True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously. The frequency of true hermaphroditism is higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymes, penes, and uteri. Histomorphologically, the testicular tissues consisted of Sertoli cells that were devoid of spermatogenic germ cells and showed proliferation of interstitial cells. However, the uteri were of normal architecture and had well-developed uterine endometrial glands. The samples were 38, XX female karyotype without the sex-determining region Y (SRY) gene. The findings of this study could contribute to the understanding of true hermaphroditism in the Korean pig industry. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

20ɑ-hydroxysteroid dehydrogenase (20ɑ-HSD) enzyme converts progesterone into biological inactive steroid, thus playing a key role in the termination of pregnancy or estrus cycle and allowing parturition and ovulation to occur in most mammalian animals. However, function and regulation of this enzyme has not known well in primate reproductive physiology. We previously demonstrated the expression level and localization of the 20α-HSD in the reproductive tissues of macaque monkeys of pre-ovulation and pre-parturition period. Also, we amplified about 2005 bp 5'-flanking region from placenta genomic DNA and examined methylation pattern and promoter activity. In present study, we focus on the analysis of molecular characterization of the promoter region by using reporter assay systems. We constructed of deleted mutants (— 890 bp; HSF-2), (— 513 bp; XFD), (— 276 bp; Ap-1) and (— 72 bp; Sp-1) and each mutants were cloned into pGL3-basic vector. These deletion mutants were transfected into CHO cells and co-transfected with Sp-1 or Ap-1 transcription factor plasmids. Compared to — 890 bp and 513 bp promoter fragments alone, transcription activity increased when these constructs were co-transfected with Sp-1 and Ap-1 factor. However, for the absence Ap-1 factor binding site in 276 bp fragment activity dramatically decreased in both transfections. Next, we constructed of 306 bp fragment which is including of Ap-1 binding site and nucleotides converted mutants of the Ap-1 factor binding site. In this result, 306 bp fragment's transcription activity was high as wild type. However, the mutant activity which converted Ap-1 site’s all nucleotide was significantly decreased. These findings are confirmed by gel-shift assay examining Ap-1 binding site on the 20 α-HSD gene upstream region and expression of Ap-1 factor was determined by RT-PCR and Western blot in pre-parturition period placenta and CHO-K1 cell line. Our results indicate that Ap-1 site (— 281 → — 274) (5'-TGTCTCAT-3') plays a crucial role for monkey 20 α- HSD gene transcription.

Interferon-tau (IFNT) is regarded, generally, to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. Although the discovery was made over two decades ago, the molecular mechanisms that regulate IFNT expression are not well understood. Previous studies demonstrated that transcription factors, caudal-related homeobox- 2 (CDX2), JUN, ETS2 and a transcriptional coactivator CREB binding protein (CREBBP) positively influenced IFNT gene expression, while OCT4 may exhibit negative regulation. We and others have observed that both CDX2 and OCT4 coexist during early stages of conceptus elongation but as development proceeds, OCT4 expression diminishes. The objective of this study was to evaluate the stimulatory and inhibitory effects of CDX2 and OCT4, respectively, on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG3 cells were co-transfected with an ovine IFNT (-654 base pair)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. When the reporter construct was co-transfected with either CDX2, ETS2 or CJUN, transcription of -654-oIFNT-Luc increased about two-fold compared to -654-oIFNT-Luc alone. When -654-oIFNT-Luc was co-transfected with both c-jun and Ets-2, activity of -654-oIFNT-Luc was increased about four-fold; cotransfection with JUN, ETS2 and CDX2 increased -654-oIFNT-Luc expression 12X, indicating that the stimulatory activity of the transcription factors was additive. OCT4, when cotransfected with -654-oIFNT-Luc, reduced expression of the later about 40% when compared to -654-oIFNT-Luc alone. When co-transfected with JUN and/or ETS2, OCT4 abolished the stimulatory effect of these transcription factors. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Therefore, when combined with the other transcription factors, CDX2 over the transcriptional inhibitory activity of OCT4. Conversely, when cells were transfected initially with OCT4 (0h) followed by transfection with CDX2, ETS2 and JUN 24 h later, -654-oIFNT-Luc expression was reduced to control (-654-oIFNT-Luc, alone) levels. Not surprisingly, 12S E1A, an inhibitor of transcriptional coactivator CREBBP, reduced stimulation of -654- oIFNT-Luc expression by CDX2, ETS2 and JUN, in combination, by about 40%, indicating that proper transcription complex formation is required for maximum expression. In conclusion, it is suggested that prior to conceptus elongation, pre-existing OCT4 may inhibit IFNT expression, but as elongation proceeds, IFNT expression increases, resulting from incremental increases in CDX2 expression, diminished OCT4 expression, and possibly proper transcription factor complex formation. Key words) Interferon-tau, CDX2, OCT4, transcription

Human protein C (hPC) is a regulator of homeostasis, suggesting its potential use as a therapy for many disease states. Protein C is a zymogen of a serine protease that is activated by thrombin. Protein C, also known