recovery rate of COCs from cultured follicles, and oocytes meiotic competence. Mechanical isolation method was superior to the enzymatic isolation because enzymatic treatment induced rupture of basement membrane. Type of macromolecule (FBS or pff) in maturation medium and coculture with follicle shell piece did not improve follicular growth. Follicular growth rate and COCs recovery was not influenced by culture media used in this study. Mature (metaphase II) oocytes were produced from PFs cultured in NCSU 23, alpha MEM and M199 but the rate was very low (about 1%). Result of the present study suggests that further improvement in PF culture system such as modification of medium composition and culture environment should be made for the successful production of mature pig oocytes from PFs. 1 This work was supported by a grant (# 20080401034072) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea. Key words: porcine, preantral follicle, in vitro maturation - 45 -

KSET-11 2007 년도한우및젖소에서수정란생산및이식실태 손동수, 최창용, 조상래, 최선호, 김현종 농촌진흥청국립축산과학원가축유전자원시험장 2007년도국내에서실시된한우및젖소의수정란이식실태를파악하기위하여전국의수의 축산분야의대학, 국 공립축산관련연구소, 농업기술센터, 생산자단체, 수정란이식개인시술소등의 187개관련기관에 2007년 1월 1일부터 12월 31일까지한우및젖소의수정란생산, 이식및임신진단결과에대하여설문서를통하여조사하였으며, 설문서를작성하여제출한 26기관의자료를분석한결과는다음과같다. 한우및젖소의수정란이식을실시하고있는기관은국립기관 2개소, 지방자치단체 9개소, 대학 1개소, 생산자단체 4소, 개인시술소 162개소로전체 178개소이었다. 과배란처리된한우 645두및젖소 175두에서수정란을채란한한우 561두및젖소 165두로부터회수된이식가능수정란은각각 3,098개및 788개로두당평균각각 5.5개및 4.8개였다. 도축한우의난소를채취하여체외수정 배양으로생산된이식가능수정란수는 27,889개이었으며, 복제수정란도 1,203개를생산하였다. 수정란이식에서한우수정란은체내수정란 1,601두, 체외수정란 6,384두, 복제수정란 42두를이식하였으며, 젖소수정란은체내수정란을 380두이식하였다. 수정란의상태별로신선수정란 7,173 두, 동결수정란 1,234두가이식되었다. 수란우품종에따른수정란이식은한우수정란을한우수란우에 1,964두, 젖소수란우 6,063두가이식되었고, 젖소수정란은젖소수란우에 380두가이식되었다. 수정란이식형태별로는수정란 1개이식 4,972두, 2개이상이식 3,160두, 인공수정후수정란추가이식 275두이었다. 수정란이식수태율은체내신선수정란 44.2%, 동결수정란 44.3% 이었고, 체외수정란에서는신선수정란 55.0%, 동결수정란 34.2% 였다. 따라서 2007년도소수정란이식은 8,407두가실시되었으며, 그중한우수정란의이식이 95.5%, 체외수정란이 75.9%, 한우수정란을젖소수란우에이식이 72.1%, 수정란 1개이식은 59.1% 이었으며, 체외신선수정란의이식에서수태율이높게나타났다. 한편, 2006년도에보고된자료와비교시과배란처리된한우및젖소공란우두수는 48.8% 가증가하였고, 한우체내수정란의생산도 72.1% 가증가하였으나체외수정란의생산은 18.1% 가감소하였다. 수정란이식두수에서체내수정란이식은 49.6% 가증가한반면체외수정란이식은 22.8% 가감소하였으며, 2개이상수정란이식은 30.0% 가감소한것으로보아우수한유전능력을갖고있는공란우에서생산된수정란이이식됨을추정할수있다. 한편, 수정란이식수태율은전년도에비해체내동결수정란과체외신선수정란에서각각 11.1% 와 9.6% 가 - 46 -

증가하였다. 사사 : 설문서자료를제출하여주신국 공립축산관련연구소, 대학, 생산자단체, 개인시술소등에대하여감사드립니다. Key words: 한우, 젖소, 수정란생산, 수정란이식, 수태율 - 47 -

KSET-12 총체보리사일리지급여가한우수란우의수태율에미치는영향 손동수, 최창용, 조상래, 최선호, 김현종, 김영근, 류일선, 김원호 1, 김일화 2 농촌진흥청국립축산과학원가축유전자원시험장, 1 농촌진흥청국립축산과학원낙농과, 2 충북대학교수의과대학 최근소사육농가에서는고가의수입건초의대체조사료원으로총체보리사일리지를급여하는농가가증가하고있다. 수정란이식에서수태율에미치는요인은매우다양하며, 한우의조기개량을위해수정란이식을희망하는농가가증가하고있으나, 아직까지수란우에서총체보리의급여효과에대한자료가없는바이를구명하고자연구가실시한결과는다음과같다. 수란우는축산과학원가축유전자원시험장에서사육하고있는 30~40개월령의한우미경산우로체중은 245~498 kg( 평균 336.8±55.8 kg) 이었다. 수란우는발정동기화 50일전부터시험 1구는 1일두당농후사료 2.0~ 2.5 kg, 총체보리사일리지 6.0~8.5 kg을시험 2구는 1일두당농후사료 3.0~3.2 kg, 건초 6.5~7.5 kg을급여하였다. 수란우의발정동기화는 1차처리에서는 day 0에 CIDR device(cidr, InterAg, Hamilton, New Zealand) 를삽입과동시에 100 ug gonadorelin(gnrh, Fertagyl, Intervet, Boxmeer, Holland) 을주사하였으며, day 7에 CIDR 의제거와 25 mg PGF 2α (Lutalyse, Pharmacia & Upjohn, Puurs, Belgium) 를주사하였고, day 9에 100 ug gonadorelin 주사하고 day 17에수정란이식을실시하였으며, 수정란이식당일또는 1일전에 1,500 IU의 hcg(chorulon, Intervet, Boxmeer, Holland) 를주사하였다. 1차발정동기화후수정란이식을실시하지못하였거나 1차수정란이식에서임신되지않은개체에대하여 2차발정동기화와수정란이식을실시하였다. 2차처리에서는 day 0에 100ug gonadorelin을주사하고, day 7에 25 mg PGF 2α 를주사하였으며, day 9에는 0.0042mg buserelin acetate(receptal, Intervet, Boxmeer, Holland) 을주사하고, 1차와같은방법으로수정란이식을실시하였다. 이식된수정란은과배란처리된한우공란우로부터회수된신선수정란이었다. 1차발정동기화와수정란이식에서시험 1구의수정란이식율과수태율은각각 50.0% 와 10.0% 였으며, 시험 2구에서는각각 55.0% 와 18.2% 였다. 2차발정동기화와수정란이식에서시험 1 구의수정란이식율과수태율은각각 50.0% 와 16.7% 였으며, 시험 2구에서는각각 54.5% 와 83.3% 였다. 따라서한우수란우에총체보리사일리지급여는건초급여에비하여수정란이식율에는영향이없으나수태율은영향을주는것으로사료된다. Key words: 청보리사일리지, 한우, 수정란이식, 수태율 - 48 -

KSET-13 Studies on the Relationship between Number of Repeat and Duration of Estrous Behavior in Hanwoo and Holstein Cattle Jun Kyu Son, Seong Jai Park, Kwang Soo Baek, You Lim Choi, Myeung Sik Lee 1, Sang Bum Kim, Hyeon Shup Kim, Seung Oh Shin, Choon Keun Park 2, Soo Bong Park 3 Dairy Science Division, National Institute of Animal Science, RDA, Cheonan 330 801, Korea 1 Hanwoo Experiment Station, National Institute of Animal Science, RDA 2 Department of Animal Biotechnology, College of Animal Life Sciences, Kangwon National University, Chuncheon 200 701, Korea 3 Planning & Coordination Division, National Institute of Animal Science, RDA The objective of this study was to investigate the relationship between behavioral signs and duration of estrous in Hanwoo and Holstein cattle for the prediction of optical insemination. Fifty two of 58 and 71 of 89 in Hanwoo and Holstein cows or heifers showed estrous exhibition within 72 h after 2 days following PGF 2α administration, respectively. The number of mounting and standing heat were 54.2 and 57.6, 59.4 and 53.5, 42.0 and 30.8 and 16.2 and 10.7 times in Hanwoo cows, Hanwoo heifers, Holstein cows and Holstein heifers, respectively. Duration of mounting were no significantly difference for Hanwoo cows (21.2± 11.3), Hanwoo heifers (19.9±11.4), Holstein cows (8.7±4.4) and Holstein heifers (16.9±8.0). Duration of standing heat were significantly (p<0.05) shorter for Holstein cows (5.4±3.4) than for Hanwoo cows (17.1±9.6), Hanwoo heifers (16.5±6.3) and Holstein heifers (15.0±7.2). Time until mounting after injection of PGF 2α were significantly (p<0.05) longer for Holstein cows (56.3±11.45) than for Hanwoo cows (42.71±10.44), Hanwoo heifers (36.6±8.21) and Holstein heifers (40.70±6.15). Time until standing heat after injection of PGF 2α were significantly (p<0.05) longer for Holstein cows (61.6±8.92) than for Hanwoo cows (46.2±11.49), Hanwoo heifers (42.7±6.06) and Holstein heifers (44.1±6.72). In the results of this study, duration of standing heat was the shortest in Holstein cows (5.4±3.4). The estimation of estrous with Holstein cows has more difficulty because to significantly shorter duration of standing heat than for Hanwoo cows, Hanwoo heifers and Holstein - 49 -

heifers. The standing heat can be a good predictor for time of ovulation but it is concluded that mounting behavior could be the best predictor for time of ovulation. Key words: Holstein cows, Hanwoo, Estrous behavior, PGF 2α - 50 -

KSET-14 Proteomic Approaches to Defining Sperm Fertility in Procine Shin Ae Oh 1, Young Ah You 1, Chan Lee 2, Kang Duk Choi 3, Myung Geol Pang 1 1 Department of Animal Science and Technology and BET Research Institute, Chung Ang University, Ansung, Gyeonggi Do 456 756, South Korea 2 Department of Food Science & Technology and BET Research Institute, Chung Ang University, Ansung, Gyunggi Do 456 756, South Korea 3 School of Biotechnology, Hankyong National University, Ansung, Gyunggi Do 456 749, South Korea To identify the proteins involved in the relation to sperm fertility, we employed a comprehensive proteomic analysis of boar sperm from small and large litter size. Protein expression levels of sperm from two group were compared using SigmaGel software. Based on the analysis, we excised from the gels a total of 357±21.37 and 328±17.03 protein spots in small and large litter size group spermatozoa, respectively. Because the standard errors of protein spot numbers for each group were not high, we regard that all the possible variations have been minimized. Total 102 protein spots were consistently observed in each sample, and these 102 spots all ranging from a molecular mass of 6.5~200 kda and a ph range of 3~10. Of these 102 spots, 25 spots showed differential expression in intensity between two group spermatozoa. In this comparison, the number of increased or decreased spots ( 4 fold mean of difference in intensity) was also detected. Two DE and subsequent LC/MS MS mass spectrometry analyses led to identification of 25 protein spots that appeared in every gel, and identified only 20 proteins. Of these 20 proteins as differentially expressed between two group. In this study, we found several proteins, however only mitochondrial trifunctional protein and one unknown protein showed increased expression and 18 proteins showed decreased expression in large litter size group. This study represents the first case where the sperm proteome map small litter size with particular condition have been compared a number of large litter size controls. This work was supported by a grant (Code # 20080401034056) from BioGreen 21 Program, Rural Development Administration, Republic of Korea. Key words: boar, sperm, fertility, protein, proteome - 51 -

KSET-15 MG132를이용한 Post Fusion 처리가돼지체세포핵이식난자의체외발육에미치는영향 유진영, 이주형, 김진영, 박준홍 1, 이은송 강원대학교수의학부대학, 서울대학교농업생명과학대학 체세포핵이식에사용되는난자의 maturation promoting factor(mpf) 수준은핵이식란의발육능에영향을미치는것으로알려져있다. 본연구에서는체세포핵이식과정에서공여핵과의전기융합후 MG132 처리에의한 MPF 수준의향상이핵이식란의 nuclear remodeling과체외발육에미치는영향을조사하였다. 돼지미성숙난자를 ecg/hcg가포함된 TCM 199 배양액에서 22시간배양한후호르몬이첨가되지않은배양액에서 17시간추가배양하여체외성숙을유도하였다. 체외성숙 39시간후 metaphase II 단계로성숙된난자를선별하여탈핵후미니돼지신생자돈에서채취한체세포를주입하였다. 재구축된난자는전기융합법 (2 DC pulses, 1.7 kv/cm, 25 μs) 을이용하여세포융합을유도하였다. 융합난자를각각 1 μm MG132과또는 0.5 mm vanadate가첨가된 TLH BSA 배양액에넣어 2시간동안처리하였다 (postfusion 처치 ). Post fusion 처치가끝난후전기자극 (2 DC pulses, 1.2 kv/cm, 60 μs) 으로난자의활성화를유도하였다. 활성화처리난자를 0.4 μg/ml demecolcine이첨가된 NCSU 23 배양액에서 4시간동안배양한후 NCSU 23 배양액으로옮겨 39, 5% CO 2, 5% O 2, 90% NO 2 의조건에서 7일간체외배양하였다. 활성화처치후 12시간에난자를고정, 1% aceto orcein으로염색하여핵상을관찰하였다. 체외배양 48시간및 7일째에각각분할및배반포로의발육을관찰하였으며, 배반포를 Hoechst 33342로염색하여형광현미경하에서세포수를조사하였다. 실험결과융합된난자를 1 μm MG132와 0.5 mm vanadate로처리한경우분할율은 71~78% 로처리군간에유의적인차이를보이지않았다. 배반포발육률은 1 μm MG132 처치군이 21% 로대조군의 11% 에비해유의적으로높은수준을보였으며 (p<0.05), vanadate 단독처리군과 vanadate 처리후 MG132로처리한군은각각 6% 와 4% 의발육률을보여대조군보다낮은경향을보였다. 배반포의세포수는대조군 (31 cell) 과 MG132처리군 (32 cell) 에비해 vanadate 처리군 (24 cell/ 배반포 ) 에서감소하는경향을보였다. 전핵형성률은대조군, MG132 처리군및 vanadate 처리군에서각각 100%, 97% 및 96% 를보여융합난자에서 MPF 수준은핵이식란의전핵형성에영향을주지않았다. 이상의결과로보아공여세포가융합된난자를높은 MPF 수준유지를위하여 MG132로처치하는것은돼지체세포핵이식란의체외발육능을증가시키는것으로 - 52 -

사료된다. This work was supported by a grant (# 20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea. Key words: 체세포핵이식, MG132, vanadate, 체외발육, 돼지 - 53 -

KSET-16 Isolation of Neural Progenitor Cells from Brain and Retina of Fetus of RFP Transgenic Cat Y. S. Lee 1, H. K. Klassen 3, J. Yang 3, X. F. Yu 1, I. K. Kong 1,2 1 Division of Applied Life Science (BK21) 2 Institute of Agriculture and Life Sciences, Graduate School of Gyeongsang National University, Jinju 660 701, S. Korea, 3 Department of Ophthalmology, School of Medicine, University of California, Irvine, CA, USA The recent demonstration that neural progenitor cells can be cultured from the mammalian brain and retina has provided new insights into the mechanisms of neural development, as well as powerful new strategies for transplantation based studies of neural regeneration. In the present study, The isolation of feline neural precursor cells followed a protocol starting with a 45 day timed pregnant Domestic short haired cat mated with a male of Turkish Angora RFP transgenic cat. The animal was placed under terminal anesthesia and the gravid uterus surgically exposed under aseptic conditions. After the embryos were removed, the RFP expression level of embryos were directly checked and the heads were collected directly that express RFP higher than the others. The brains and eyeballs were removed by dissection and the brains were broke with blade and retinas were separated from eyeballs. Neural tissue was enzymatically digested and the resulting cell suspension washed repeatedly, and brains for brain progenitor cells (BPC) and retina for retinal progenitor cells (RPC) were cultured individually at high density in fibronectin coated flasks containing Ultra Culture Medium (Lonza); EGF (20 ng/ml); bfgf (40 ng/ml); PDGF AB (20 ng/ml, Peprotech, Rocky Hill, NJ, USA); and antibiotics. FBS (5% by volume, Hyclone, Logan, UT, USA) was included overnight to promote adherence, and the medium completely changed to serum free medium the next day. Subsequently, cells were fed by medium exchange every 2 to 3 days and passaged at confluence using a Hank s based cell dissociation buffer (Gibco, a subsidiary of Invitrogen Corp., Carlsbad, CA, USA) and gentle trituration with a flame polished glass pipette. The isolated BPC and RPC were successfully survived and cultured at least 4-54 -

passage after at least 4 passage (day 15) post isolation showing high level of RFP expression. In conclusion, establishment of feline RFP neural progenitor cells would be useful in terms of functional transplantation studies, given the extensive literature on retinal and cortical electrophysiology in the cat, as well as the identified feline models of retinal dystrophy. Supported by KOSEF (#M10525010001 05N2501 00110). YS Lee were supported by the Post BK21 Program. Key words: neural progenitor cells, brain progenitor cells, retinal progenitor cells, isolation, establishment - 55 -

KSET-17 Methylene Blue를이용한간편정액진단키트와이를이용한돼지간편품질진단액상정액개발 이장희 1, 백순화 2, 지달영 1, 박달영 3, 김관국 4 1 바이오컬쳐 ( 주 ), 2 백석문화대학, 3 천안시농업기술센터, 4 충남농업기술원 본연구는 methylene blue(mb) 용액과정액을혼합하여혼합된정액의변화된색상에의하여육안적으로정액의품질 ( 활력및생존성 ) 을간편하게진단할수있는키트를개발하고자하였다. 채취된종모돈정액에대한품질평가는메틸렌블루환원시간 (MRT) 과색상변화에대한색상기준표를적용하였다. 간편품질평가용정액진단키트는그림 1과같이투명한정액스트로우내에부피비 (v/v) 로공기층 0.1~ 0.4, 희석액층 0.04~0.4, 공기층 0.01~0.04, 메틸렌블루용액층 0.01~0.04로흡입하고나머지부분은공기층으로구분하여위치시킨다음흡입되었던입구를밀봉하여정액품질검사키트를제작하였다. 정액진단키트의사용은입구를절단하고검사하고자하는정액시료 0.04~0.4를흡인한후밀봉하여흔들어서시료들을혼합시켜메틸렌블루환원반응을진행시킨후색상기준표와대비하여정액의품질을간단히평가하였다. 한편, 이와같은원리를응용하여정액스트로우내에부피비 (v/v) 로 Androhep 보존액으로희석된정액층 0.1~0.4, 공기층 0.01~0.04, 차단액층 0.01~0.06, 공기층 0.01~0.04 및구연산나트륨이첨가된 Methylene blue 용액층 0.01~0.04를구분하여차례로위치시고밀봉하여진단용샘플정액을제조하고이를그림 2와같이동일한대용량의액상정액하단에위치간편품질진단돼지액상정액을제조하였다 ( 특허출원번호 : 10 20080098136. 출원일 : 2008.10.07) 그림 1. 정액진단키트 ( 색상기준표별도 ) 그림 2. 간편품질진단돼지액상정액 - 56 -

종모돈 10두의정액을 10일간보존한정액 300샘플에대해서정액진단키트를이용하여색상기준표와대비한결과수정가능색상과는 100% 수준에서정확성을나타내었으며, 이를활용한간편품질진단돼지액상정액의경우에도같은결과를나타내었다. 본연구는농촌진흥청 2008 년농업기술센터연구개발지원사업으로수행되었음. Key words: methylene blue, 돼지, 활력, 색상기준표, 간편품질진단액상정액 - 57 -

KSET-18 Methylene Blue 를이용한정액의간편품질식별기술개발 이장희 1, 백순화 2, 지달영 1, 박달영 3, 김관국 4 1 바이오컬쳐 ( 주 ), 2 백석문화대학, 3 천안시농업기술센터, 4 충남농업기술원 본연구는 methylene blue(mb) 용액과정액을혼합하여혼합된정액의변화된색상에의하여육안적으로정액의품질 ( 활력및생존성 ) 을간편하게진단할수있는방법을개발하고자하였다. 종모돈 10두로부터채취된정액에대한품질을조사하기위하여메틸렌블루환원시간 (MRT) 과색상변화를칼라코드와비교확인하였다. 채취된정액은 3.6 g sodium citrate가첨가된 methylene blue(50 mg per 100 ml) 용액과희석하였으며, 희석배율은원정액의경우는 1:1, 희석된정액은최종 1:10~40의범위로하였다. 메틸렌블루용액과혼합된정액의색상에대해서칼라코드에의한색상기준표설정하고정액과 methylene blue 용액의혼합후 45 항온수조에침지하여나타나는색상을색상기준표와육안적으로비교하여간편하게정액의품질을평가하고활력을조사한결과는다음과같았다. 표 1. 정액과메틸렌블루용액과의혼합후정액품질판정색상기준표 칼라코드색상범위단계활력범위판정결과 1 단계 ( 혼합직후 ) 10% 이하수정불가 2 단계 10~30 수정불가 3 단계 31~50 의 심 4 단계 51~70 의 심 5 단계 71~90% 이하 수정가능 6 단계 90% 이상 수정가능 채취직후의 10두의원정액과 MB와의혼합후의 MRT는 3~6분이었으며, 평균 3.8분이었다. 채취된정액을 Androhep 희석액과 1:1 이상희석한후정자의농도는최소 5 x 10 7 spermatozoa/ml 조정하여 10일동안 17 정액보관고에보관하였을때보존기간별 MRT는 1일 ( 채취직후 ), 3일 (72시간후 ), 5일, 7일및 9일에각각 3.8, 3.9, 4.2, 4.8 및 6.5였다. 한편, 정액과 methylene blue 용액과의혼합후정액의품 - 58 -

질판정기준색상표는표 1과같았으며, 이와같은결과는메틸렌블루환원반응에의하여대부분의정액에대해서품질 ( 활력기준 ) 을간편하게추정할수있을것으로기대되었다. ( 특허출원번호 : 10 20080098136. 2008.10.07 출원 )) 본연구는농촌진흥청 2008 년농업기술센터연구개발지원사업으로수행되었음. Key words: methylene blue, 정액, 활력, 품질평가, 색상기준표 - 59 -

KSET-19 형태학적품질이낮은돼지미성숙난자의체외성숙능, 단위발생능및체세포핵이식후의체외발육능 이주형, 유진영, 김진영, 박준홍 1, 이은송 강원대학교수의학부대학, 1 서울대학교농업생명과학대학 체외성숙에공여되는미성숙난자로는일반적으로난구세포가치밀하고세포질이균질한난자가사용되며, 형태학적인등급이낮은많은수의난자가폐기된다. 본연구에서는형태학적등급이낮은돼지미성숙난자를체외성숙한후난자의크기에따른난자의성숙능및단위발생능을검토하였다. 미경산돈의난소의직경 3~8 mm 의난포로부터난자를채취한후난구세포가세층이상으로치밀하게부착되어있고난세포질이균질한난자 (A군) 와나머지난자 (B군) 를구분하여체외성숙에공여하였다. 체외성숙종료후난구세포를제거한다음 B난자의크기를육안적으로관찰하여상대적인크기에따라크기가작은난자 (BS군) 와큰난자 (BL군) 를 1:1의비율로구분하였다. 각군의난자 (A, BS, BL) 를대상으로제1극체가배출된난자만을선별하여본실험실의표준적인방법 (Journal of Embryo Transfer, 2007, 22: 235 243) 에준하여단위발생란및체세포핵이식란을작성하였다. 단위발생란과체세포핵이식란을 porcine zygote medium 3 배양액에넣어 39, 5% CO 2, 5% O 2, 90% NO 2 의조건에서 7일동안체외배양하였다. 체외배양 48시간에분할율을, 168시간후에배반포로의발육및세포수를조사하였다. 실험결과체외성숙난자의평균직경은 A 군과 BL군, BS군에서각각 115 μm, 118 μm, 112 μm로측정되었다. 난자의체외성숙률은 A, BL, BS군에서각각 92%, 92% 및 71% 로 BS군에서유의적으로낮은성숙능을보였다. 단위발생란의분할율은 A, BL, BS군에서각각 93%, 90% 및 73% 로 BS군에비해 A와 BL군에서유의적으로높게나타났으며, 배반포발육율은각각 41.5%, 44.8%, 34.2% 로 A군과 BL군이 BS군에비해유의적으로높은발육능을보였다. 배반포의평균세포수는 35~38개로유의적인차이가없었다. 체세포핵이식과정에서난자 세포의융합율은 72~78% 를보였으며, 핵이식란의분할율은 82~88% 고관찰되었다. 배반포발육률은 A, BL, BS군에서각각 22%, 19% 및 13% 로 A군과 BL군이 BS군에높은것으로나타났으며, 배반포의평균세포수는 32~40개로관찰되었다. 이상의결과로부터형태학적등급이낮은미성숙난자도체외성숙배양후난자의크기가클경우형태학적등급이높은난자와동등한체외발육능을보이는것으로생각된다. 따라서형태학적등급이낮은미성숙난자도체외성숙배양후난자의크기가클경우체외수정란또는체세포핵이식란의생산에난자공급원으로활용될수 - 60 -

있을것으로사료된다. This work was supported by a grant (# 20080401034072) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea. Key words: 체외성숙, 난자크기, 체세포핵이식, 돼지 - 61 -

KSET-20 장기불임우의수정란이식성적 정연길 1, 설현석 1, 백천규 1, 김상희 1, 배남철 1, 정경일 3, 김혜진 2, 권은정 2, 송해범 2 이티바이오텍 ( 주 ) 1, 현대가축인공수정소 3, 대구대학교동물자원학과 2 번식우를사육하는농가는대부분인공수정을실시하고있으나불임우가 10% 전후되어수태율이낮아번식우농가의경제적손실이매우큰실정이다. 인공수정으로수태가되지않는장기불임우의치료방안으로수정란이식이최근에활발하게연구되고있다. 본보고는현장의인공수정사및수의사와팀을이루어인공수정에서수태되지않는장기불임우를대상으로한우의체외수정란을수정란이식한결과를요약하였다. 현장의인공수정사와수의사가 4~6회의인공수정에서수태되지않는한우와젖소를장기불임우로판정하고, 장기불임인한우 15두와젖소 30두에한우의체외수정란을신선란과동결란을각각수정란이식하였고, 수정란이식한후 60일에직장검사법으로수태율을조사하였다. 표 1. 한우와젖소의장기불임우에한우체외수정란을수정란이식한결과 수란우 체외수정란 이식두수 수태두수 ( 수태율 ) 한우 동결란 15 9(60.0) 젖소 신선란 30 20(66.7) 표 1에서보는바와같이한우체외수정란중동결란과신선란을장기불임인한우와젖소에각각수정란이식한경우 9/15두 (60.0%) 와 20/30두 (66.7%) 가각각수태되어수태율이비교적좋았다. 일반적으로일정한주기에정확하게발정은오지만인공수정을 3회이상실시하여도수태가되지않는장기불임우는난관의이상, 항정자항체의존재등과특히젖소는유전적인비유능력의상승과사료급여의영양적인불합리등여러가지요인으로번식효율의저하를가져오지만수정란이식이장기불임우의치료방법으로효과가있는것으로생각되며, 특히젖소의경우에는장기불임우에대한한우체외수정란의수정란이식은번식효율이개선되고한우송아지의생산으로농가소득에도기여할것으로생각된다. Key words: 장기불임우, 체외수정란, 동결란, 신선란, 수정란이식 - 62 -

KSET-21 한우체외수정란의 Cytoplasm Color 가배발달에미치는영향 정우재, 조수진, 이영수, 권태현, 공일근 경상대학교농업생명과학대학응용생명과학부 Division of Applied Life Science (BK21) 본실험에서는도축장유래난소를이용하여채란된난자를이용하여난자의 Cytoplasm Color에따른배발달율을조사하였다. Cytoplasm Color에따라 Pale color (PC), Brown color (BC) 및 Dark color (DC) 3 Group으로나누어 10% FBS, 1 μg/ ml β estradiol, 10 μg/ml FSH, 0.6 mm Cystein, 0.2 mm Na Pyruvate가첨가된 TCM 199 배양액 (IVM medium) 에 38.5, 5% CO 2 incubator에서 22~24시간동안성숙시켰다. 성숙된난자는난구세포를깨끗이제거한후, Leica Application Suite 2.7.1 R1(build 1384) Program을이용하여각그룹의 Cytoplasm Intensity를측정하였고, Mean Red(124.2±3.8, 115.1±4.0, 111.2±1.6), Mean Green(127.9±3.7, 116.3±4.0, 111.2±1.4), Mean Blue(131.1±4.2, 122.8±4.4, 121.1±1.3) 값을통해각그룹간 oocyte color 를확인하였다. 또한각그룹간 Mitochondria의분포를조사하기위해 Mitochondrial Activity가가장높은성숙 22 h 난자를 Mitotracker Green staining을실시하였고, 동일한기기를사용하여 Mean Green Intensity 값을측정한결과, DC group 이다른그룹에비해상대적으로높은수치를나타내었다 (105.5±25.3, 137.5±30.8 vs. 170.1±31.2). 수정율은 DC group이다른그룹에비해높았으나 (53.5±23.4, 73.0±4.6 vs. 83.1±2.8), 배발달율은 PC group은다른그룹에비해낮았으나, BC와 DC group 은차이가없었다 (10.2±6.4, 18.1±4.0, 17.5±6.5). * Supported by KOSEF (#M10525010001 05N2501 00110). Key word : bovine, in vitro, embryo development, ooplasm intensity - 63 -

KSET-22 흑소정액의동결보존후생존성 조상래 1, 김현종 2, 최창용 1, 최선호 1, 손동수 1 1 농촌진흥청국립축산과학원, 2 농촌진흥청법무감사담당관실 한우의품종중흑소는현재보존의두수가적어멸종의위험을가지고있는실정이다, 그러므로한우품종들의정액종결보존은매우중대한사항이다. 따라서흑소의정액을이용한동결보존기술의확립은유전자원의보존뿐만아니라, 종의번식과복원력에도매우중요한기술이다. 현재의연구를흑소에서채취한신선정액을이용하여동결보존한후융해하였을때그생존성을조사하였다. 공시축은가축유전자원시험장보유흑우 1두를시험장내안전정액채취시설내에서채취하였으며, 정액동결방법은김등 (2008) 의방법에준하여실시하였다. 채취된정액을 37 온장고에넣어신속하게실험실로이동하였다. 이동후 BTS로희석하여 1,500 rpm으로 15분간원심분리후상층액을제거하고 Andromed로 1ml로희석한후 4 로콜드챔버에서 30분간격으로두배로희석하여최종정자농도가 1 10 8 개 /ml 농도로희석한후 4시간동안평형하였다. 평형후 0.5ml 스트로우에밀봉하고액체질소상단 5 cm 5분, 5 cm 10분, 10 cm 10분으로유지한후바로액체질소에침지하고동결보존후융해후활력과생존율을조사하였다. 정액의융해는공기중에약 10 초간융해한후 37 온수에서 20초간스트로우를침지하여융해하였다. 정액의활력과생존성의평가는 IX 70 현미경하에서정자의활력을평가하였으며, 정자의생존율은 MicroLux 현미경하에서 MARKER CHAMBER에서생존율을평가하였다. 실험의결과로서는정액을채취한후의생존율과활력은 70% 와 75% 로나타났으며, 동결융해후의각생존율은 40%, 20%, 20%, 그리고 35% 와활력은 65%, 60%, 60%, 그리고 65% 의활력을보였다. 이상의결과를살펴볼때정액의희석재로이용되는 Andromed를이용한흑소정액의동결보존후생존율은다소낮은경향을보였으며, 활력은 60% 의수준으로나타났다. 동결융해후의생존성을높일수있는방법과생존율향상을위한방안을더모색하고동결정액을이용한수정란의생산효율성에도관한연구가병행되어야할것으로보인다. 따라서 Andromed를이용한흑소정액의동겨보존에서정액의봉입정자수를좀더높이고생물제재를사용하지않고서도장기간유전자원으로서보존밀활용이가능할것으로사료된다. Key words: 흑소, Andromed, 정액동결, 융해, 보존, 생존성 - 64 -

KSET-23 한우체내 외수정란의동결융해후의생존성 조상래 1, 김현종 2, 최창용 1, 최선호 1, 손동수 1 1 농촌진흥청국립축산과학원, 2 농촌진흥청법무감사담당관실 소수정란을효과적으로이용하기위해서수정란의동결보존의필요성은대두되어왔다. 그러나수정란을동결하여융해하였을때수정란이다시회생할수있는비율이낮아송아지까지생산되기까지는다양한원인이존재한다고알려져있다한우의우수한유전능력을지닌개체와멸실위험에속한종들과토산종들의유전자원보존측면에서생식세포동결보존은중요한미래자산이다. 따라서이러한개체들을복원에이용될때까지생식세포들의보다안전한방법으로보존하기위한방법들이연구되어오고있다. 생식세포의유전자원보존방법으로서는주로초자화및완만동결을주로이용하게된다. 동결방법간의장, 단점이있으나본연구에서는완만동결법을이용한소의미성숙난포란을동결 융해후생존성을조사하여유전자원보존방법의효율성을조사하고자연구를실시하였다. 체외수정란을이용하기위해서소의난포란은도축난소를이용하였다. 난포란의체외성숙을위해서는 TCM 199(Sigma) 배양액에 FSH(10 μg/ml), LH(10 μg/ml) 와 5% FBS(Gibco) 를첨가하여 5% CO 2 인큐베이터에서 22시간배양후체외수정에공시하였다. 체외수정은 BO 배양액에서 6시간동안체외수정을실시하였으며, 체외배양은 serum free 배양액에서배양하여 7, 8일째까지발달된수정란을이용하였다. 체내수정란생산은손등 (2007) 의방법에준하여실시하였다. 수정란의동결에사용된동결보호제조성은 1.8 M Ethylene Glycol(EG) + 0.05 M Trehalose + 10% FBS가첨가된동결보호제에서동결을실시하였다. 수정란동결방법은프로그램화된동결기 (CL 863) 를이용하였다. 동결기챔버에넣기전동결보호제에서약 20분간평형시킨후 0.25 ml 동결스트로우에장착하여챔버에옮겨넣었다. 동결속도는 0.3 / 분으로 -35 까지실시하여, -35 에도달후약 3분이경과한다음액체질소에침지하여동결보존하였다. 동결후수정란의평가와생존율확인을위해서융해는공기중에서약 10초, 37 온수에서 20초간융해후 HEPES TCM 199 배양액에서수정란을 3회세척한다음체외배양액으로옮겨체외배양을약 12시간이상배양한후수정란의재확장및부화된수정란을생존성이있는것으로평가하였다. 체내수정란의융해후생존율은 80%, 체외수정란은 77.8% 로나타났다. 체내수정란이체외수정란보다는다소높은생존율을보였다. 그리고체내수정란을동결융해후생존한수정란을수란우 5두에이식하였을때약 60일후직장검사법에의하여 3마리에서임신을확인하여 60% 의수태율을확인하였다. Key words: 한우난포란, 완만동결, 초자화동결, 체내 외수정란 - 65 -

KSET-24 한우체내 외수정란의발육단계별산소소비량변화 최창용, 조상래, 김현종, 김재범, 손동수 농촌진흥청국립축산과학원 FTA 대응방안으로고능력한우증식을위한수정란이식기술의접목이요구되고있으며, 한우의능력개량및브랜드추진을위한우수한우수정란의수요가급증하고있다. 그러나이식가능체내수정란회수율, 수정란이식수태율등과같은국내소수정란이식기술성적이선진국에비하여저조한실정이다. 수정란의질을판단하는등급판정의기준은국제수정란이식학회 (IETS) 의형태학적인등급판정에의하여이루어지고있으나, 그판정이검사자에따라주관적으로이루어질수있다. 수정란이배반포까지발달하면서세포수가증가함에따라수정란의산소소비량도증가하게되므로수정란의산소소비량을측정할수있다면과학적이고, 객관적인수정란의질을판정할수있을것이다. 따라서본연구는한우체내 외수정란의발달단계에따라수정란의산소소비량을측정함으로써수정란의질을객관적으로판정할수있는방법을강구하고자수행하였다. 도축장에서회수한난자를이용한한우체외수정란과발정동기화및과배란처치후회수한체내수정란을수정란호흡장치 (FHK, HV 405, Japan) 를이용하여수정란의발육단계별산소소비량 (10 14 /mol s -1 ) 과한우동결수정란의융해후산소소비량을측정하였다. 도축장회수난소에서채취한난자의산소소비량은채란직후난자와체외성숙후난자에서각각 2.3±0.3과 3.1±0.7을나타내어체외성숙된난자가미성숙난자에비해산소소비량이증가하였다. 체외성숙된난자를체외수정한후발육단계별산소소비량을측정한결과 4세포, 8세포, 상실배, 배반포, 확장배반포및부화배반포에서각각 1.9±0.3, 1.2±0.2, 2.4± 0.3, 5.4±1.6, 5.2±0.7 및 7.6±1.6을나타내었는데, 초기배수정란은난자의산소소비량과별차이를나타내지않았으나배반포수정란의경우산소소비량이급격하게상승하는것을확인하였다. 배반포와확장배반포를이용하여한우체내수정란과체외수정란의산소소비량을측정한결과, 배반포에서체내수정란 4.3±1.0, 체외수정란 5.4±1.6을나타내고, 확장배반포에서체내수정란 5.5±0.5, 체외수정란 5.2±0.7을나타내어체내수정란의산소소비량이더높을것이라는기대와달리유의적인차이를나타내지는않았다. 한우체외수정란의동결 융해후산소소비량을측정한결과융해후생존한수정란은 4.7±0.3을나타낸반면퇴축된수정란은 1.0±0.2를나타내어퇴축수정란은산소소비량도낮아지는것을수치상으로확인하였다. 수정란이살아있고, 세포수가많다면산소소비량도증가할것이라는추측을객관적인수치로확 - 66 -

인하게되었는데, 이를바탕으로좀더지속적이고, 폭넓게연구를수행하여등급이좋은수정란을이식한다면수태율을향상시킴으로써한우사육농가의경제성을높일수있는좋은활용방안이될수있을것으로사료된다. Key words: 한우, 수정란, 산소소비량, 수정란호흡장치, 수태율 - 67 -

KSET-25 Vitrification of Canine Oocytes Y. Abe 1, D. S. Lee 1, H. Suzuki 1 and S. K. Kim * College of Veterinary Medicine, Chungnam National University, Daejeon 305 764, South Korea 1 Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan 702 701 Korea The objective of the present study was to compare the vitrification method for cryopreservation of canine oocytes. Canine cumulus oocytes complexes (COCs) were collected from ovaries, and were vitrified by ethylene glycol based (E30S) or DMSO based (DAP213) methods. In the E30S method, COCs were exposed to the vitrification solution, composed of 30% ethylene glycol and 0.5 M sucrose, strep wise transferred onto a cryotop holder, then plunged directly into liquid nitrogen. In the DAP213 method, COCs were exposed to 1.0 M DMSO and DAP 213 solution in a cryotube, and thereafter plunged directly into liquid nitrogen. Although vitrified warmed COCs in the E30S method showed fewer morphological abnormalities, and higher viability than those in the DAP213 method, there was no significant difference in between. The recovery rates of COCs in the DAP213 and E30S groups were 97.5% and 92.7%, respectively. In both the vitrification methods, about 60% of the vitrified warmed oocytes showed normal morphology. However, there was a tendency for the viability of oocytes, as assessed by P1 stain, in the step wise group vitrified with E30S to be higher than those in the DAP213 group (17.6% and 5.1%, respectively, p>0.05). These results indicate that either method of vitrification is available and statistically comparable for cryopreservation of canine oocytes. Key words: canine oocytes, vitrification, cryopreservation * (J, Mamm. Ova. Res., 25:32 36) - 68 -

KSET-26 Identification of Differentially Expressed Gene in Corpus Luteal Stage using Random Amplified Polymorphism DNA(RAPD-PCR) D. W. Jung 1, S. H. Kim 1, H. J. Lee 2, J. T. Yoon 3, K. S. Min 1, S. Y. Hwang 1 1 Major in the Animal Biotechnology The Graduate School of Biology & Information Technology Hankyong National University. 2 Research Center of Genetic Engineering, Hankyong National University. 3 Department of Animal Life Research, Hankyong National University. This study was conducted to investigate the specific expression gene in the corpus luteal stage. Corpus luteal is an internal secretion organ with one phase of estrus cycle. So, an endocrinology mechanism is studied most, but it isnʹt still enough research of a estrus cycle mechanism. Corpus luteal type in the estrus cycle analysed the polymorphism of mrna by RAPD. Analysis samples were used the corpus luteal of non pregnancy bovine. This samples were classified with five stage(ch2, CH3, CL3, CL2, CL1). The result of RAPD analysis which used 100 type random primer in the CH2, CL2 polymorphism, fragment was found two types(pr11 and pr88) specific genes expressing in corpus luteal CH2 stage. Sequence analysis of these gene showed 99% homology to a previously reported ESTs from a bovine. To analysis mrna expression of these gene, SQ RT PCR and real time PCR was conducted. This gene was expressed in the whole corpus luteal stage. And analysis result of expressed pattern by real time PCR was the most highly expressed in CH2 stage and CL3 stage, CH3 stage was lowly expression and progressively decreased from stage CL2 to CL1. Therefore, this gene was thought highly expressed in cellulosity stage or Cellular change stage. Key words: corpus luteum, RAPD - 69 -

KSET-27 Study on In Vitro Maturation Improvement of Porcine Oocytes Y. H. Kim, M. H. Rhee 1, S. K. Kim 1 College of Veterinary Medicine, Chungnam National University, Daejeon 305 764, South Korea College of Veterinary Medicine, Kyungpook National University, Daegu 702 701 Korea This study was carried out to investigate the effects of the supplementation of glutamine, glucosamine and glutathione on the porcine oocytes on IVM rates. Cocs were incubated in NCSU 23 supplemented with at 2.0~10.0 mm glucosamine, 0.5~4.0 mm glutamine and 0.1~1.0 mm glutathione for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered with mineral oil and cultured in a CO 2 incubator (38, 5% CO 2, 95% air). The IVM rates of oocytes cultured in NCSU 23 supplemented with 0.5, 1.0, 2.0 and 4.0 mm glutamine for 48 hrs were 46.0±4.5%, 52.0±4.8%, 50.0±4.2% and 44.0±4.5%, respectively. The IVM rates of oocytes cultured in NCSU 23 supplement with 2.0, 5.0, 7.0, 10.0 mm glucosamine for 48 hrs were 44.0±4.5%, 42.0±4.5%, 38.0±4.6% and 24.0±4.8%, respectively. The IVM rates of oocytes cultured in NCSU 23 supplemented with glucosamine were no significantly increased compare to the control (42.5±4.0%). The IVM rate of oocytes cultured in NCSU 23 supplement with 3.0, 5.0, 7.0, 10.0 mm glutathione for 48 hrs were 40.0±3.2%, 54.0±4.2%, 48.0±4.5%, 44.0±4.8%, respectively. The IVM rate of oocytes cultured in NCSU 23 supplement with glutamine and glutathione were significantly increased compared to those control (42.5±4.0%). Glucosamine did not affect the IVM rates of oocytes. IVM rates of oocytes cultured in NCSU 23 medium for 48 hrs were significantly increased compared to the cultured for 40 hrs. Key words: porcine oocytes, glucosamine, glutamine, glutathione, IVM rate - 70 -

KSET-28 In Vitro Development of Vitrified Immature Porcine Oocytes Following ICSI B. K. Lee, M. H. Lee 1, S. K. Kim College of Veterinary Medicine, Chungnam National University, Daejeon 305 764, South Korea 1 College of Veterinary Medicine, Kyungpook National University, Daegu 702 701 Korea In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrified thawed porcine oocytes were examined. Also, the developmental capacity of vitrified thawed immature porcine oocytes following ICSI was investigated. Oocytes were cultured in NCSU 23 medium supplemented with 5% FBS at 38 in 5% CO 2 and air. The in vitro maturation rate of vitrifiedthawed oocytes (24.1±2.5%) was lower than that of the control (46.0±3.2%, p<0.05). The in vitro maturation rate of vitrified thawed oocytes treated with 1.0~5.0 ug CB+NCSU 23 medium were 22.2±3.0%, 30.7±3.2%, 46.3±3.1%, 38.5±3.2%, respectively. The in vitro maturation rate (46.3±3.4%) of the vitrified thawed oocytes treated with 3.0 μg CB for 30 min was the highest of all vitrification groups, When the in vitro developmental rates of the vitrified thawed (with EDS and EDT) oocytes following ICSI were 18.5±2.5%, 16.4±2.1%, respectively. This results were lower than the control group (24.0±2.5%). Key words: porcine oocytes, vitrification, cytoclacin, in vitro developmental rates - 71 -

KSET-29 EFfects of Pre-Treating In Vitro Matured Bovine Oocytes with Taxol as a Cytoskeletal Stabilizer and Either Sucrose or Trehalose as a Cryoprotectant on Spindle Morphology and Embryonic Development after Cryopreservation S. H. Park, J. H. Lee, I. S. kim, M. J. You, J. H. Park, J. K. Kwon, K. C. Lee, J. H. Kim, I. J. Yu College of Veterinary Medicine, Chonbuk National University, Jeonju, Korea The purpose of this study was to determine the efficacy of pre treating in vitro matured bovine oocytes with Taxol as a cytoskeletal stabilizer and either sucrose or trehalose as a cryoprotectant on spindle morphology and embryonic development after cryopreservation. Bovine oocytes were matured in TCM 199 supplemented with 10% Fetal Bovine Serum (FBS), 5 ng/ml Epidermal growth factor (EGF), 0.01IU/ml luteinizing hormone (LH), and 1ug/ml estradiol for 22h in 39, 5% CO 2. We prepared the vitrification solutions as followings: TCM 199 HEPES with 20% FBS was used as basic medium of vitrification solution (BM). Vitrification solution 1 (VS1) was composed of 1.6M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM. Vitrification solution 2 was composed of 0.5M sucrose, 3.2M EG and 2.6M DMSO (VS2S) or 0.5M trehalose, 3.2M EG and 2.6M DMSO (VS2T) in BM. We set six experimental groups: oocyte with cumulus (C1), oocyte without cumulus (C2), Taxol+sucrose(TS), Taxol+trehalose (TT), sucrose(s), and trehalose (T). Before oocytes cryopreservation, oocytes were exposed to maturation medium supplemented with 1uM Taxol for 15min in 39, 5% CO 2. Oocytes were equlibrated in TCM 199 HEPES with 20% FBS for 10~15min, exposed to VS1 for 20sec, and continued to expose to VS2S or VS2T for 30sec. Oocytes were frozen by solid surface vitrification (SSV). Vitrified oocytes were stored in cryotubes in LN 2 for 1 day~2 weeks. Vitrified oocytes were thawed by transferring them into TCM 199 HEPES supplemented with 0.5M, 0.25M, and 0.1M sucrose or 0.5M, 0.25M, and 0.1M trehalose at 38, respectively. TT, T, and S groups showed the higher normalities of spindle than that of TS (47%, 50%, 49% vs. 28%). Blastocyst rates and cleavage rates of T, S, TT, and TS groups were lower than those - 72 -

of controls. In conclusion, Taxol pre treatment before cryopreservation was not effective on normalities of spindle chromosomes and embryonic development. However, trehalose might be replaced as a cryoprotectant for bovine oocytes matured in vitro. Key words: taxol, sucrose, trehalose, meiotic spindle, embryonic development - 73 -

KSET-30 The Effects of Dibutyryl camp on Meiotic Maturation and Developmental Competence of In Vitro Matured or Parthenogenically Activated Porcine Oocytes S. H. Park, J. H. Lee, I. S. kim, M. J. You, J. H. Park, J. K. Kwon, K. C. Lee, J. H. Kim, I. J. Yu College of Veterinary Medicine, Chonbuk National University, Jeonju, Korea The effects of dibutyryl camp(dbcamp) on maturation and developmental competence of in vitro matured or parthenogenically activated porcine oocytes were determined. To determine the optimal concentration of dbcamp to arrest and resume meiosis of porcine oocytes, porcine cumulus oocyte complexes (COCs) were cultured in North Carolina State University (NCSU) 23 supplemented with different concentration of dbcamp, 0.5 mm, 1mM, 5mM, 10mM for 22h and additional 22h after removal of dbcamp at 39, 5% CO 2. To determine the optimal culture time in dbcamp, porcine COCs were cultured in 0.5 mm dbcamp for different incubation time, 17h, 22h, 27h, and 42h, respectively and additional 22 h. The nuclear status was examined according to different concentrations at 22 h and 44h, and different incubation times. Oocytes were cultured for 22 h with 0.5 mm dbcamp and additional 22 h without dbcamp to assess oocyte developmental potential after in vitro fertilization(ivf) or parthenogenetic activation(pa). The optimal concentration for meiosis arrest and resumption of oocytes was 0.5 mm and 1 mm. The optimal culture time for meiosis arrest of oocytes was between 17 h and 22 h. Oocytes cultured from 39h to 49h showed more than 80% meiotic resumption. The rate of 2 pronuclei in dbcamp group was lower (p<0.05). However, there were no significant differences of cleavage rate and blastocyst rate among IVF treated with dbcamp group, IVF non treated group, PA treated with dbcamp, and PA non treated group. In conclusion, dbcamp was effective on meiotic arrest of porcine oocytes. However, it did not improve embryonic development in IVF and PA. Key words: porcine oocytes, meiotic arrest, dbcamp, embryonic development, parthenogenic activation - 74 -

KSET-31 Toxicity Test of Sucrose and Trehalose as a Cryoprotectant in Immature Bovine Oocytes S. H. Park, J. H. Lee, I. S. kim, M. J. You, J. H. Park, J. K. Kwon, J. H. Kim, I. J. Yu College of Veterinary Medicine, Chonbuk National University, Jeonju, Korea The purpose of this study was to examine the toxicity of sucrose or trehalose as a cryoprotectant in immature bovine oocytes. We prepared cryoprotectant solutions as followings: TCM HEPES with 10% Fetal Bovine Serum (FBS) was used as a basic solution (BS). Cryoprotectant solution (CS)1 was composed of 0.1 M sucrose (S) and 1.6M ethylene glycol (EG) or 0.1M trehalose (T) and 1.6M EG in BS. CS2 was composed of 0.25M S and 3.2M EG or 0.25M T and 3.2M EG in BS. We set three experimental groups: oocytes non exposed to CS(C), oocytes exposed to sucrose (S), and oocytes exposed to trehalose (T). Immature bovine oocytes were exposed to 0.1 M S and 1.6M EG or 0.1M T and 1.6 EG for 3min and then 0.25M S and 3.2M EG or 0.25M T and 3.2M EG for 1min. Treated oocytes were warmed by transferring them into TCM199 HEPES supplemented with 0.25M sucrose or trehalose for 5min and then 0.1M S or T for 5min at 3 8. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor (EGF), 0.01IU/ml luteinizing hormone (LH), and 1ug/ml estradiol for 24h in 39, 5% CO 2. Nuclear maturation was assessed by stainning oocytes with 1% aceto orcein. MⅡ rates were not different among groups (S: 88.8% vs. T:87.9% vs. C:86.1%). Oocytes were fertilized with bovine spermatozoa of 6 10 6 /ml. The cleavage rates, blastocyst rates, and cell numbers in blastocyst were assessed. The cleavage rate of trehalose group (73.3%) was higher than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was higher in trehalose (34.3%). However, there were no significant differences among experimental groups. The cells numbers in blastocyst were not different among experimental groups (C:84.9% vs. S:82.3% vs. T:84.2%). In conclusion, sucrose and trehalose were not toxic to use as a cryoprotectant in immature bovine oocytes. Key words: immature bovine oocytes, sucrose, trehalose, nuclear maturation, embryonic development - 75 -

KSET-32 Searching Examination of Apoptosis Mechanism by the Cell Block S. C. Son 2, S. H. Kim 1, D. W. Chung 1, H. J. Lee 3, S. Y. Hwang 1, K. S. Min 1, J. T. Yoon 3 1 Major in the Animal Biotechnology The Graduate School of Biology & Information Technology, Hankyong National University. 2 Department of Genomic Engineering, Hankyong National University. 3 Research Center of Genetic Engineering, Hankyong National University. This report was studied the expressed apoptosis genes (bovine BNIP 3, CASP 3, Bax, BCL 2 apoptosis family member) to clarify the expression of apoptosis regulatory mechanism of cell block. Oocytes were separated into five groups (experimental groups 1: Maturation Oocyte (MO), 2: 2~16 Cell Death (CD), 3: 4~16 cell block (CB), 4: death blastocyst (DB), 5: blastocyst (BL)) and cultured in TCM 199 and co culture by cumulus cell. RT PCR and real time PCR was conducted to analysis gene expression of BNIP 3, CASP 3, Bax, BCL 2 gene. The expression of BAX gene was significantly higher pattern in cell block group than those of any other groups, but other genes were low pattern. Expression of Bcl2 gene was significantly higher pattern in death blastocyst group than those of any other groups. Expression of BNIP 3 gene was significantly higher pattern in cell death group than those of any other groups. Expression of Casp3 gene was significantly high pattern in death blastocyst group than those of any other groups. Cell block was no significant effect on the expressions of BCL2, Casp3 and BNIP3 mrna. Transcripts level of apoptotic gene BAX was also significantly high in the cell block. As a result the cell block was not extinction by CASP3 developed by BNIP3. These results suggest cell block was observed development suspension phenomenon by high expression of BAX. Key words: apoptosis, Casp 3, BNIP 3, BAX, BCL2, cell block - 76 -

KSDB-33 Cell permeable Peptides Fused with EGFP Offers an Efficient Delivery Method to Achieve Short term Regulation of Gene Expression in Mammalian Dells Sojung Kwon, Hyunjung Lim Dept. of Biomedical Science & Technology, RCTC, IBST, Konkuk University, 1 Hwayang dong, Kwangjin gu, Seoul 143 701 Korea A novel class of membrane translocation agents, cell permeable peptides (CPPs), have been tested and used for cellular delivery of various proteins. Some CPPs have been shown to be non cytotoxic and are also effective in vivo in delivering macromolecules. While these CPPs have endless potentials to be used as primary tools for ʺProtein Therapeuticsʺ, their effectiveness has not been tested in reproductive systems such as germ cells. Thus, we sought to find effective and usable CPP systems for oocytes and cells of reproductive organs. We chose 9 known CPP peptides and prepared CPP cargo(gfp) fusion proteins. We first tested efficacy of these CPP GFP fusion proteins in several cell lines including the AN3CA human uterine adenocarcinoma cells. Transduction into cells was assessed by the fluorescence signal in the cells compared to that of cells treated with GFP protein. We found that MPG is the most effective CPP GFP in various cell lines. TAT GFP has already been shown to effectively enter cells and this was again confirmed in our system. We tested if MPG GFP can enter the mouse oocytes. Our initial observation shows that MPG GFP may be effective in zona free oocytes, but this requires further experiments. Through the confirmation of the EGFP fluorescence signal in transduced cells, we showed that MPG is a highly efficient system to carry protein as a form of the CPP cargo fusion form. In further study, we will confirm whether the transduced molecules using MPG system can perform the expected biological functions correctly in the cell. This work was supported by the Korean Science and Engineering Foundation (KOSEF) grant funded from the Korean Government (MOST) (No. R01 2006 000 10501 0). Key words : cell permeable peptide(cpp), protein therapeutics, transduction - 77 -

KSDB-34 Bioceramic poly D,L lactic co glycolic Acid(PLGA) Scaffold 에접종한인간지방조직에서유래된중간엽줄기세포의골형성 김동윤 1, 강유미 1, 홍순갑 1, 도병록 2, 이준영 1 1 충북대학교자연과학대학생물과학부, 2 휴림바이오셀 Mesenchymal stem cells(mscs) 를이용한조직공학에서 3D 세포배양은필수적인요소이다. 생분해성 scaffold는생물학적안정성을가져야하기때문에 PLA, PLGA, gelatin, collagen, elastin과같은 biomaterials이 scaffold의물질로사용되며, 물리적 생리학적자극을통하여줄기세포의증식과분화를유도한다. 이에본실험에서는 bioceramic 을첨가하여만든다공성 poly D,L lactic co glycolic acid(plga) scaffold에의한인간지방조직에서유래된중간엽줄기세포 (human adipose tissue derived mesenchymal stem cells, ATMSCs) 를접종하여 ATMSCs의골형성과정에미치는영향을알아보고자수행하였다. ATMSCs의증식률은기본배양액군에서는지속적인세포증식을보인반면 OI배양액처리군은세포접종후 14일 (0.81±0.03) 까지활발한세포증식을보이다 21일 (1.05± 0.05) 이후세포의증식이둔화되는양상을보였다. OI배양액군의 ALP활성은세포배양 21일 (1.95±0.01) 까지꾸준한증가를보이다 28일 (1.72±0.01) 에는감소한반면기본배양액군은배양동안계속감소하는양상을보였다. OI배양액군의 ATMSCs 골형성에있어서 2D 배양환경은 14일이후세포군집이형성되어 nodule을형성하기시작하여배양시간에따라 nodule의크기가점점증가하였다. 3D 배양은 bioceramic PLGA scaffold와 PLGA scaffold를사용하였고 SEM을통한 scaffolds의표면에서 bioceramic에의해 pore size가작게형성되었으며, 세포부착에있어서대조군에비해 bioceramic이첨가된실험군에서다소높은부착율을나타냈다. 세포접종후배양 21 일에대조군 (2.18±0.09), 실험군 (2.50±0.11) scaffold 모두에서높은 alkaline phosphatase (ALP) 활성값을보였고, 28일이후대조군 (1.70±0.01) 과실험군 (1.77±0.01) 은급격한감소를나타냈으며, 대조군보다실험군에서높은 ALP 활성을보였다. 56일간생쥐의등쪽피하에이식한 scaffold내에합성된칼슘과인의함량은배양시간에따라증가하였고, 칼슘은 2배이상높은함량을나타냈다. Alizarin red염색을통한조직학적검사에서도대조군에비해실험군에서칼슘함량증가를확인하였다. 위의실험결과들을종합해볼때 ATMSCs의골형성능은 well plate보다 scaffold 가더효과적이며, scaffold 제작시 bioceramic의첨가는 scaffold의 pore size를감소시켜서표면적을증가시키므로세포의부착과증식을향상시키고, 골형성분화에따른광물화단계의 scaffold 내칼슘과인의함량을증가시켜 ATMSCs의골형성을촉진시키는효과적인물질로사료된다. Key words: ATMSCs, PLGA, ALP activity, osteogenic induction(oi), alizarin red - 78 -

KSDB-35 인간조혈모줄기세포의냉동보존에미치는항산화제의영향 김응배 1, 홍순갑 1, 도병록 2, 김경숙 3, 이준영 1 1 충북대학교생명과학부, 2 휴림바이오셀생명공학연구소, 3 코어스템 Reactive oxygen species(ros) 에의한산화적손상은냉동보존과정과체외배양과정중세포생존률감소의주된요인중하나이며, 특히줄기세포의경우냉동보존후쉽게분화하거나사멸하는경향이있음이잘알려져있다. 비타민 A, C, E등과같은항산화제는소난자의체외성숙과정동안발생되는산화적스트레스로부터난자를보호한다고보고되었으며, 생쥐배아줄기세포의체외배양시 paraquat의처리는세포의생존율감소와 ROS 생성을 2배로증가시켰고 ascorbic acid를처리함으로써세포의생존율과 ROS 생성도가대조군과유사한값으로회복되었다고보고되었다. 따라서본연구는체외배양된인간조혈모줄기세포의냉동보존시선별된항산화제를처리하여항산화제가줄기세포의생존및자동분화에미치는영향을조사하고자하였다. 항상화제를처리한조혈모줄기세포를냉동한뒤해동시킨후세포의생존률은 α tocopherol(10 300uM) 과 ascorbic acid(0.1 1mM) 처리군이대조군 (62.7±8.0%) 에비해높은생존율을보였고, 그중 150uM α tocopherol 처리군 (70.5±7.0%) 이가장높은생존률을보였다. LIVE/DEAD assay kit를사용하여세포막손상을측정한결과대조군및실험군모두에서세포막손상을확인할수없었다. 항산화제처리후냉동보존, 해동된줄기세포를배양하여자동분화율을측정하였으나자동분화율에있어서는모든실험군에서대조군 (10.1±1.6%) 과유의한차이를보이지않았고, 그중 150uM α tocopherol(7.3±2.6%) 처리군에서가장낮은자동분화율을나타내었다. 위의연구결과로보아항산화제는인간조혈모줄기세포냉동보존시생존율을향상시키며특히 α tocopherol은인간조혈모줄기세포의냉동보존과정동안효과적인항산화제로작용할것이라사료된다. Key words: ROS, cryopreservation, antioxidant, α tocopherol - 79 -

KSDB-36 인간조혈모줄기세포의냉동보존에미치는냉동보존제의영향 김응배, 홍순갑, 도병록, 김경숙, 이준영 충북대학교생명과학부, 휴림바이오셀생명공학연구소, 코어스템 냉동보존과정은탈수와결빙에의한세포막및세포내소기관의물리화학적손상을야기시킨다고알려져있으며, 특히냉동된줄기세포의경우해동후자동분화를일으키거나생존율저하, ECM 분포변화및세포막의미세구조변화를야기시킨다고보고되었다. 세포표면의단백질이나 ECM 기능이중요한줄기세포의경우각각의성체줄기세포에맞는다양한냉동보존제의개발이매우절실하다. 이에본실험에서는 membrane permeable additive와 non permeable additive가인간조혈모줄기세포의냉동보존에미치는영향을알아보고자실험을수행하였다. 기본냉동보존제 ( 대조군 ) 는 5%DMSO+20%FBS가함유된 DMEM L를사용하였고, 실험군은 permeable additive로 5 10% dimethyl sulfoxide(dmso), 5 10% ethylene glycol(eg), 5 10% prophanediol(proh) 와 non permeable additive인 50mM trehalose 를첨가하였다. 냉동보존제로냉동보존후해동된조혈모줄기세포의생존율은 10% DMSO(83.43±11.05%) 와 5% EG+5% DMSO(79.37±7.30%) 처리군과 trhalose가첨가된 10% DMSO(84.64±5.62%) 와 5% EG+5% PROH(85.14±10.16%) 처리군에서각각의대조군 (78.88±13.18%, 82.75±9.58%) 에비해높은생존율을보였다. 냉동된세포의해동후배양 28일동안세포증식율은 10% EG와 10% PROH 처리군을제외한나머지실험군에서대조군보다낮은증식율을나타냈다. ALP 활성 peak는배양 7일에나타났으며 10% PROH(27.20±3.89), 5% EG+5% DMSO(27.30±3.34), 5% EG+5% PROH(39.20±5.25), 5% DMSO+5% EG+5% PROH(30.80±5.04) 처리군은대조군 (16.20±3.21) 에비해현저히높은 ALP 활성값을보였다. 세포미세구조변화에서는 trehalose를첨가하지않은실험군에서전체적으로뚜렷한세포질확산을관찰할수있었으며, trehalose를첨가한 10% DMSO, 5% EG+5% DMSO 처리군에서대조군세포질확산과많은차이를보였다. 위의실험결과들로보아 10% DMSO는세포의미세구조에서대조군과다소차이를보였지만, 대조군을비롯하여다른실험군에비해높은생존율과증식율을보여인간조혈모줄기세포의적절한냉동보존제라사료되며, 10% DMSO 단독사용보다는 50mM trehalose를첨가함으로써더욱향상된줄기세포의생존율을보일것이라사료되다. Key words: cryopreservation, DMSO, trehalose - 80 -

KSDB-37 Expression Pattern and Cloning of Erf Gene in Ascidian, Halocynthia roretzi Jung Eun Kim, Gil Jung Kim Applied Marine Biotechnology and Engineering, Kangnung National University In ascidian embryos, FGF signaling induces notochord, mesenchyme, and briain formation. Although a conserved FGF/Ras/MAPK/Ets pathway is known to be involved in this signaling, the mechanisms of regulation of this signaling pathway are not well understood. The Ets family of transcription factors is characterized by a conserved DNA binding domain and involved in many developmental processes. Members are classified into at least six subfamilies, ETS, YAN, ELG, PEA3, TCF, and ERF by their structure and amino acid sequence similarity. In the present study, we have isolated Hr Erf, an ascidian homolog of vertebeate Erf, to elucidate the interaction of transcription factors involved in the FGF signaling pathway in ascidian embryos. The Hr Erf cdna encompassed 2154 nucleotides including 25 adenyl residues at the 3ʹ end and encoded a predicted polypeptide of 580 amino acids. The polypeptide contained Ets domain in the amino terminus of the protein. The overall degree of amino acid identity between the Hr Erf and vertebrate Erf genes was approximately 30%, and Ets DNA binding domain was highly conserved (82%). We determined the expression pattern of Hr Erf gene in the various adult tissues and embryos using QPCR. In adult animals, Hr Erf mrna was predominantly detected in the muscle and stomach, and at lower levels in ganglion, gill, gonad and hepatopancreas. During embryogenesis, Hr Erf mrna was detected from egg to early developmental stage embryos, whereas the transcripts levels were decreased from neurula stage. To further examine the spatial expression of Hr Erf mrna, we carried out whole mount in situ hybridization at various stages. Similar to the QPCR results, a significant amount of maternal transcripts of Hr Erf was present in the fertilized egg and early developing embryos. Hr Erf expression was gradually lost from the neurula stage. Zygotic expression of Hr Erf was first detected in most blastomeres of the 32 cell stage embryos. At gastrula stage, Hr Erf was specifically expressed in the precursors of brain and mesenchyme cells These results suggest that zygotic Hr Erf products involve in specification of mesenchyme and brain cells. It remains to be determined roles of the Hr Erf genes during ascidian embryogenesis. Key words: Erf, FGF signaling, Ets, ascidian - 81 -

KSDB-38 Spermatozoa Motility in Yellow Croaker Larimichthys Polyactis: Effects of Dilution Ratio, ph, Temperature and Cations Minh Hoang Le 1, Han Kyu Lim 2, Byung Hwa Min 2, Jung Uie Lee 2 Young Jin Chang 1 1 Dept. of Aquaculture, Pukyong National University, Busan 608 737, 2 Aquaculture Management Division, National Fisheries Research and Development Institute, Busan 619 705, Korea Spermatozoa motility is a prerequisite factor determining semen quality and fertilizing capacity in fish. The effects of environmental factors including ph, temperature and cations as well as the role of dilution ratio on spermatozoa motility parameters (SMPs) in yellow croaker Larimichthys polyactis were examined. Males of yellow croaker (22.5 24.2 cm of fish length and 126.3 132.2 g of fish weight) were kept together unexposed to females in a tank (2 m 3 ) supplying seawater with a water temperature of 17 21, salinity of 32 33 psu and 5 6 mgo 2 /L at a flow rate of 0.2 L/s. Semen was collected individually by serial waves of abdominal pressure and then kept on ice until analysis. SMPs (spermatozoa velocity, movable spermatozoa ratio and total duration of spermatozoa motility) were measured immediately after initiation of semen activation until 100% of spermatozoa were immotile. All experiments were performed in triplicate of three males at room temperature (20 22 ) using light microscopy under 200X magnification setting video camera and video timer to connect with video recorder and player. The best conditions for SMPs were ph 8.0, temperature 10 and dilution ratio 1:100. Spermatozoa were immotile in distilled water. Cationic factors can stimulate the initiation of spermatozoa activation. The maximum spermatozoa velocity, movable spermatozoa ratio and total duration of spermatozoa motility were observed in each solution containing 0.4 M NaCl, 0.4 M KCl, 0.2 M CaCl 2 and 0.2 M MgCl 2. The present study provides us with some basic knowledge about yellow croaker spermatozoa biosensitivity to cationic effects. A sensitivity of yellow croaker was observed after induction of activation of spermatozoa motility in solutions containing cations. Concentrations more than 0.4 M Na +, more than 0.4 M K +, more than 0.2 M Ca 2+ and more than 0.2 M Mg 2+ had negative effects on spermatozoa motility. Key words: larimichthys polyactis, yellow croaker, spermatozoa motility, dilution ratio, temperature, ph, cations - 82 -

KSDB-39 Effect of Dilution Ratio, Diluents and Temperature on Semen Storage in Yellow Croaker Larimichthys polyactis Minh Hoang Le 1, Han Kyu Lim 2, Byung Hwa Min 2, Mi Seon Park 2 Young Jin Chang 1 1 Dept. of Aquaculture, Pukyong National University, Busan 608 737 2 Aquaculture Management Division, National Fisheries Research and Development Institute, Busan 619 705, Korea Storage temperature is a major factor that effects the viability of fish gametes in vitro studies. The objective of the present study were to find the best condition for semen storage in yellow croaker Larimichthys polyactis. The effects of dilution ratio, diluents and temperature on semen storage in yellow croaker were estimated in terms of spermatozoa activity parameters: spermatozoa activity index (SAI), movable spermatozoa ratio (MSR) and spermatozoa velocity (SV). Males of yellow croaker (22.5 24.2 cm of fish length and 126.3 132.2 g of fish weight) were kept together unexposed to females in a tank (2 m 3 ) supplying seawater with a water temperature of 17 21, salinity of 32 33 psu and 5 6 mgo 2 /L at a flow rate of 0.2 L/s. Semen was collected individually by serial waves of abdominal pressure and then kept on ice until use. The dilution ratio experiment was estimated through out semen diluted with artificial seminal plasma (ASP) at a ratio of 1:1, 1:3 and 1:5 in refrigerator set to 0. In addition, semen was diluted at a ratio of 1:3 with ASP and marine fish Ringerʹs solution (MFRS) into 1.5 ml Eppendorf tubes and stored at 0, 2 and 4 in refrigerator. Spermatozoa activity parameters were tested in triplicate after semen storage and at interval of 2 or 3 days until 100% spermatozoa were immotile at room temperature (20 22 ) using light microscopy under 200X magnification setting video camera and video timer to connect with video recorder and player. The best condition for semen cold storage was dilution ratio of 1:3 in ASP at 0 and the stored spermatozoa sustained the active state for 14 days. This result provides us with basic knowledge about effects of dilution ratio, diluents and temperature on semen storage of yellow croaker. Condition for semen cold storage more than 0 temperature, more than 1:3 dilution ratio of semen to diluents and MFRS diluent significantly decreased semen activity parameters after semen cold storage in yellow croaker. Key words: Larimichthys polyactis, yellow croaker, dilution ratio, diluents, temperature, semen storage - 83 -

KSDB-40 Expression of Gangliosides during Neuronal Differentiation of Human Bone Marrow derived Mesenchymal Stem Cells In Vitro Jae sung Ryu, Jung woo Jin, Hyo jung Yang, Eun jung Jung, Chin hyoung Cho, Young kug Choo Division of Biological Science, Wonkwang University, Iksan, Jeonbuk, Korea The roles of glycosphingolipids, particularly gangliosides, have been studied for several years in neurons, glia, and many cell lines. These lipids are involved in a variety biological process, including neuritegenesis, synaptic function, neural repair, proliferation, and cell adhesion. In the nervous system, gangliosides have attracted particular attention as they occur at high levels and their levels change in a developmental regulated program, and have been shown that maintain of gangliosides biosynthesis improve neurite outgrowth. In this study, the relationship between gangliosides expression and neuronal cell development was investigated using an in vitro model of neuronal differentiation from human bone marrow derived mesenchymal stem cells (hmscs) that are able to differentiate into a range of specific cell types in vitro and in vivo. First, to verify the isolated population contained pure hmscs, we performed positive and negative characterization by fluorescence activated cell sorting (FACS) analysis, thus we identified pure hmscs. Next, hmscs were differentiated into neuronal cell with α MEM/10% FBS +10 ng/ml bfgf for 24h and transferred to neuronal induction media, containing α MEM/10% FBS/2% DMSO +200 um β hydroxyanisole (BHA) for 24h. Neuronal induced cells maintenance with DMEM/F 12 media containing 10ng/mL bfgf +10ng/mL EGF +25ng/mL NGF for 2 weeks. High performance thin layer chromatography (HPTLC) showed that gangliosides GM3, GM2 and GD1a expressed in differentiated into neuronal cells for 2 weeks, especially GD1a increased comparison to control cells. Immunofluorescence staining also agreed with the results of HPTLC assay. These differentially expressed gangliosides suggest that gangliosides may have specific functions in stem cells and during neuronal differentiation. Therefore, these results also suggest that regulation of gangliosides expression have used as the marker for differentiated neuronal cell from hmscs. This study was supported by a grant from the Korea Research Foundation (KRF 2007 C00560) and a Grant (M10619010001 08N1901 0010) from the Korea Science and Engineering Foundation. Key words: human mesenchymal stem cells (hmscs), gangliosides, neuronal differentiation - 84 -

KSDB-41 Expansion and Hatching of Blastoceol through Lipid Metabolites or Concanavalin A Ja Young Maeng, Mihee Oh, Yong Pil Cheon Department of Biology, Institute of Basic Sciences, Sungshin Womenʹs University, 249 1, Dongseondong 3ga, Seongbuk gu, Seoul 136 742, Korea Blastocoel formation and expansion are primitive qualification for implantation and embryonic differentiation. Blastocoel expansion is requirement for hatching in mouse blastocyst. In this study, we inspected the possible regulator of blastocyst expansion and hatching using concanavalin A (ConA), PGF 2 α, PGE 2 or ouabain. ConA stimulated intracellular free calcium releasing in embryo stage specific manner: ConA trigered the free calcium increasement in blastocyst but it could not induce the calcium increase in morula. Most of the blastocyste exposed to ConA developed to expanded stage embryo but did not progress to the hatching stage. Most of the expanded embryos were still stayed at the expanded stage until 144 hr post hcg injection. A known modulator of intracellular free calcium, calcium ionophore and prostaglandin F 2 α (PGF 2 α) also could stimulate expansion. Progression to the hatching stage was also significantly lower in those groups like with that ConA. However, ConA effects were removed by adding the PGs and the embryos regained the developmental potency. It resulted in hatching of ConA pretreated blastocyst with the same levels of control. Pretreatment of ConA and treatment of PGF 2 α induced the oscillation of the intracellular free calcium in PGF 2 α. Ouabain showed a tendency to delayed progression of blastocyst to the hatching stage but did not inhibit the expansion or hatching. These results means that calcium is a regulator of blastocoel expansion and hatching of the blastocyst rather than Na + /K + ATPase. It is suggested that expansion is essential indirect factor in hatching in mouse embryo. Key words: blastocoel, concanavalin A, PGE 2, PGF 2 α, expansion - 85 -

KSDB-42 멍게의세포주기제어인자클로닝과발현양상 박민영, 이호수, 김길중 강릉대학교해양생명응용공학과 동물의발생은세포분열, 세포분화및형태형성등의과정이규칙적이고조화롭게구성된현상이다. 동물의발생에서수정이후가장뚜렷한현상은활발한세포분열이다. 왕성한세포증식을통하여배아는우선세포수를늘리고점차적으로세포분화가일어나필요한종류의세포들이형성된다. 발생과정에서세포분열은질서있고조화로운발생을위하여발생의시간적기준과각세포의발생운명에따라조절되는것으로알려져있다. 즉, 세포분화를조절하는신호가세포분열에영향을미치는것으로해석되는데현재까지발생과정에서자세하게연구된예가드물다. 멍게의초기발생에서 FGF signaling에의하여척삭과간충직이유도된다. FGF signaling이억제되면척삭과간충직으로분화할세포는각각신경삭과근육세포가된다. 이때신경삭세포는척삭에비하여더많은세포분열을하고, 근육세포는간충직에비하여적은수의세포분열을한다. 이와같은결과는세포분화와세포분열이밀접하게연관되어있다는것을시사한다. 본연구에서는멍게의단순한초기발생과정을이용하여세포분화를조절하는 FGF signaling이어떠한방식으로세포분열에영향을미치는지세포주기제어인자에대한해석을중심으로조사하고자한다. 현재까지멍게 (Halocynthia roretzi) 에서 cyclin A, cyclin B, cyclin E, cdk2, cdk6 및 wee1 등의세포주기조절인자유전자를클로닝하였으며, cyclin D, cdk1 및 cdc25 등은클로닝중이다. 클로닝된유전자중에서 cdk2와 cyclin A의예상아미노산배열은척추동물의각상동체와 79% 와 52% 의높은상동성을나타냈다. 클로닝된유전자의전사수준발현을 whole mount in situ hybridization을통하여조사했다. 모든유전자에서 maternal transcripts의존재가확인되었다. 전체적으로낭배기의간충직전구세포에서 zygotic expression이시작되었고, wee1의경우는 16세포기부터신경배기까지 zygotic expression이뚜렷하게관찰되었다. 앞으로이들세포주기제어인자의발현양상이 FGF signaling에의하여영향을받는지, 세포주기제어인자의발현변화가세포분화에도영향을미치는지등을조사할예정이다. Key words: 세포분열, 세포분화, cdk, cyclin, wee1, FGF signaling, Halocynthia roretzi - 86 -

KSDB-43 광염성경골어류넙치 (Paralichthys olivaceus) Stanniocalcin 2(STC 2) 의조직별유전자발현 신지혜, 손영창 강릉대학교대학원해양응용생명공학과 스타니오칼신 (stanniocalcin 1, STC 1) 은어류의신장내에매몰된스타니우스소체에서최초로발견된이량체당단백질로서, 칼슘과인산염의항상성을유지시키는항과잉칼슘호르몬이다. 포유류는 STC 1과 paralog 유전자인 STC 2가주로심장, 근육, 비장, 간, 신장, 정소, 장등의조직에서검출되었다 (Ishibashi et al. 1998). STC 1 은자가분비 / 곁분비를통하여다양한조직에서광범위하게발현되어지며, 포유류의조골세포분화의촉진 (Yoshiko et al. 1999) 과지방세포분화에있어특이적발현양상을나타낸다고보고된바있다 (Serlachius et al. 2004). 현재까지 STC 2는난소에서스테로이드성호르몬의발현을조절한다는보고가있으나 STC 1에비해명확한생리적작용이밝혀진바없으며, 어류에서는현재제브라피쉬와복어에서만 STC 2 의유전자분석이이루어졌다 (Luo et al., 2005). 본연구에서는광염성어류인넙치의 cdna 유전자분석과다양한조직내 mrna의발현패턴을조사하였다. 넙치 STC 2 cdna는전체염기서열 1501 bp, 번역부위는 861 bp (286 aa) 인것을확인하였으며, 유전자염기의상동성은복어 (89.0%), 제브라피쉬 (77.0%), 인간 (57.7%) 과높은상동성을보였으나, 넙치의 STC 1(24.3%) 과는낮은상동성을나타냈다. RT(reverse transcription) PCR 분석을통하여넙치의다양한조직에서 STC 2는광범위한발현양상을보였으며, 이를바탕으로향후어류의 STC 2의생리학적기능과발현조절메카니즘을규명하고자한다. Key word: 넙치, 항과잉칼슘호르몬, cloning, RT PCR, stanniocalcin 2-87 -

KSDB-44 Expression of Formin Family of Proteins in Mouse Oocytes during Meiosis I Hyejin Shin, Sojung Kwon, Hyunjung Lim Dept. of Biomedical Science & Technology, RCTC, IBST, Konkuk University, 1 Hwayang dong, Kwangjin gu, Seoul 143 701 Korea To achieve an asymmetric cell division in the vertebrate oocyte, the chromosomespindle complex formed at the metaphase needs to be migrated to the egg cortex. This process is thought to involve complex interactions among cytoskeletons and associated proteins. While several proteins, such as formin 2 (Fmn2), are shown to be crucial for this process, the mechanism is still unclear. Formin(Fmn) proteins are conserved across evolution and contain characteristic formin homology (FH) domains. FH1 and FH2 domains possess the actin nucleation activity. mdia is a formin protein of the diaphanous subfamily. There is a growing body of evidence that mdia1 and related mdia2 are involved in the stabilization of microtubules via slowing microtubule depolymerization in certain cells. While the role for Fmn2 during meiosis is well documented, expression of mdia1 and mdia2 in the mouse oocytes is not known. In this study, we examined expression of these two formin proteins in the maturing mouse oocyte in vitro by immunofluorescence staining. We observed that both mdia1 and mdia2 exhibit dynamic expression patterns during meiosis I. For example, mdia2 is first localized inside the nuclear envelope as spots during the prophase I, and it is later localized in the spindle poles by 8~9 hr in culture. These results suggest that mdia proteins may be involved in regulating cytoskeleton directed cellular events during the oocyte maturation. Further studies are required to examine functional aspects of mdia proteins in the oocytes. This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080045). Key words: mdia1, mdia2, Fmn2, oocyte, meiosisⅠ - 88 -

KSDB-45 Sex related Expression of Thyroid Hormone Receptor α (TRα) and β (TRβ) in Black Porgy, Acanthopagrus schlegeli during the Process of Sex Change Myung In An, Kwang Wook An, Cheol Young Choi Division of Marine Environment & Bioscience, Korea Maritime University Thyroid hormones (THs) regulate growth, development, differentiation, metabolism and the maintenance of homeostasis in vertebrates. THs act by binding specific receptors, thyroid hormone receptor (TR) α and TRβ. TRs exist in teleost gonad cell nuclei and are known to relate with determination of the sex of teleost. To clarify the function of TRα and TRβ during sex change process from male to female, we examined the expression pattern of TRs in each gonadal developmental stages (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary and mature ovary), and to identify the effects of gonadotropin releasing hormone analog (GnRHa) and 17β estradiol (E 2 ), we examined the expression pattern of TRs in immature black porgy injected with GnRHa and E2. In this study, we isolated TRα and TRβ cdna using the Rapid amplification of cdna race techniques and then cloned into the pgem T Easy Vector. The colony formed by transformation was cultivated in DH5α competent cell. TRα (GenBank accession no. EF605273) and TRβ (EF605274) full length cdna contained 1251 and 1188 nucleotides, including an open reading frame that was predicted to encode a protein of 416 and 395 amino acids, respectively. We also measured the levels of TRα protein expression by western blot analysis during the process of sex change. During sex change process, TRα mrna expression was highest in mature ovary, and western blot analysis was the same as mrna expression. Similarly, TRβ was only expressed high in mature ovary. And in GnRHa treated fish, TRα levels were highest at 12 h post injection but TRβ levels decreased during the experimental period. In E2 treated fish, TRα levels were highest at 6 d post injection and TRβ were highest at 9 d post injection. Therefore, it is concluded that both of TRα and TRβ were involved in sex change of the protandrous black porgy. Key words: black porgy, sex change, TRα, TRβ - 89 -

KSDB-46 Induction of Estrogenic Activity in MCF 7 Cell by Silkwarm (Bombyx mori) Pupa Water Extracts Hyo Jung Yang 1, Jae Sung Ryu 1, Jung woo Jin 1, Ye Seul Na 2, Yong Il Park 2, Kyung Soo Nam 3, Young Kug Choo 1 1 Dept. of Biological Science, College of Natural Sciences, Wonkwang University, Iksan, Jeonbuk, Korea 2 Dept. of Biotechnology and The Biomaterial Engineering Research Center, The Catholic University of Korea, Bucheon, Gyeonggi do, Korea 3 Bogo F&D, 936 Jinmiparagon, 13 Yeouido dong, Yeongdeungpo gu, Seoul, Korea In the present study, we have shown was to investigate the effects of silkworm pupa water extracts on estrogen activity. Freeze dried silkworm pupa was extracted with distilled water (D.W.) for 6h at room temperature, and then these resource was freeze dried to filtration (0.45 μm) It was then dissolved in D.W. and further fractionated through Dowex 50W 8X (H + )column. The flow through and wash with D.W. were pooled together and freeze dried (Sample 1). The extracts were treatmented with 70% ethanol. After centrifuged the sample, the supernatant was freeze dried by evaporator for ethanol eliminating (Sample 2), or the pellet was freeze dried (Sample 3). These samples were examined for their effects on the growth of MCF 7 cells, human breast cancer cell line, by colorimetric 3 (4,5 dimethylthiazol) 2,5 diphenyltetrazolium bromide (MTT) assay. In addition, the estrogenic activity of these resources was investigated by competition binding assays with estrogen receptor α (ERα) and estrogen receptor β (ERβ). Among there sample, especially sample1 was exposed the highest binding to ERα (IC50 1.019 μg/ml) and ERβ (IC50 1.210 μg/ml). The viability of MCF 7 cells was regulated according to various sample and concentration. Interestingly, growth rates of MCF 7 cells were difference with samples that sample1 showed the significantly highest growth that increased about 2 fold(703.2%, at 62.5 μg/μl concentration), compared with control (450.37%). Taken together, this study suggests that the silkworm pupa water extracts may be useful as potential phytoestrogens for the climacteric. This study was supported by a grant (20080201 033 015 001 01 00) from the Korea Rural Development Administration. Key words: silkworm pupa water extract, estrogen, estrogen receptor, MCF 7 cells - 90 -

KSDB-47 Optimal Culture Condition to Maintain Motility and Viability Up to 5 Days in Normozoospermic Males Young Ah You, E. A. Mohamed, Shin Ae Oh, Myung Geol Pang Dept. of Animal Science & Technology, and BET Research Institute, Chung Ang University, Ansung, Gyunggi Do 456 756, South Korea The preservation of human sperm motility and viability in processed semen samples is important to transport of sperm to distant laboratories and to the advancement of assisted reproductive technologies, including intra uterine insemination (IUI), in vitro fertilization (IVF), intra cytoplasmic sperm injection (ICSI), and sperm cryopreservation. The aim of this study was to evaluate the effect of different temperature and media ph on sperm motility and viability for 5 days in processed specimens. Semen samples with normal sperm parameters, defined according to World Health Organization criteria, were used. Sperm motility were analyzed using computer assisted sperm analysis, viability were analyzed using hypoosmotic swelling test, and capacitation status were analyzed using chlorotetracyclin test. Ejaculated semen gently washed to remove seminal plasma in HEPES buffered Tyrodʹs Albumin Lactate Pyruvate medium (HTALP). Each 5ml of HTALP+0.3% albumin with a final sperm concentration of 1 10 6 /ml were incubated for 5 days in 3 different temperature (4, 22 and 37 ) combined with 3 different ph (ph 6.5, 7.5 and 8.5). The optimal culture condition to maintain sperm motility and viability was the sperm kept at room temperature (22 ) in ph 7.5 medium. This group sperm were best in capacitation status for IUI, IVF, and ICSI. In the results of this study, HTALP can be used a basic medium for culture and longevity preservation, and sperm kept at room temperature is beneficial. Key words: optimal sperm culture condition, longevity, preservation - 91 -

KSDB-48 Survival Competition between X and Y bearing Sperm Young Ah You, E. A. Mohamed, Shin Ae Oh, Myung Geol Pang Dept. of Animal Science & Technology, and BET Research Institute, Chung Ang University, Ansung, Gyunggi Do 456 746, Korea The studies on the viability of sperm and/or detection of chromosome constitution in human sperm have been carried out in various conditions by several researchers. We hypothesized that there is difference in the survival between X and Y bearing sperm. To assess these characteristics, it is necessary to measure both functional and cytogenetic parameter simultaneously in single spermatozoon. To assess the survival difference between X and Y bearing sperm, we performed the hypoosmotic swelling test (HOST) and three probe (chromosome 18 and sex chromosomes) three color fluorescence in situ hybridization (FISH) simultaneously which gave an insight into viability and cytogenetic characteristics together. The human sperm were cultured up to 5 days in 3 different temperature conditions (4, 22 and 37 ) combined with 3 different ph conditions (ph 6.5, 7.5 and 8.5). The survival of X and Y bearing sperm was significantly higher in culture at 22 in ph 7.5. The survival of X bearing sperm was significantly higher than that of Y bearing sperm with different significantly in all culture conditions. From the results of this study, it seems that the X bearing sperm survives longer than Y bearing sperm in any possible condition. Key words: survival, X and Y bearing sperm, fluorescence in situ hybridization - 92 -

KSDB-49 Tissues Distribution of Melanocortin 4 Receptor and Vinculin Genes in Rainbow Trout, Oncorhynchus mykiss Jong Man Yoon Dept. of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University, Gunsan 573 701, Korea Increased melanocortin receptor stimulation following leptin administration plays an important role in leptin induced hypophagia and increased sympathetic nervous system activity and is partly responsible for leptin induced weight loss. Fish agouti family genes have also been identified in some fish such as goldfish, zebra fish and puffer (Cerda Reverter & Peter, 2003; Song et al, 2003; Kurokawa et al., 2006). Despite the importance of these systems in various organisms health, there are as yet no basic information reported for any of the members of agoutirelating protein, melanocortin 4 receptor and vinculin genes in rainbow trout, Oncorhynchus mykiss or coldwater fishes. Liver, muscle, brain, heart, pituitary, kidney, intestine, spleen and gill tissues, respectively, were obtained from rainbow trout (20 cm long, 1 year old fingerlings) anesthetized by 100 mg/kg tricaine methanesulfonate. Real time reactions were carried out with PCR master mix (Applied Biosystems, Foster City, CA, USA). The tissue distributions of the cdna of O. mykiss three genes were analyzed using quantitative real time PCR with primer sets for tissue expression analysis. A dissociation curve was made at the end of each run to make sure that there was no non specific amplification. PCR efficiency was calculated from each linear regression of standard curves. Complimentary DNA (cdna) solutions were used as the template for quantitative real time PCR assay along with agouti relating protein (AgRP), melanocortin 4 receptor I (MC4R I), melanocortin 4 receptor II (MC4R II) and vinculin genes. Further, the Ct of each DNA was compared. After several experiments with four individual genes of rainbow trout, author estimated a variation ratio of the mean Ct value of the DNA extracted using the comparative CT method was 37.27, and the standard deviation was 5.33. Key words: agouti relating protein, melanocortin 4 receptor, mrna, tissue distribution, vinculin - 93 -

KSDB-50 Variation of Shell Color in Three Geographic White Clam (Meretrix lusoria) Populations of West Sea Jong Man Yoon 1, Jong Yeon Kim 2 1 Dept. of Aquatic Life Medicine 2 Faculty of Marine Life Sciences, College of Ocean Science and Technology, Kunsan National University, Kunsan, 573 701, Korea In general, the species classification of rockfish is based on morphological variations in shell type, shell color, shell length, body weight and shell size. Genomic DNAs (gdnas) were isolated from the hard clam (Meretrix lusoria) population of Gunsan located in the West Sea of Korea peninsula. DNA extraction and/or purification was performed as described previously (Yoon and Kim, 2003). Genetic distances among different individuals of the LSCP (light shell color population) population of hard clam (lane 1 11), GSCP (grey shell color population) population of hard clam (lane 12 22) and DSCP (dark shell color population) population of hard clam (lane 23 33), respectively, were generated using the CLA SSIFICATION option in Systat version 10 (SPSS Inc., Chicago, IL, USA) according to the bandsharing values and similarity matrix. A hierarchical clustering tree was constructed using similarity matrices to generate a dendrogram, which was facilitated by the Systat version 10. The dendrogram, generated by seven reliable oligonucleotides primers, indicates three genetic clusters. The longest genetic distance (0.801) was found to exist between individuals in the two populations, between individuals no. 33 of the DSCP population and no. 06 of the LSCP population. Three hard clam populations can be clearly distinguished, especially by their morphological characters and PCR based approach. Fig. 1. PCR generated electrophoretic profiles of individual hard clam (Meretrix lusoria). DNA isolated from LSCP population of hard clam (lane 1 11), GSCP population of hard clam (lane 12 22) and DSCP population of hard clam (lane 23 33) were amplified by oligonucleotides primers. Key words: genetic distance, hard clam, Meretrix lusoria, morphological variation, shell color - 94 -

KSDB-51 ERa Regulates CYP17 Expression in the Ovary Sung Eun Lee 1, Chemyong Ko 2, Yong Pil Cheon 1 1 Department of biology, Sungshin Women s University, Seoul, South Korea 2 Center of Excellence in Reproductive Sciences, University of Kentucky, Lexington, KY 40536 In the ovary, estrogen regulates steroid hormone synthesis through estrogen receptors, ER alpha (ERα) and beta (ERβ). In the theca cell, estrogen inhibit the steroid synthesis but the regulatory mechanisms are not well understood. ERα located in theca/interstitial cells and oocytes and ERα total KO mouse is sterile with hemorrhagic cyst. To know the role of ERα in ovarian physiology, we used ERα theca/interstitial specific KO mice (ERαflox/flox Cyp17icre or tierαko). Ovaries and serum were collected at diestrus stage from 2, 4, and 6 months old control or tierαko and applied various analytical examination. Compared with the 2 month old mice, the length of diestrus of tierαko was increased in 4 month old mice, and 6 month old mice increased up to 92.9%. FSH level was slightly increased in KO mice independently to the age: tierαko shows 4.3 mg/ml to 7.3 mg/ml serum level but control mice show 3.4 mg/ml to 5.6 mg/ml. Surprisingly, we could not detect basal LH in blood level in the tierαko mice (Control: 0.37 0.48 ng/ml) but their testosterone was kept the normal range. The expression profiles of CYP17 were analyzed with imuunohistochemical analysis and real time PCR which is androgen producing enzyme. The level of CYP17 mrna was dramatically decreased in control mice both in hcg administered or diestrus stage wild mice, but it just decreased only half level in the tierαko mice. In the control, the level of CYP17 protein was changed similar with mrna. However, in the tierαko mice, the levels of this protein was not changed. CYP17 protein specific signal was also detected specifically in corpus lutea of tierαko. From these data, it is cleared that the regulatory role of ERα in androstenedion (testosterone) production is independent to LH. And there regulatory role is important to fertility and hormonal physiology. Key words: estrogen receptor α, theca cell, CYP17, androgen - 95 -

KSDB-52 Exposure to Di(2 ethylhexyl) Phthalate Affects the Maturtation of Vas deferens in Prepubertal Male Rats Won Yong Lee, Sung Ho Lee Department of Life Science, Sangmyung University, Seoul 110 740, Korea Di(2 ethylhexyl) phthalate (DEHP) is used in numerous consumer products, mainly imparting flexibility and durability to polyvinyl chloride (PVC) based plastics. Due to its anti androgenic effects, DEHP has been known as reproductive and developmental toxicant in male rodents and human. The present study was undertaken to examine whether prepubertal exposure to DEHP can make any alteration during the maturation process in male rats. Four week old male rats (SD strain) were received DEHP (low and high dose, 20 and 200 mg/kg/day, respectively) by oral gavage for 17 (till 6.5W), 21 (till 7W) and 24 days (till 7.5W), and the rats were sacrificed on the next day, respectively. Body weight (BW), the wet weight of tissues were measured. Histological studies were performed to assess the structural alterations. Serum testosterone (T) levels were measured by radioimmunoassay. As a result, no sign of considerably disturbed puberty onset was observed. There was no significant change in BW, serum T levels and tissue weights except prostate gland in DEHP treated animals compared to vehicle treated ones. Treatment with DEHP significantly decreased the weight of ventral prostate at 7W (control vs low dose group = 171.00±18.85 mg vs 107.92±17.36 mg, P<0.05; control vs high dose group = 171.00±18.85 mg vs 119.85±9.02 mg, p<0.05). Interestingly, morphological changes in histology of Vas deferens were apparent when the low dose DEHP treated samples were compared with control. In the DEHPtreated samples(at 6.5W), hyperplasia of both smooth muscle cells and lamina propria cells, reduced ruminal volume and thickened connective cell layer compared to those of control were observed. The hypoplastic states were continued for 7.5W. In conclusion, the present study demonstrated that (i) adverse effect(s) of DEHP on sexual maturation during prepubertal period could be limited, (ii) vas deferens is a sensitive target for DEHP in immature rats, (iii) the effect of the low dose DEHP on Vas deferens might be mediated through mechanism other than antiandrogenic pathway. Key words: DEHP, low dose, prepubertal, Vas deferens, morphology - 96 -

KSDB-53 Expression of Placenta related Genes (Cdx2 and GATA6) in Natural Mating Porcine Development Byung Hyun Cha 1,2, Sung Hun Bae 1, Pyung Hee Kim 1, Seongsoo Hwang 1, Byong Chul Yang 1, Gi Sun Im 1, Myong Jik Kim 1, Hee Tae Cheong 2, Hwan Hoo Seong 1, Yeoung Gyu Ko 1 1 Animal Biotechnology Division, National Institute of Animal Science 2 School of Veterinary Medicine, Kangwon National University Abnormal development and fetal loss during whole implantation period is concerns for production of somatic cell nuclear transferred animals including other many technology. So our hypothesis is that the reasons of problems with produced cloned animal offspring are also related with interactions between porcine conceptus and their endometrial environment. In the present studies, we investigated the expression pattern of the formation of placenta related genes (Cdx2 and GATA6) in whole in vivo pre implantation, peri implantation embryos and each tissues of normal fetus at day 25, 35, 55 by quantitative mrna expression analysis using realtime PCR. As showed in the our result, Cdx2 and GATA6 mrna expression levels in pre, peri and post implantation, our evidence suggests a differential expression pattern between Cdx2 and GATA6. Interestingly, expression level of both genes were significantly opposed at day 18 around. Accordingly, it would seem that these gene makes up for the each others. Thus we suggest that it can be postulated that aberrant expression of Cdx2 and GATA6 genes in endometrium and extraembryonic tissue at pre, peri and postimplantation may be closely related with the lower efficiency of clone production, especially placental abnormalities. (This work was supported by grant No. 20070301034040 from bio organ, Republic of korea.) Key words: Cdx2, GATA6, porcine early development - 97 -

KSDB-54 Inhibitory Effect of Benzo[a]pyrene on Spermatogenesis and Steroidogenesis in Male Mice Ji Young Choi 1, Won Hee Song 3, Sang mi Lee 1, Jungwon Seo 1, Min Sun Sung 1, Young Ah You 3, E. A. Mohamed 3, Yoo Jin Park 3, Shin Ae Oh 3, Young Ju Kim 2, Myung Geol Pang 3, Inho Jo 1 1 Department of Molecular Medicine and 2 Obstetrics and Gynecology, Ewha Womans University School of Medicine, Seoul, South Korea 3 Department of Animal Science and Technology and BET Research Institute, Chung Ang University, Ansung, Gyeonggi Do, South Korea Exposure to benzo[a]pyrene (BaP) has been shown to promote various diseases including cancer by increasing oxidative stress and DNA adduct formation. In this study, we investigated the effects of BaP on spermatogenesis and steroidogenesis. Five week male mice were administrated everydaywith 1, 10 mg/kg of BaP or vehicle (corn oil) for six weeks. One week after the last treatment, the mice were anesthetized,and blood and organs were collected. Western blotting analysis revealed that high dose of BaP increases significantly in the levels of CYP1A1 and reduces the capacity of antioxidant in liver, suggesting a detrimental drug effect. The levels of testosterone in blood decreased in BaP treated mice. All organ weights tested per body weight were not different between BaP treated mice and corn oiltreated controls. However, histotological analysis demonstrates that high dose of BaP significantly decreased the spematogenic area, the ratios of seminiferous tubles with elongated spermatid, and the number of Leydig cells. Our data suggest that BaP decreases spermatogenic capacity and testicular function. This research was supported by a grant (08512KFDA418) from Korea Food & Drug Administration in 2008. Key words: benzo[a]pyrene, spermatogenesis, testis, steroidogenesis - 98 -

KSDB-55 Pubertal Development Characteristics and Artifical Masculinization on Juvenile Longtooth grouper, Epinephelus bruneus Sung Pyo Hur 1, Yong Woon Ryu 1, Bong Soo Lim 1, Young Bo Song 1, Goo Yeon Hyun 2, Se Jae Kim 3 and Young Don Lee 1 1 Marine and Environmental Research Institute, Cheju National University, Jeju 690 986, Korea 2 Tamra Marine Products Industry, Jeju 697 904, Korea 3 Department of Life Science, Cheju National University, Jeju 690 756, Korea Puberty is the period which animals obtain the ability of reproduction for the first time. An examination about pubertal development is needed for understanding of sexual maturation characteristics. Longtooth grouper is protogynous hermaphrodite and undergoes sex reversal from female to male. But the physiological mechanism of sex change remains largely unknown. In this study, we researched that characteristics of pubertal development in immature female and induce masculinization in juvenile longtooth grouper. Experimental fish were classified into three groups (about 1, 2, 3 kg) by the body weight (BW). On the non breeding season, 1, 2 kg groups showed perinucleous stage while vitellogenesis was observed in 3 kg group. However, on the breeding season, 2, 3 kg groups showed vitellogenesis while 1 kg group showed perinucleous stage. These results indicate that longtooth grouper are able to classify three stages; pre puberty (1 kg), dummy run (2 kg) and puberty (3 kg). Sexually immature pre pubertal (1.04±0.04 kg) were treated with GnRHa and GnRHa+IGF I. After 20 days, GnRHa and GnRHa+IGF I groups showed vitellogenesis stage. FSHβ mrna levels were highest in the GnRHa treatment group. Serum levels of E2 were incresaed in GnRHa treatment groups at after 5 days. These results indicate that GnRHa and GnRHa+IGF I treatment have more effects for inducing on set of puberty in pre pubertal individuals. Also, expressions of FSH β have more important effects than LHβ during sexual maturation and oocyte development. In masculinization of juvenile longtooth grouper(113±17 g), The fish were injected with a non steroidal aromatase inhibitor (AI, Fadrozole) in a dose of 3 and 5 mg/kg BW and 17α methyltestosterone (MT) in a dose of 5 mg/kg BW. Serum levels of 11 KT was significantly increased in AI 5 mg treatment groups at 7th and 21st weeks after the injection. Expression of FSHβ mrna was increased only AI 5 mg injected groups after 21weeks. These results suggest that In first stage of sex change, oocytes were degenerated cause by 11 KT stimulate. And in second stage of sex change, spermatogenesis occurred cause by 11 KT and FSHβ mrna stimulate. Key words: longtooth grouper, puberty, sex change, GtH subunits - 99 -

KSAR-56 Microtube Culture System Promotes The Expression of Oct4 And Igf2 in the Parthenogenetic Murine Embryos Hoin Kang, SangKyu Park, Sangho Roh School of Dentistry, Dental Research Institute, CLS21, Seoul National University, Seoul 110 749, Korea Mammalian embryonic in vitro culture systems are still under development. Over time, many modifications of embryo culture methods have been established, such as culturing embryos with oviduct epithelial cells, called co culture system, the use of 3D culture space for change physical environment called 3D embryo culture system, and the use of paired media in sequence, called sequential media. In murine embryo culture, micro droplet culture system is generally used. Moreover, mineral oil is an indispensable material in micro droplet embryo culture because its transparency allows culture monitoring and prevent evaporation of the medium. However, lipophilic factors in the medium can be absorbed into the oil overlaid, and conversely, deleterious factors from oil can diffuse into the medium. In previous studies, we established a novel oil free microtube culture (MTC) system. Parthenogenetic murine embryos were placed into a 0.2 ml thin wall flat cap PCR tube and cultured to the blastocyst stage. The embryos in MTC exhibited a higher blastocyst formation rate and larger populations of inner cell mass in the blastocysts compared with micro droplet counterpart. Here in this study, we analyzed the expression of Oct4, Nanog, Igf2, Fgf2 and Egf in those blastocysts by real time RT PCR. The expression levels of Oct4 and Igf2 in MTC group were significantly increased when compared with micro droplet counterpart whereas the level of Nanog, Fgf2 and Egf were not significant. Elevated expression of Oct4 and Igf2 might have positive effects on embryonic development. As Oct4 plays a role for maintenance of pluripotency in the embryonic stem cells (ESC), culturing embryos in MTC may be supportive to ESC generation. Comparably higher expression of Igf2, a imprint gene, in MTC group indicate that MTC system might activated Igf2 expression in murine parthenogenetic blastocysts. In conclusion, MTC system promoted the expression of Oct4 and Igf2 in the pathenogenetic murine embryos. This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant (M10641000001 06N410000110 and R01 2007 000 10316 0) - 100 -

KSAR-57 Tetracycline inducible Expression Systems in the htpo Transgenic Chickens Mo Sun Kwon, Bon Chul Koo, Ji Yeol Roh, Min Ji Kim, Hyuna Lee, Teoan Kim Dept of Physiology, Catholic University of Daegu School of Medicine, Daegu, Korea Unregulated over expression of foreign gene products may also have unwanted physiological or toxic effects in the transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline inducible promoter. The Tetracycline regulatory systems (Tet system) are currently the most widely used regulatory systems for conditional gene expression. However, gene expressions in the Tet system are not completely regulated but a little leaky due to the inherent defects in Tet based systems. A more tightly controllable regulatory system can be easily achieved when the advanced versions (rtta2 S M2) of rtta and an ideal minimal promoter in responsive components (ptretight) are used combination therein. In this study, we tried to produce human thrombopoietin (htpo) from various target cells and transgenic chickens using the retrovirus vector combined with tetracycline inducible gene expression system. htpo is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher htpo expression was observed in CEF, while tighter expression control was obtained in HeLa. We also measured the biological activity of the htpo using Mo7e cells whose proliferation in dependant on htpo. The biological activity of the recombinant htpo in CEF was higher than both its commercial counterpart and htpo in other target cells. The recombinant retrovirus was injected beneath the blastoderm of non incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation and 8 hatched chicks were found to express vectorencoded htpo gene. When the Go transgenic chicken was fed with doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, htpo concentration of the transgenic chicken blood was 200 ng/ml. These results are informative in order to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins. ʺThis work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD)ʺ(KRF 2005 050 C00004) and by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (grant No. R11 2002 100 04005 0). Key words: tetracycline inducible expression system, transgenic chickens, human thrombopoietin (Htpo), bioreactor - 101 -

KSAR-58 Analysis of Mtdna in Blastomeres of Porcine Mouse Interspecies Embryos Myung Youn Kim, Yan Shi Quan, Kenji Naruse, Su Min Choi, Rong Xun Han, Chang Sik Park, Dong Il Jin Research Center for Transgenic Cloned Pig, Chungnam National University Interspecies somatic cell nuclear transfer (iscnt) is an valuable tool for studying interactions between the cytoplasm and nucleus. we generated porcine mouse reconstructed embryos via iscnt using porcine oocytes as recipient cytoplasm and mouse fatal fibroblast as doner cells. In this study we analyze the distribution of different stage of mitochondrial (mtdna) preimplantation development. Porcine cumulus oocyte were obtained from follicles of ovaries and matured in TCM 199. After 22h of maturation culture, the oocytes were transferred to maturation medium without hormone supplementation, and incubated for an additional 22 h. Enucleation was accomplished the first polar body and cytoplasm and a single donor cell was individually microinjected into vitelline space of the enucleated oocyte. The reconstructed embryos were electrically fused with two DC pulses of 1.1 kv/cm for 30 μ sec in 0.3 M mannitol medium. The activated embryos were cultured in different medium for 7 days. we obtain to 2 cell stage from blastocyst in porcine mouse reconstrcted embryos. the cleavage rate is significantly different between intraspecies reconstructed embryos and interspecies reconstructed embryos. However the rate of blastocyst is similar in both reconstructed embryos. Mitochondria DNA via iscnt embryos was identified as heteroplasmy of donor cell and recipent cytoplasm mtdna. in which mtdna derived from mouse somatic cells and porcine oocytes cytoplasm was found in porcine mouse cloned embryos from 2 cell stage to blastocyst stage developmental. These results suggest that porcine cytoplasm may have species preference as a recipient in iscnt and the mtdna of porcine mouse cloned were existed in heteroplasmy from 2 cell to the blastocyst stages. Key words: Interspecies somatic cell nuclear transfer, mitochondiria - 102 -

KSAR-59 Immune System Evaluation of Human Erythropoietin(hEPO) Transgenic Pigs Min Ji Kim 1,2, In Sul Hwang 1, Sung Jun Byun 1, Hwi Cheul Lee 1, Seung Hoon Lee 1, Won Kyoung Chang 1, Jong Ki Cho 2, Sang Tae Shin 2, Jin Ki Park 1 1 Animal Biotechnology Division, National Institute of Animal Science, RDA 2 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea As use of transgenic(tg) animals as bioreactors of therapeutic proteins is becoming more actual, health, reproduction and viability of TG animals will become the major issues of concern to produce proteins persistently. However, numerous reports list the abnormlity obtained from TG animals. We previously identified human erythropoietin (hepo) pigs have abnormality such as lung injury. The immune system is the important factor affecting disease resistance and the number and function of immune cell is influenced by physiological and pathological status. Therefore this study were performed to assess the immune system of hepo TG pigs. The immune system was evaluated serum Ig M, A, G levels and determining the population of helper T cells (CD4), cytotoxic T cell (CD8). Immunoglobin levels were analyzed by sandwich capture ELISA. Also, distribution of CD4 T cell and CD 8 T cell was analysed by Flow cytometry. Hematological analysis indicates that hepo TG pigs have polycythemia and thrombocytopenia. However, there were no significant differences between hepo TG pigs and wild type(wt) pigs in the immunoglobin analysis. The number of CD 4 and CD 8 T cell in hepo TG pigs was lower than in WT pigs, but there was no significant differences. From this study, we detected significant changes of blood cell parameter, but didnʹt indication of an impaired immune system in hepo TG pigs. It appears that hepo TG pigs have normal immune system similar to WT pigs. Key words: human erythropoietin(hepo), transgenic, pig, immune system,cd4 T cell, CD8 Tcell, immunoglobin - 103 -

KSAR-60 Efficient Modification of Male Germline Stem Cells Using Lentiviral Transduction in Pig Byung Gak Kim 1, Bang Jin Kim 1, Yong An Lee 1, Ki Jung Kim 1, Yong Hee Kim 1, Chul Geun Kim 2, Kwan Sik Min 3, Buom Yong Ryu 1 1 Department of Animal Science & Technology, Chung Ang University, Ansung, Korea, 2 Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Korea. 3 Animal Biotechnology, Graduate School of Bio. & Information Technology, HanKyong National University, Ansung, Korea Donor derived spermatogenesis after spermatogonial transplantation to recipient animals could serve as a novel approach to manipulate the male germ line in species where current methods of genetic modification are still inefficient. This study was designed to investigate stem cells activity and modification methods of male germline stem cells using lentiviral transduction and xenotransplantation of spermatogonial stem cells in pigs. To obtain insight into stem cell activity and the behavior of pig germ cells, the pattern and kinetics of pig spermatogonial colonization in recipient seminiferous tubules were analyzed following transplantation. Donor testis cells were harvested from testes obtained at castration of 10 to 14 days old boars using a sequential enzymatic digestion procedure with collagenase, hyaluronidase, and trypsin. Subsequent sequential isolation procedure with laminin and geletin showed that approximately 80% of the purified donor cells were gonocytes. Purified pig gonocyte were transplanted into the testes of immunodeficient recipient mice in which endogenous spermatogenesis had been depleted by busulfan. Germ cells transplantation study showed that germ cells labeled by PKH26 could reside on the basal membrane of the seminiferous tubule of nude mice. At the time of transplantation, the PKH26 labeled cells were randomly distributed throughout the tubules, which eventually led to the formation of colonies after 2 months. Using a lentiviral vector, an enhanced green fluorescent protein (egfp) transgene was introduced into the genome of fresh porcine gonocytes, which were microinjected into the testes of immunodeficient - 104 -

mice to assess modification efficiency. An average of 16% transduced germ cell colonies were developed from gonocytes transplanted into the seminiferous tubules of a recipient testis. The spermatogonial stem cells colonization pattern was analyzed for 6 months following transplantation, and an increase in colony length could be observed. These results indicate that the donor germ cells from pigs can successfully proliferate and colonize within the recipientʹs seminiferous tubules via lentiviral transduction. Therefore lentiviral transduction can be used to efficiently modify germline stem cells of pigs and possibly other domestic animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals. This work was supported by a grant (20070101034007, 20070401034012) from BioGreen 21 Program, Rural Development Administration, Republic of Korea. Key words: spermatogonial stem cells, spermatogenesis, gonocyte, porcine, lentivirus, - 105 -

KSAR-61 Parthenogenetic Induction of Canine Oocytes by Electrical Stimulation and Ca EDTA Bong Soo Kim, Zae Young Ryoo School of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kv/cm) for 50 μsec with single DC pulse following 1 mm Ca EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kv/cm after 1 mm Ca EDTA treatment, the rate of pronucleus and 2 cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca EDTA treatment could cause canine oocytes to be degenerated. However, 2 cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested thatelectrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed. Key words: electrical stimulation, parthenogenesis, canine, oocyte, Ca EDTA - 106 -

KSAR-62 Expression Analysis of 20alpha Hydroxysteroid Dehydrogenase in the Maintenance of Pregnancy in the Bovine S. H. Kim 1, Y. S. Shin 1, H. J. Lee 2, K. S. Min 1, S. Y. Hwang 1, J. T. Yoon 3 1 Major in the Animal Biotechnology, The Graduate School of Bio. & Information Technology 2 Research Center of Genetic Engineering 3 Department of Animal Life Research, Hankyong National University Progesterone was known an essential role in estrus cycle and pregnancy. The precise timing of both the synthesis and degradation of this steroid hormone is crucial for reproductive success. In the corpora lutea of rats and mice, 20α HSD is considered to be involved in functional luteolysis. It is also distributed in other tissues including the placenta, endometrial epithelia and fetal skin, although the roles in these tissues remain to be elucidated. In the present study, we investigated the expression pattern of 20α HSD during pregnancy in bovine tissues (caruncle, metrium and corpus luteal). To determine the function of 20α HSD gene during pregnancy, we collected the bovine pregnant tissues (caruncle, metrium and corpus luteal) on 30, 60 and 90 days of pregnancy. A mrna expression of 20α HSD gene, RT PCR and real time PCR were conducted. The protein expression was detected by immunohistochemistry and western blotting method. Expression of 20α HSD mrna in the caruncle progressively decreased from day 30, 60 to 90 in the pregnancy. 20α HSD was the most highly expressed in the corpus luteal day 90. And the metrium progressively increased from day 30, 60 to 90. Analysis 20α HSD expression of protein was different from mrna. Expression of 20α HSD protein in the caruncle progressively increased from day 30, 60 to 90 in the pregnancy. 20α HSD was the most highly expressed in the corpus luteal day 30 and 90. And the metrium progressively increased from day 30 to 60 and then decreased day 90. These results suggest that maternal 20α HSD plays a role in maintaining normal pregnancy at least partially by controlling the progesterone hormone concentrations in bovine ovary and placenta during pregnancy. Key words: 20α HSD, bovine pregnancy, corpus luteal, carucle, metrium - 107 -

KSAR-63 Effect of Progesterone receptor in Apoptosis Regulatory Mechanism of Bovine Pregnancy Tissues S. H. Kim 1, D. W. Chung 1, H. J. Lee 2, Y. H. Chung 3, S. Y. Hwang 1, J. T. Yoon 4, K. S. Min 1 1 Major in the Animal Biotechnology, The Graduate School of Bio. & Information Technology 2 Research Center of Genetic Engineering 3 Department of Companion Animal and Animal Resources Science, Joongbu University 4 Department of Animal Life Science, Hankyong National University This was studied the identification of a expressed gene for the bovine BNIP 3, CASP 3, Bax, BCL 2 apoptosis family member, apoptotic protein Casp 3 and progesterone receptor to identify the apoptosis regulatory mechanism of caruncle, metrium and corpus luteal. Analysis gene expression of BNIP 3, CASP 3, Bax, BCL 2 gene, RT PCR and real time PCR was conducted, and apoptotic expression of Casp 3 antibody detected Immunohistochemistry(IHC) and western blot. Expression of progesterone receptor mrna progressively increased in the caruncle. The BNIP 3 and Casp 3 were progressively decreased from day 30 to 90 in the pregnancy. Analysis result of Casp 3 protein and apoptosis in the bovine pregnancy tissue were progressively decreased. Expression of progesterone receptor mrna in the corpus luteal progressively increased from day 30 to 60, but decreased day 90. Analysis result of Casp 3 protein and apoptosis was the most highly expressed in the corpus luteal day 30 and 90. The metrium expression pattern was appeared like corpus luteal apoptosis pattern. Progesterone receptor was no significant effect on the expressions of BCL2 and BAX mrna. Expression of progesterone receptor also increased BNIP3, and activated Casp 3. These results suggest that progesterone receptor of pregnancy tissue plays a role in maintaining normal pregnancy. Key words: apoptosis, Casp 3, BNIP 3, progesterone, BCL2, - 108 -

KSAR-64 Reprogramming of Human Somatic Cells into Induced Pluripotent Stem Cells Eun Young Kim 1,2, Hyo Young Park 1,2, Hyung Min Chung 3, Se Pill Park 1,2,4 1 Mirae Biotech, 2 Cheju National University Stem Cell Research Center 3 Pochun CHA University, 4 Cheju National University Human induced pluripotent stem (hips) cells will be a useful source for the patient specific cell therapy. This study was to examine the reprogramming of human somatic cells by defined factors into hips cells. Human fetal lung fibroblasts (IMR90) were infected using self inactivating human immunodeficiency virus type 1 based lentiviral vectors containing four transcription factors (Oct4/Sox2/ Nanog/Lin28). Transfection level was confirmed by expression of the VENUS protein reporter gene and transfected cells were cultured on mouse embryonic fibroblast feeder in human embryonic stem (hes) cell culture medium. 19~21 days later, two ES like colonies were picked and separately cultured under the existing hes cell culture environment. hips cells were shown typical hes cell morphology and growth pattern. By RT PCR, two hips cell lines were confirmed exogenous gene expression with endogenous gene expression, and this result was certainly different compared to positive hes cell (CHA4) and parental IMR90 cells. Also, hips cells expressed hes cell marker including alkaline phosphatase, stage specific embryonic antigen 3, 4, tumor rejection antigen 1 60 and 1 81. In addition, these hips cells have normal karyotype (46, XX) and DNA finger printing indicates that two hips cell lines were derived from the parental IMR90 cells. When the pluripotency of hips cells was examined, embryoid bodies were formed and three embryonic germ layered in vitro differentiation was confirmed by immunocyto chemistry (alpha feto protein, smooth muscle actin and tublinbeta). These results demonstrated that normal hips cells can be derived from human somatic cells using defined factors, and this system is very stable to maintain the hips cell characteristics and pluripotency. Key words: human ips cell, Oct4/Sox2/Nanog/Lin28, lentiviral vector, transfection - 109 -

KSAR-65 돼지인공수정용유통정액의기술적인현황 김인철, 유재원, 홍준기, 조규호, 우제석, 정일병, 정기화 1, 배상종 2, 강권 2, 김상현 2, 김대실 2, 김시주 2 농촌진흥청국립축산과학원, 1 진주산업대학교, 2 한국돼지유전자협회 국내의돼지인공수정은 1994년도에본격적으로보급되기시작한후지난 15년간급속한발전을하여왔다. 2008년 7월부터 9월까지국내돼지인공수정현황에대한실태조사를실시하였다. 2008년 10월현재등록되어있는센터수는 52개소였으며, 정액보급량은년간 179만 2천두분이고센터당평균 39,000두분으로전국모돈두수대비소요량의 90% 를차지하였으며 2004년도보급량 154만 9천두분에비해 15.5% 증가하였다. 정액판매가격은 13,000~14,000원이었으며, 대부분의센터는단일개체정액 (72.3%) 을판매하고있으며, 혼합정액 (12.8%) 단일정액과혼합정액혼용은 14.9% 였다. 센터별사용하는보존액의종류는 13종이었고 Modena(10%), Androhep(6%), Gene sperm(6%), BTS(5%) 순으로조사되었다. 정액제조시보존액내항생제를별도로첨가하는센터는 8개소 (18.2%) 였고, 항생제를첨가하지않는센터는 36개소 81.8% 로조사되었다. 1 dose당정자농도는 30 10 8 /100 ml가 43.8%, 25 10 8 이 37.5% 로각각조사되었다. 정액포장용기는팩 (87.5%), 병 (2.1%), 튜브 (2.1%) 및기타방법 (8.3%) 으로조사되었다. 이상의결과로볼때돼지 AI센터의기술적인부분이질적으로많은향상이있었음을알수있었다. Key words: 돼지인공수정, 보존액, 혼합정액, 정액포장용기 - 110 -

KSAR-66 Effect of Flavonoid Treatment on In Vitro Survival of Frozen thawed Bovine Parthenogenetic Embryos Jae Youn Kim 1,2, Kyoung Ha So 1,2, Min Jee Park 1,2, Hyo Young Park 1,2, Yeon Ok Kim 1,2, Eun Young Kim 1,2, Se Pill Park 1,2,3 1 Mirae Biotech, 2 Cheju National University Stem Cell Research Center, 3 Cheju National University This study was to investigate the in vitro survival of frozen thawed bovine parthenogenetic embryos according to treatment of flavonoid before freezing or/ and after thawing. In vitro produced bovine parthenogenetic day 2 ( 2 cell) embryos were treated w/wo 10 μm flavonoid for 6 days. For freezing, parthenogenetic expanding blastocysts were pretreated in 10% ethylene glycol (EG) (v/v) in D PBS for 5~10 min, exposed in EG 30 for 30 sec, put on the inner wall of 0.25 ml straw, and then straw was directly plunged into LN 2. Thawing was taken by 5 step procedures, thawed embryos were exposed in 1.0 M sucrose (MS), 0.5 MS, 0.25 MS, 0.125 MS and then D PBS without sucrose for 1 min in each step. In non treatment (-) or flavonoid treatment (+) group before freezing, thawed blastocysts were divided into four groups according to the treatment of flavonoid after thawing (-/-, -/+, +/-, +/+). Also, these results were compared with frozen thawed IVF group. Among the frozen thawed parthenogenetic embryo groups, irrespective of flavonoid treatment before freezing, in vitro survival rates of flavonoid treatment groups after thawing were higher (-/+, 58%; +/+, 65%) than those of non treatment group (-/-, 47%; +/-, 55%) after thawing. However, all of those results were not better than that of frozen thawed IVF group (75%). TUNEL assay of DNA fragmentation indicated apoptotic indices were also decreased in flavonoid treatment group (-/+, 13.6%; +/+, 13.5%) after thawing compared to non flavonoid treatment group (-/-, 24.8%, +/-, 23.7%), irrespective of flavonoid treatment before freezing. These results demonstrated that flavonoid addition in culture medium before freezing and after thawing brings beneficial effects on in vitro survival of bovine frozen thawed parthenogenetic embryos and especially flavonoid treatment after thawing mainly affect the in vitro survival of them. Key words : bovine embryo, parthenogenetic, freezing, flavonoid, TUNEL - 111 -

KSAR-67 Cell type Specificity of Interleukins 1α and 1β on Prostaglandin and Plasminogen Activator Production in Bovine Endometrial Cells Tae Shin Kim 1, Soo Bong Park 2, Choon Keun Park 1, Dong Seok Lee 3 1 Division of Animal Biotechnology, College of Animal Life Science, Kangwon National University, Chuncheon 200 701, Republic of Korea 2 National Institute of Animal Science, Suwon 441 706, Republic of Korea 3 School of Life Sciences & Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702 701, Republic of Korea Interleukin (IL) 1 is a potent stimulator of prostaglandin production in bovine endometrium, and IL 1 affects plasminogen activator (PA) activity in several types of cells. In this study, we determined the effects of IL 1α and IL 1β on production of the prostaglandins PGF 2α and PGE 2 and on PA activity in cultured bovine endometrial epithelial and stromal cells. We also determined the effects of PGE 2 and PGF 2α on PA activity in these cells. Finally, we used RT PCR to examine the expression of IL 1α, IL 1βand IL 1 receptor type 1 (IL 1R) mrna in cultured bovine endometrial cells. This analysis revealed that IL 1α mrna was present only in the stromal cells, whereas IL 1β and IL 1R mrnas were present in both cell types. When cultured cells were exposed to IL 1α and IL 1β at concentrations ranging from 0.006 to 3 nm for 24 h, IL 1 and IL 1 were found to dose dependently stimulate PGEα and PGF 2α production in stromal cells (p<but not in epithelial cells. On the other hand, exposure to IL 1α and IL 1β dosedependently increased PA activity in the epithelial cells, whereas neither stimulated PA production in the stromal cells. When cells were exposed to IL 1α and IL 1β at concentrations ranging from 0.06 to 3 nm for 24 h, the two IL 1s differed in their effects on both PGE 2 and PGF 2α production in stromal cells and had significantly differed in their effects on PA activity in epithelial cells. Exposure to PGE 2 and PGF 2α did not affect PA activity in either stromal or epithelial cells (p> 0.05). Taken together, these results suggest the possibility that both IL 1 and IL 1 are produced by the stromal cells, that IL 1β is produced by the epithelial cells, and that IL 1α is a far more potent stimulator than IL 1β of prostaglandin and PA production in cultured bovine endometrial epithelial and stromal cells. This work was supported by a grant (Code# 20070401034015 and Code# 2007 0301034040) from BioGreen21 Program, Rural Development Administration. Key words: interleukin 1, prostaglandins, plasminogen activator, endometrial cells, cattle - 112 -

KSAR-68 Stimulation of Plasminogen Activator Activity by Free Radicals in Boar Spermatozoa Tae Shin Kim 1, Young Seung Lee 1, Soo Bong Park 2, Choon Keun Park 1, Dong Seok Lee 3 1 Division of Animal Biotechnology, College of Animal Life Science, Kangwon National University, Chuncheon 200 701, Republic of Korea 2 National Institute of Animal Science, Suwon 441 706, Republic of Korea 3 School of Life Sciences & Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702 701, Republic of Korea Plasminogen activators (PAs), commonly found on the membrane of spermatozoa, convert plasminogen into plasmin and may participate in mammalian fertilization. Correlations have been reported between reactive oxygen species (ROS) and spermatozoa function, although the relationship between PA activity and ROS is unknown. We used an in vitro model of free radical generation whereby boar spermatozoa were preincubated in xanthine and xanthine oxidase (X XO) and PA activity was then measured. The acrosome reaction of boar spermatozoa was significantly promoted by 100 mu/ml plasmin (p<0.01), similar to levels achieved when stimulated with the positive calcium (2 mm) control. The addition of plasminogen to the fertilization medium significantly promoted both spermatozoa binding (157.5± 14.0 spermatozoa/oocyte) and the percentage of oocytes with a male pronucleus (74.5± 6.4%) compared with control (98.4±21.8 spermatozoa/oocyte and 51.4±5.3%, respectively; p<0.05). The acrosome reactions of spermaotozoa were significantly higher when incubated with calcium (2 mm; 60.2±2.7%), calcium (2 mm)+edta (6 mm; 29.4± 4.2%), sodium nitroprusside (0.1 mm; 38.0±4.2%), H 2 O 2 (100 mm; 56.0±3.0%), and X XO (0.5 mm and 0.05 U/mL, respectively 31.8±3.7%) compared with non capacitation medium as control (19.0±2.7%; p<0.05). However, when spermatozoa were incubated with only X XO, PA activity was significantly higher than with other treatments (p<0.05). Moreover, the addition of the antioxidant superoxide dismutase to the X XO system significantly blocked the PA activity of spermatozoa (p<0.05). The PA activity of spermatozoa treated with X XO was significantly reduced by the addition of MEK inhibitor (55.2±5.6 ng/ml) and p38 inhibitor (57.4±2.7 ng/ml), but not PI3K inhibitor, compared to the control (X XO; 68.0±5.8 ng/ml p<0.05). The - 113 -

induction of PA activity in boar spermatozoa by free radical generation suggests the PA/plasmin system plays a role in mammalian fertilization. This work was supported by a grant (Code# 20070401034015 and Code# 2007 0301034040) from BioGreen21 Program, Rural Development Administration. Key words: plasminogen activators, boar spermatozoa, acrosome reaction, free radical antioxidants - 114 -

KSAR-69 소임신초기혈장단백질발현양상분석 김평희 1,2, 박원종 1, 형남웅 1, 김점순 1, 고응규 1, 양병철 1, 김명직 1, 진동일 2, 성환후 1, 황성수 1 1 국립축산과학원동물바이오공학과분자제어연구실, 2 충남대학교 본연구의목적은임신또는비임신한우의혈액을이용하여착상시기동안임신관련단백질의발현양상을살펴보기위하여실시하였다. 3~5년생한우암소 (4~7년생) 에동기화처리를한후인공수정을실시하여임신과비임신으로구분하여본연구에공시하였다. 인공수정을실시한날을첫회로하여 3, 4, 5, 8 및 12주에각각채혈을실시하였다. 혈액으로부터원심분리로혈장을분리하여사용시까지 80 냉동고에보관하였다. ELISA Kit을이용하여 progesterone 농도를측정하였다. 이차원전기영동법을이용하여발굴된 17개의특이발현단백질 (haptoglobin, ferrochelatase, paraoxonase 1, hemopexin 및 fibrinogen 등 ) 중에서임신과직접적연관이있다고판단되는 2~3개의유전자에대하여 western blotting 방법으로발현차이를확인하였다. 임신특이적단백질로알려진 PSP(pregnancy specific protein) 는임신군에서지속적으로증가하는경향을나타내었으며, 혈관형성과직접적으로관련하는것으로알려진 Angoipoietin 1과Angiopoietin 2 간의비율은임신군과비임신군간에차이가나타나는것을확인할수있었다. 면역체계급성기반응에의해유발되는것으로알려진 haptoglobin은임신에서는발현이거의나타나지않았으나, 비임신군에서는강하게발현하는것을확인할수있었다. 한편 oxidative stress로인한손상을회복시키는기능을하는것으로알려진 paraoxonase(pon) 1은임신 1~2주까지는차이가나타나지않았으나, 3주차에서부터임신군에서강하게발현하는것으로확인되었다. 이상의결과를종합하여보면임신초기다양한종류의단백질이임신과관련하여역할을하고있다는것을확인할수있었다. 따라서이들단백질에대한기능분석등에대한추가적인연구가필요한것으로사료된다. Key words: 소, 혈장단백질, 임신, 이차원전기영동, western blotting - 115 -

KSAR-70 Human Fas Ligand Expression on Pig Cells can Inhibit Xenogeneic Antibody dependent Cell mediated Cytotoxicity K. W. Park 1,2, K. M. Choi 1,3, S. H. Kim 1, S. P. Hong 1, J. Y. Yoo 1, H. M. Choi 1, Y. C. Park 1, M. Y. Park 1, Y. J. Yoon 1, J. G. Seol 1 1 MGEN, Inc., 2 Sunchon National University, 3 Chungnam National University Although the birth of homozygous α 1,3 galactosyltransferase gene knockout pigs raises hopes for an imminent breakthrough in xenotransplantation, human CD8 + cytotoxic T lymphocyte (CTL) mediated rejection still remains as a immunological barrier to solve. The cytotoxicity of human CD8 + CTL against pig cells is highly detrimental and mediated at least in part by the Fas/FasL pathway. Apoptosis inducing Fas ligand (FasL) is a typeⅡ membrane protein, predominantly expressed in the activated T cells. FasL is cleaved by putative metalloproteinase to produce a soluble form. In this study, we blocked the shedding of human FasL by deleting its cleavage site. To generate a membrane bound form of human FasL, expression plasmids for a series of deletions at the cleavage site were constructed; cytofasl and mutantfasl are FasL deletion mutants in which the amino acids 8 to 69 and 110 to 134 were deleted, respectively. To prevent CTL mediated xenocytotoxicity, we overexpressed the mutant human FasL by means of binding Fas antigen on human CD8 + cells for the common receptor, human Fas. We transfected the mutantfasl plasmid into minipigʹs fetal fibroblasts and established two trasngenic clonal cell lines. The integration of human FasL gene was confirmed by PCR and expression levels were measured by FACS and confocal assay. The levels of membraned bound FasL was increased relative to the control. These molecules may represent a step forward toward preventing CD8 + CTL mediated Xenograft rejection. This work was supported by a grant (code # 2007040103403100804) from Bio Green21 Program Rural Development Administration, Republic of Korea. cytotoxic T lymphocyte (CTL), hu Key words: discordant xenotransplants, human CD8 + man fas ligand, minipig - 116 -

KSAR-71 Flavonoid Treatment Improves the In Vitro Developmental Capacity of Bovine Somatic Cell Nuclear Transfer Embryos Min Jee Park 1,2, Jae Yeon Kim 1,2, Kyoung Ha So 1,2, Hyo Young Park 1,2, Yeon Ok Kim 1,2, Eun Young Kim 1,2, Se Pill Park 1,2,3 1 Mirae Biotech, 2 Cheju National University Stem Cell Research Center, 3 Cheju National University Flavonoid serves as radical scavenger was thought that have beneficial effects on embryonic development in vitro. This study was to examine the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos. For the activation, in vitro matured or SCNT bovine oocytes were treated in 5 um ionomycin for 5 min and 6 DMAP for 3 h. To optimize the flavonoid concentration, when the parthenogenetic day 2 ( 4 cell) embryos were treated with varying concentrations of flavonoid (0.5, 1, 10, and 20 um) for 6 days, in vitro development rate of 10 um treatment group was higher (57.1% blastocyst) than that of any other groups (control, 49.5; 0.5 um, 46.7; 1 um, 54.3; 20 um, 37.5%). In differential staining, ICM cell number was significantly higher after 10 um flavonoid treatment than control or 20 um flavonoid treatment (p< 0.05), while total cell was slightly increased after 10 um treatment. Also, DNA fragmentation was significantly decreased after 10 um flavonoid treatment (p<0.05). The relative abundance of transcripts for internal anti oxidant (Mn SOD) gene was significantly higher after flavonoid treatment (p<0.05), while oxidant (SOX) gene expression was lower than control. Transcripts level of anti apoptotic (Bax inhibiter, Suvivin) gene and growth (In Tau, Glu 5) gene was also significantly high after flavonoid treatment (p<0.05), while apoptotic (caspase 3, Bax) gene expression was low after flavonoid treatment. Thus, when we cultured SCNT bovine embryos in 10 um flavonoid, blastocyst production rate (30.7%) was higher than control (26.0%). In addition, cell numbers of total cell and ICM cell (164.3± 13.5, 50.0±5.8) of day 8 SCNT blastocysts produced after 10 um flavonoid treatment were significantly higher than those of control (135.6±6.3, 36.9±6.0). These results demonstrated that flavonoid treatment can improve the in vitro developmental capacity of bovine SCNT embryos by anti oxidant and anti apoptotic effect. Key words: bovine embryo, flavonoid, anti oxidant, anti apoptotic, SCNT - 117 -

KSAR-72 Pregnancy Rate of In Vitro Produced Elite Hanwoo Embryos according to Transport Time Course Hyo Young Park 1,2, Eun Young Kim 1,2, Jae Yeon Kim 1,2, Kyoung Ha So 1,2, Min Jee Park 1,2, Yeon Ok Kim 1,2, Young Hun Kim 3, Seong Ho Mun 3, Chang Eon Oh 3, Ho Jin Hwang 3, Se Pill Park 1,2,4 1 Mirae Biotech, 2 Cheju National University Stem Cell Research Center 3 Jeju Livestock Promotion Center of Jeju Special Self Goverming Province 4 Cheju National University This study was to investigate pregnancy rate of IVM/IVF/IVC elite Hanwoo (registered in government) embryos according to transport time course. For the production of elite embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen thawed bull semen (purchased from LIMC, KPN# 497) were used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of cleavage (>2 cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocysts at day 8, healthy embryos were freshly transferred on production day and frozen embryos were direct transferred on appropriate day. These IVM/IVF/IVC embryos were produced in Seoul, embryo transfer (ET) was planned in 10 areas of Cheju island by airplane. Thus, we examined the pregnancy rate in recipient cow according to transport time course. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 h, pregnancy rate in ET group required less 4 h (60%, 9/15) was significantly higher than that required more 6 h (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in Thermos (straw container) before ET was measured by TUNEL assay, apoptotic index was increased with storage time dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial. Key words: bovine embryo, embryo transfer, transport time, pregnancy - 118 -

KSAR-73 Analysis of SLA DQA1 Expression in the Uterus during the Estrous Cycle and Pregnancy in Pigs Heewon Seo, Yohan Choi, Hakhyun Ka Department of Biological Resources and Technology, Yonsei University, Wonju, 220 710, Republic of Korea Successful pregnancy is associated with intrauterine immunosuppression to prevent rejection of the conceptus, which is a semiallograft within the uterine environment. Previously, we identified swine leukocyte antigen (SLA) DQA1, a MHC class II gene, as a differentially expressed gene on day (D) 12 of pregnancy compared to genes expressed on D12 of the estrous cycle. Because MHC molecules play critical roles in immune responses to foreign antigens, SLA DQA1 might be involved in preventing immune disruption of pregnancy. We examined expression of SLA DQA1 during the estrous cycle and pregnancy. Uterine endometrial tissue samples were collected from D12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Northern blot analysis of endometrial RNA determined that SLA DQA1 mrna was detectable in the uterine endometrial tissues during the estrous cycle and pregnancy of all stages. Immunohistochemical experiments demonstrated that SLA DQA1 protein was localized to stroma, mainly on the cells underlying luminal epithelial layer and blood vessels during the estrous cycle and pregnancy. Results in this study show that SLA DQA1 is present in the uterine endometrial stroma and blood vessels during the estrous cycle and pregnancy. These results suggest that active immune reactions may occur in the uterine endometrium to support pregnancy in pigs. This work was supported by the BioGreen 21 Program (#20070301034040), Rural Development Administration, Republic of Korea. Key words: pig, pregnancy, uterus, SLA DQA1-119 -

KSAR-74 Analysis of EDG2 Expression in the Uterus during the Estrous Cycle and Pregnancy in Pigs Heewon Seo 1, Mingoo Kim 2, Yohan Choi 1, Chang Kyu Lee 2, Hakhyun Ka 1 1 Department of Biological Resources and Technology, Yonsei University, Wonju, 220 710 2 Department of Food and Animal Biotechnology, Seoul National University, Seoul 151 921, Republic of Korea Lysophosphatidic acid (LPA), a simple phospholipid derived mediator implicated in diverse biological actions, acts through the specific G protein coupled receptors EDG2, EDG4, EDG7, and GPR23. Our previous study showed that EDG7 is cell type and stage specifically expressed in the uterine endometrium and LPA via EDG7 increase PTGS2 expression in the uterine endometrium during the period of implantation. Although EDG7 is considered to be predominant LPA receptor in the uterine endometrium, other LPA receptors might play a role to mediate LPA functions in the uterine endometrium during pregnancy. Among EDG2, EDG4, and GPR23, we investigated expression of EDG2 during the estrous cycle and pregnancy. Uterine endometrial tissue samples were collected from D12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Real time RT PCR of endometrial RNA determined that EDG2 mrna was constitutively expressed in the uterine endometrial tissues during the estrous cycle and pregnancy of all stages. Analysis by immunoblotting revealed that EDG2 proteins were present in the porcine uterine endometrium with a similar expression pattern to that of mrna. Immunohistochemical experiments demonstrated that EDG2 protein was localized to endometrial epithelium and stromal cell, specifically nulei of these cell types. Results in this study show that EDG2 is constitutively expressed in the uterine endometrium during the estrous cycle and pregnancy. These results suggest that LPA EDG2 signaling system may also play a role in the uterine endometrium throughout pregnancy in pigs. This work was supported by the BioGreen 21 Program (#20070301034040), Rural Development Administration, Republic of Korea. Key words: pig, pregnancy, uterus, LPA2, LPA - 120 -

KSAR-75 The Atioxidant Potent of Apple Poly to Support In Vitro Produced Bovine Parthenogenetic Embryos Kyoung Ha So 1,2, Jae Youn Kim 1,2, Min Jee Park 1,2, Hyo Young Park 1,2, Yeon Ok Kim 1,2, Eun Young Kim 1,2, Se Pill Park 1,2,3 1 Mirae Biotech, 2 Cheju National University Stem Cell Research Center, 3 Cheju National University The culture of embryos with high concentration oxygen produces free oxygen radicals which have been implicated as major cause of in vitro embryo development arrest and cell death. This study was to investigate the effect of phytochemicals Apple poly (AP) as an anti oxidant on the bovine parthenogenetic embryo development in vitro. Bovine parthenogenetic embryos were produced from activation procedure of in vitro matured bovine oocytes in 5 um ionomycin for 5 min and 6 DMAP for 3 h. To optimize the treatment concentration of AP, day 2 ( 2 cell) embryos were treated with varying concentrations of AP (0.01, 0.05 and 0.1 %) for 6 days, 0.05 % AP treatment group was indicated higher development rates (58.8 %) than any other groups (control, 47.1; 0.025 % AP, 49.7; 0.1 % AP, 50.2 %). Total cell and ICM cell number in 0.05 % AP treatment group was also higher than those in other treatment groups. This result was proved that the cystic blastomere formation in 0.05 % AP group was appeared earlier than that in other groups. TUNEL assay of DNA fragmentation presented that apoptotic index was significantly low in 0.05 % AP treatment group than other groups (p<0.05). The relative expression levels of anti oxidant (MnSOD), anti apoptotic (Survivin, Bax inhibitor) and growth (In tau) genes were significantly higher after AP treatment (p<0.05), while oxidant (Sox), apoptotic (Caspase 3, Bax) expression levels were after AP treatment low than control. This result demonstrated that the addition of AP in culture medium can support the better in vitro developmental capacity of bovine parthenogentic embryos. Key words: apple poly, anti oxidant, bovine embryo, parthenogenetic - 121 -

KSAR-76 Improved Developmental Capacity of Bovine Embryos by TUDCA Treatment Bong Seok Song 1,2, Ji Su Kim 1, Kyu Sun Lee 1, Cheol Hee Kim 2, Kyung Kwang Lee 1, Deog Bon Koo 1 1 Center for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305 806, Korea 2 Department of Biology, Chungnam National University, Daejeon 305 764, Korea Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported that it attenuates ER stress mediated cell death by interrupting classic pathways of apoptosis. Therefore, in the present study, we explored the anti apoptotic effect of TUDCA on ER stress induced apoptosis in preimplantation bovine embryos. After in vitro maturation and fertilization, presumptive bovine zygotes were cultured in CR1 aa medium supplemented with TUDCA (0, 10, 50, and 100 μm) for 7 days at 38.5, 5% CO 2 in air. To assess the embryonic quality, we investigated the allocation of embryonic cells and apoptotic patterns in the blastocyst stage. We also investigated the effect of TUDCA in the ER stress induced bovine embryos by tunicamycine. In this study, a higher proportion of bovine embryos (34.3%) treated with 50 μm TUDCA developed to the blastocyst stage as compared to the other groups (21.9, 20.2, and 19.1%, p<0.05). Nuclei number (103.5±5.2, n=15) of blastocysts in the TUDCA (50 μm) treated group were higher than that (81.8±5.3, n=13) of the control group (p<0.05). While, developmental capacity of embryos treated with tunicamycine (2 μg/ml) was significant decreased as compared with non treated group (6.7% vs 22.5%, p<0.05). However, when embryos were cultured with both TUDCA (50 μm) and tunicamycine (2 μg/ml), development potential was slightly improved (10.4%). Also, rate of TUNEL positive nuclei were reduced in the blastocysts produced by TUDCA treatment. In conclusion, our results indicate that TUDCA improves the developmental competence of bovine embryos by preventing the ER stress induced apoptosis during preimplantation stage. Key words: taurorsodeoxycholic acid, TUNEL, tunicamycine, bovine embryos - 122 -

KSAR-77 The Impact of Benzo{a}pyrene on Male Fertility Won Hee Song 1, Ji Young Choi 2, Young Ah You 1, E. A. Mohamed 1, Yoo Jin Park 1, Shin Ae Oh 1, Sang Mi Lee 2, Jung Won Seo 2, Min Sun Sung 2, Young Ju Kim 2, In Ho Jo 2, Myung Geol Pang 1 1 Department of Animal Science and Technology and BET Research Institute, Chung Ang University, Ansung, Gyeonggi Do 456 756, South Korea 2 Department of Molecular Medicine and OB/GYN, Ewha Womans University School of Medicine, Seoul 158 710, South Korea. Benzo[a]pyrene (BaP) affects human health via disruptions of endocrine and male reproductive system. The objective of this study was to predict the capacity of male fertility using computer assisted sperm kinematics, chlortetracycline (CTC) assay for capacitation status, hypo osmotic swelling test (HOST) for membrane integrity and in vitro sperm penetration assay using zona free hamster oocytes for sperm fertility evaluation (SPA). Five week old C57BL/6 male mice were administrated daily with oral dose of 1, 10 mg BaP/kg body weight or vehicle (corn oil) during six weeks. One week after last administration, sperm cells were collected from cauda epididymis. The sperm count and motility were significantly decreased in 1 and 10 mg/kg BaP treated groups. CTC assay showed that AR pattern (acrosome reacted sperm) was significantly increased in 1 and 10 mg/kg BaP treated groups. However, B pattern (capacitated sperm, acrosome intact sperm) was significantly decreased in 1 and 10 mg/kg BaP treated groups. From result of HOST, non swelling pattern (dead or sperm with damaged membrane) was significantly increased in 1 and 10 mg/kg BaP treated groups. Finally, significantly decreased sperm fertility was observed in 1 and 10 mg/kg BaP treated groups in SPA. Our data suggest that BaP significantly disrupts male fertility with decreased semen parameters, increased premature acrosome loss, increased damaged sperm membrane and decreased sperm fertility. This research was supported by a grant (08152 KFDA 418) from Korea Food & Drug Administration in 2008. Key words: benzo[a]pyrene, sperm, function, fertility - 123 -

KSAR-78 Responsiveness Differences to Estrogenic Activities in Capacitation Dong Ha Shin, Won Hee Song, Yoo Jin Park, Shin Ae Oh, Myung Geol Pang Department of Animal Science and Technology and BET Research Institute, Chung Ang University, Ansung, Gyeonggi Do 456 756, South Korea This study investigated possible effects of 17β estradiol (E2), progesterone (P4), and two environmental estrogens, genistein (Gen) and 4 tert octylphenol (OP), on bovine, porcine and mice sperm capacitation status in vitro. Frozen thawed bovine, porcine liquid and epididymal mice sperm suspensions were incubated with 0.001~100 μmol/l E2, P4, Gen and OP for either 15 or 30 min at 39 and then assessed using chlortetracycline (CTC) fluorescence. A Concentration independent stimulation of capacitation was observed, with more cells with B pattern (capacitated, acrosome intact) being observed in E2 at 0.001~100 μmol/l compared with controls and other compound treatments for both 15 and 30 min in each sample. E2 at 0.001~100 μmol/l also stimulated the frequencies of AR pattern cells (acrosome loss) compared with controls and other compound treatments for both 15 and 30 min in porcine and mice spermatozoa. P4 stimulated cells with AR pattern with concentration dependency for 15min and B pattern for 30min in mice sperm suspensions. B and AR pattern in porcine sperm and F (intact) and AR pattern in mice sperm were accelerated by Gen for 30 min sperm suspensions. However, OP had not effect on cells with B pattern, but it stimulated acrosome reaction. All four compounds effectively stimulated capacitation and acrosome reaction in three mammal spermatozoa. However, when spermatozoa were incubated for 30 min, capacitation status and acrosome reaction were higher than 15 min in all compounds. However, the responsiveness of bovine, mouse and porcine sperm to E2, P4, Gen and OP were significantly different. Porcine sperm appeared to be even more sensitive. Our data suggest that porcine sperm are suitable tool for screening of endocrine disruptors, because porcine sperm have more sensitive responsiveness than other sperm. Key words: estradiol, progesterone, endocrine disruptor, capacitation, acrosome reaction - 124 -

KSAR-79 Defects in the Vestibular Systems in the Circling Mouse Mi Jung Shin, Zae Young Ryoo School of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea We have identified deafness of circling mice because of its inner ear abnormalities; degenerated cochlea and reduced cellularity in the spiral limbus. Circling mice become hyperactive at approximately 7 days of age and then begin to exhibit circling behavior. The circling behavior of circling mice might be highly correlated with the vestibular system. The inspection of the epithelia and ganglion neurons in vestibule revealed mild abnormalities: some hair cells in the ampulla tended to have longer stereocilia and the numbers of ganglion cells were remarkably reduced compared to heterozygous mice. The reduced cell density in the vestibular neurons is thought to be the main reason for the phenotypic abnormalities. The circling mutant cerebellum is grossly normal, with characteristic lobulation and lamination in cresylviolet stained sections. The thickness of the granular layer, distributions of stellate cells and basket cells, and morphology of Purkinje cells of the circling homozygous mutant were normal. This suggests that the abnormal behavior of the circling (cir/cir) mouse is not the result soft hemorphological defects in the cerebellum. Key words: circling mouse, transgenic mouse, tmie, inner ear - 125 -

KSAR-80 Expression of Apoptosis Genes in Porcine Nuclear Transfer Embryos Fused/Activated with Sperm Cytosolic Factor (SCF) Joo Hyun Shim, In Sun Hwang, Mi Rung Park, Hyo Jin Moon, Dong Hoon Kim, Hwan Hoo Seong, Boh Suk Yang, Gi Sun Im Animal Biotechnology Division, National Institute of Animal Science, RDA, Suwon 441 706, Korea During mammalian fertilization, the sperm introduces oscillatory changes in the eggʹs intracellular concentration of free calcium ([Ca 2+ ]i) that are essential for initiating egg activation and subsequent embryo development. This study investigated the development of porcine nuclear transfer embryos fused/activated in the presence of a porcine sperm cytosolic factor (SCF). Matured oocytes for 40~ 44 h were enucleated, transferred with a fetal fibroblast, and then fused/activated with two electric pulses (DC) of 1.2 kv/cm for 30 us. Control group of embryos was fused/activated in the fusion medium supplemented with 1.0 mm CaCl 2 2H 2 O. Treatment (SCF group) of embryos was fused/activated in the fusion medium supplemented with 0.1 mm CaCl 2 2H 2 O and 100 ug/ml SCF. Fused embryos were cultured in PZM 3 under 5% CO 2 in air at 38.5 for 6 days. In vivo blastocysts were collected from synchronized 7 to 11 month old Landrace gilts. Apoptotic cell death was analyzed by using a TUNEL assay. Expression of Bax α, Bcl xl, Caspase3 and p53 gene in the blastocysts was analyzed by real time RT PCR. SCF group showed significantly higher blastocyst formation rate than that of control group (23.0% vs. 15.2%; p<0.05). SCF group showed significantly lower frequency of apoptosis compared to the control (6.37% vs. 8.18%; p<0.05). The relative abundance of Bax α mrna expression was significantly higher in in vivo or control group than that of SCF (p<0.05). For Bcl xl, control or SCF groups showed lower mrna expression than that of in vivo (p<0.05). The relative abundance of Bax α/bcl xl was clearly higher in the control than that of in vivo or SCF group (p<0.05). Also, the relative abundance of p53 and caspase3 mrna expression were significantly higher in the control than that of in vivo or SCF group (p<0.05). These results show that the supplementation of SCF at fusion/ activation can improve the quality of porcine nuclear transfer embryos. - 126 -

KSAR-81 Imprinting Status Analysis of Igf2/H19 Locus in Mouse Male Germline Stem Cells Shin Hye Oh,, Hyun Seung Lee, Jung Ho Hwang, Myung Rae Park, Sung Jun Uhm, Hoon Taek Lee Department of Animal Biotechnology, Bio organ Research Center, Konkuk University Spermatogonial stem cells (SSCs) isolated from adult mouse testis and these cells can become ES like cells respond to culture conditions. In this study, we analyzed DNA methylation and expression status of insulin like growth factor Ⅱ (Igf2) and H19, which reciprocally expressed imprinted genes in multipotent adult germline stem cells (magscs). Expressions of these genes areregulated by DNA methylation of imprinting control region (ICR) at the 2~4 kb upstream of H19 gene. To compare imprinting status according to culture conditions, we separate cultured in only GDNF contained medium and GDNF+LIF contained medium. Quantitative RT PCR results showed that expression of H19 was approximately 3 fold lower in all magscs compared to mouse embryonic stem (ES) cells, while those of Igf2 was 2.5 and 1.7 fold higher in GDNF and GDNF+LIF treated magscs. Through the bisulfite genomic sequencing, DNA methylation results showed that four CTCF binding sites within the ICR in GDNF treated magscs wereall 100% (9/9, 9/9, and 8/8, respectively) hypermethylated strands. However, GDNF+LIFtreated magscs were slightly different that CTCF binding sites 1, 2, and 4 were 100% (10/10, 9/9) hypermethylated strands and CTCF binding site 3 was 66.7% (8/12) hypermethylated strands. These results suggest that although in GDNF+ LIF treated magscs partially demethylated, in the mass, magscs were hypermethylated at H19ICR and have androgenetic expression status. Therefore, magscs were epigenetically stable for H19 ICR with different culture conditions. Key words: DNA methylation, genomic imprinting, germline stem cells - 127 -

KSAR-82 Ectopic Expression of tmie Transgene Induces Various Recovery Levels of Behavior and Hearing Ability in the Circling Mouse Dong Hoon Yu, Zae Young Ryoo School of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea The circling (cir/cir) mouse is one of the murine models for human non syndromicdeafness DFNB6. The mice have abnormal circling behavior, suggesting a balanced disorder and profound deafness. The causative gene was trans membrane inner ear (tmie) gene of which the mutation is a 40 kilobase genomic deletion including tmie gene itself. In this study, tmie over expression trasngenic mice were established. Individuals with germline transmission have been mated with circling homozygous mutantmice (cir/cir) in order to produce the transgenic mutant mice (cir/cir tg) as agenetherapy. After the genotyping, phenotypic analyses were performed so that the insertion of the new gene might compensate for the diseases such as hearingloss, circling behavior, or swimming inability. Some individuals exhibited complete recovery in their behavior and hearing but the others did not show any amelioration in behavioror hearing. Individual mice had very different levels of tmie transgene expression in the cochlea. These results clearly indicate that tmie protein plays an important role when the appropriate expression level of tmie was expressed in the innerear. The protein levels were variable in each individual and these are thought to induce the differences in disease amelioration levels. Key words: circling mouse, transgenic mouse, tmie, inner ear - 128 -

KSAR-83 수퇘지에서통마늘분말급여가정액및혈액내 Poly phenol 농도에미치는영향 유재원, 최은지, 홍준기, 조규호, 우제석, 전기준, 이성대, 지상윤, 정현정, 박준철, 김인호 1, 김인철, 정일병 농촌진흥청국립축산과학원, 1 단국대학교동물자원학과 마늘 (garlic: Allium sativum) 은백합과 (Lilliaceae) 파속 (Allium) 에속하며, 비늘줄기가있는다년생식물이다. 마늘은전통적으로혈액순환을촉진하며, 피로회복, 체력증진, 살균작용빈혈증, 냉증에좋다고알려져왔다. 마늘의주요성분인 alliin {CH 2 = CHCH 2 S(O)CH 2 CH(NH 2 )COOH[(+) S allylcy steine sulfoxide} 은마늘조직파쇄시 alliinase에의해서 allicin으로분해되어 diallyl disulfide와함께마늘의독특한냄새를낸다. 마늘의유효성분중마을껍질에는육질과마찬가지로 polyphenols 이나 flavonoids, 항산화비타민등항산화성분이다량포함되어있다. 이러한항산화물은정자와난자수의증가를가져오고고환을발달시킨다고하였으며 (Yuriko 등, 2001), 마늘내의항산화물이번식능력향상에영향을줄수있음을시사하고있다. 따라서본연구에서는양돈용사료에통마늘분말급여가혈중 poly phenol 농도변화와정액성상에미치는영향을구명코자실시하였다. 공시축은 12개월령튜록수퇘지 9두를대조구 4두와통마늘분말 0.3% 급여구 5두로나누어시험에배치하여 13주간사육하였다. 정액채취는시험시작일로부터종료일까지 1주간격으로 13주간채취하였고, 채혈및혈중항산화물농도에미치는영향은 1, 3, 6, 9, 12 및 13 주 6회분석하였다. 통마늘분말을급여후총정자수는통마늘분말급여후 6, 7 및 8주에서대조구보다높게조사되었다. 통마늘분말급여후정장내폴리페놀농도분석결과대조구와차이가없었으며, 혈액에서는 1, 3, 및 6주에서는대조구와차이가없었으나 9, 12 및 13주에는대조구에비해유의적으로높게조사되었다. 따라서통마늘급여는수퇘지의혈청내에항산화물농도증가시켜정액성상개선에효과가있는것으로사료되었다. Key words : 마늘, 항산화, 정액성상, 돼지 - 129 -

KSAR-84 Effect of Bacteria Eliminated Sperm by Percoll Method on Sperm Quality and In Vitro Embryo Development in Pig Han Jun Yoo 1, Jun Myeong Jeon 1, Yong Seung Lee 1, Hee Tae Cheong 2, Boo Keun Yang 1, Choon Keun Park 1 1 College of Animal Life Science and 2 School of Veterinary Medicine, Kangwon National University Bacteriospermia is a frequent finding in ejaculated boar semen and can result in detrimental effects on semen quality and longevity. It causes bacterial contamination of reproductive duct and spread of disease in pigs. The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and in vitro embryo development in pig. Ejaculated semen from miniature pig was collected by gloved hand method into a pre warmed (37 ) thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with developmental rate of embryo after IVF using separated and un separated sperm by percoll. The result, number of bacteria in ejaculated semen was decreased rapidly with 65% percoll separation. Abnormality of separated sperm was lower than un separated sperm. Also, the percentage of F patterned separated sperm was higher, while the percentage of AR patterned was lower than un separated sperm on CTC analysis. Developmental rate of embryo using separated sperm by percoll had a higher percentage than with un separated sperm. In conclusion, sperm separated by percoll method showed improvement in sperm quality and developmental rate of embryo than un separated sperm in miniature pig. This work was supported by a grant (Code#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea. Key words: miniature pig, spermatozoa, percoll, CTC, embryo development - 130 -

KSAR-85 The Anti oxidative and Anti apoptotic Effects of Various Antioxidants on the Development of Parthenogenetic Porcine Embryos Hyung Soo Yuh, Zae Young Ryoo School of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, we examined the anti oxidative and anti apoptotic effects of the three antioxidants, β mercaptoethanol (β ME), α tocopherol and extracellular superoxide dismutase (EC SOD), onthe development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1, 19.1, 14.6, 5.0, 17.2, 17.5, 12.0 and 4.0 % for control, 1, 3, 5 μm β ME, 50, 100 μm α tocopherol, EC SOD Tg MEF and EC SOD NTg MEF conditioned medium at day 3, respectively. Here, β ME, α tocopherol and EC SOD Tg MEF conditioned medium at day 3 increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (p<0.05). The average number of total cells and apoptotic cells at the blastocyst was were analyzed at the optimal condition of the three antioxidants. The three antioxidants did not increase the average number of total cells at the blastocyst, but they decreased apoptotic cells at the blastocyst as compared to control without supplementation (p<0.05). When the reactive oxygen species (ROS) level in 2 cell embryos after 1 μm β ME treatment was examined, it was decreased (p< 0.05). In conclusion, we concluded that the three antioxidnats, β mercaptoethanol, α tocopherol and EC SOD, can play a role as a strong stimulator inthe development of parthenogenetic porcine embryos. Key words: parthenogenesis, porcine, embryo, antioxidant, apoptosis - 131 -

KSAR-86 Porcine XBP 1 is a Transcription Factor Involved in ER Stress Response Kyu Sun Lee, Jin Yu Zhang, Ji Su Kim, Kyung Kwang Lee, Kweon Yu, Deog Bon Koo Center for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305 806, Korea The unfolded protein response (UPR) is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER) stress. The occurrence of chronic ER stress has been extensively described in a wide variety of diseases linked to protein misfolding and aggregation. However, the genes involved in the UPR pathway of porcine have not been reported, which take part in the response to a challenge with ER stress. Transcription factor X box binding protein 1 (XBP 1), a key component of the ER stress response, is required for development of specific lineage of cells and maintenance of cellular functions. In this study, we describe the cloning strategy and the functional characterization of porcine Xbp 1 (pxbp 1). We analyzed the molecular characters of pxbp 1 to establish its role in response to ER stress using porcine embryonic fibroblast (PEF) cells. DTT and tunicamycin, well known ER stressors, lead to activation of UPR marker, IRE 1 and BiP in PEF cells. The pxbp 1 mrna undergoes IRE 1 mediated unconventional splicing in the response to ER stress and produces two protein isoforms, active pxbp 1 and inactive pxbp 1. In normal condition, pxbp 1 proteins predominantly localize in cytoplasm, whereas pxbp 1 proteins translocate into nuclei by ER stress. These results demonstrate that pxbp 1 could be use as a specific UPR marker in porcine system and open new opportunities for examining the UPR in vitro development of porcine embryos. Key words: endoplasmic reticulum stress, unfolded protein response, xbp 1, porcine - 132 -

KSAR-87 The Pregnancy Rate Effects of an Estrus Synchronization Protocol using MGA in Korean Cattle Myeung Sik Lee, H. J. Jung, J. S. Ko, S. D. Lee, H. I. Yoon, S. B. Choi, S. G. Im, S. J. Moon 1, B. Y. Ryu 2, M.G. Pang 2 Hanwoo Experiment Station, National Institute of Animal Science, RDA, 1 ChunNam University, 2 Chung Ang University According to breeding size magnification of the cow the time and effort become disturbance plentifully in estrus detection and complicating they minimize and a estrus detection efficiently they will control and propagation at the time of height should have been boiled the necessity is coming to be high in the time. The research which shows in consequently accomplished about MGA+PGF 2 α (control 1) comes under rescuing should have boiled the estrus synchronization methods in compliance with MGA+GnRH +PGF 2 α (control 2) and MGA+Ov synch methods affect in estrus synchronization and pregnancy rate of the Korean cattle. Estrus detection rate about PGF 2 α (control) with 71.7% and from treated with MGA+Ov synch appears highly with 95%, the first service pregnancy rate from control group compares in 51.6% and from treated group appears consider highly with 73.3%, and also, when insemination 3 in standard, the pregnancy rate compares in control group 86.7% was the tendency which from treated group is high with 95%. In cattle and cow to given with MGA+Ov synch in the serum P4 level after and from MGA treated on 4 day after that increases from the condition which with 4.18 ng/ml and 5.43ng/ml was increase respectively, about the at 7 day is maintained, after treated with PGF 2 α depreciated simultaneously at 10 days the estrus was induced and according to the aspect which was respectively to 1.09 ng/ml and 1.25 ng/ml level seemed and estrus synchronization appropriately could be controlled. It was therefore conducted that the treated with MGA+ PGF 2 α the which follows in the estrus synchronization in compliance with controls and MGA+GnRH+PGF 2 α and MGA+Ov synch controls appeared to 91.3%, 90.8% and 95.0%, respectively. In addition, the first treated with MGA+ PGF 2 α which follows in the estrus synchronization controls in compliance with MGA+ GnRH+PGF 2 α and MGA+Ov synch controls was 64.2%, 65.1% and 73.3%, respectively, the pregnancy rate appeared with 92.6%, 93.3% and 95.0%, respectively. Key words: estrus synchronization, ovulation synchronization, conception rate, Hanwoo - 133 -

KSAR-88 Production of hupa Expressing Transgenic Chicken by Retroviral Vector System Sung Ho Lee 1, Heo Young Tae 1, Kyung Tae Lee 1, Seongmin Kim 1, Ji Hyung Lee 1, Teoan Kim 2, Hoon Taek Lee 1 1 Department of Animal Biotechnology, Bio organ Research Center, Konkuk,University Korea, 2 College of Medicine, Daegu Catholic University, Daegu, Korea Transgenic poultry has been expected to be an excellent bioreactor system for producing of pharmaceutical proteins. Plasminogen activators (PA) because of description below are used the function which disjoints plasminogen with plasmin the thrombus solvent. Urokinase type PA (upa) is relation of tissue PA and streptokinase and also itself accomplishes the function. Human upa responsible for prevention and cure a thrombus. Paticulally, it used all type of blood colt, pus and medical sugery. The productive method uses the animal cell, e coli. HuPA demands are increasing cannot be sufficient that demand gradually. So, the new method which and powerful is productive is necessary. This study investigated the possibility of bioreactor system which produce human upa using avian species. Recombinant retrovirus was produced using replication defective MLV based retrovirus vectors encapsidated with VSV G glycoprotein and was injected beneath the blastoderm of non incubated chicken embryos (stage X, at laying). Results showed that, following 21 days of incubation, hatching rate of gene injection group (RSV promoter 5 %, CMV promotor 2.8%) was significantly lower than normal group (80.2 %). PCR and RT PCR analysis of gdnasisolated from different organs showed that out of 20 (RSV 12, CMV 8) of 23 (87%) chicks expressed vector encoded hupa gene in diverse organs with specific primer set. Therefor, These data demonstrate that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, could not only be an efficient bioreactor for the production of pharmaceutical drugs. Specially, it is shows the possible to that chicken could be used as bioreactors to produce hupa. Key words: transgenic, upa, retrovirus vector, chicken - 134 -

KSAR-89 유비쿼터스환경에서새끼돼지하중감지식자동분만알림시스템개발 이장희 1, 백순화 2, 지달영 1, 연승호 3 1 바이오컬쳐 ( 주 ), 2 백석문화대학, 3 KT 컨버전스본부 본연구는분만관리의효율을높이고분만관리시대기하는인력및시간을절감시키고자돼지의분만직후에분만상태를자동으로통보받을수있는시스템을개발하기위해 KT 컨버전스본부및백석문화대학과공동으로수행하였다. 하중감지센서를활용한자동분만알림장치의원리는돼지의분만시출산된신생자돈은모돈의유두쪽으로이동한다는사실에입각하여자돈이이동하는길목에새끼의체중정도의하중을감지하는센서를장치하여새끼가이동하게되면이를분만개시신호로하여관리자에게통보하는시스템이다. 이를위하여후보돈 5두에대해서인공수정을실시하고 NESPOT 기반 (KT, 한국통신 ) 에서의모니터링시스템과무선랜을적용한실시간 data 전송시스템및센서모듈을구현하기위한방법은그림 1과같았다. 초산돈 5두의분만직후의통보및확인을위해서네스팟기반의모니터링시스템을설치하고분만예정일 3~7일이전에하중감지장치가장착된깔판을자동이동통로에설치하여관찰하였다. 그림 1. 하중감지식자동분만알림장치의모식도 그림 2. 하중감지식의도면및자동분만알림장치의통보결과 - 135 -

실험결과분만된초산돈 5두중 4두에서분만직후분만상태를자동으로통보받았으며, 1두에서는하중감지센서의불량을나타내었다 ( 자동통보율 80%). 분만된초산돈 4두의분만상태통보시간은분만개시 ( 첫새끼만출시간 ) 후 ( 前 ) 각각 15 분, 26분, 38 및 25분으로평균 26분이었다. 본실험결과하중감지식자동분만알림장치는돼지의분만관리에매우유용한수단을제공할것으로사료되었다 ( 실용신안출원번호 : 제2008 0079785호. 2008.08.14 출원 ) Key words: 돼지, 분만관리, 하중감지센서, 자동분만알림장치, 센서모듈 - 136 -

KSAR-90 유비쿼터스환경에서온도센서를활용한자동분만알림시스템개발 이장희 1, 백순화 2, 지달영 1, 연승호 3, 홍완표 3 1 바이오컬쳐 ( 주 ), 2 백석문화대학, 3 KT 컨버전스본부 본연구는야간분만이나공휴일분만등분만관리의효율을높이고분만관리시대기하는인력및시간을절감시키고자돼지의분만직전에분만상태를자동으로통보받을수있는시스템을개발하기위해 KT 컨버전스본부및백석문화대학과공동으로수행하였다. 온도센서를활용한자동분만알림장치의원리는온도센서가질내에삽입되어있는동안에는체온을유지하다가양수파열또는태아만출시에는동반탈락된센서가체온보다낮은온도 (30 전후 ) 을감지하게되면이를분만개시신호로하여관리자에게통보하는시스템이다. 이를위하여후보돈 5두에대해서인공수정을실시하고 NESPOT 기반 (KT, 한국통신 ) 에서의모니터링시스템과무선랜을적용한실시간 data 전송시스템및센서모듈을구현하기위한방법은그림 1과같았다. 초산돈 5두의분만전분만상태의통보및확인을위해서네스팟기반의모니터링시스템을설치하고분만예정일 3~7일이전에온도센서가장착된삽입장치를질내에삽입하여관찰하였다. 그림 1. 자동분만알림장치의모식도 그림 2. 자동분만알림장치의분만상태통보과정및결과 - 137 -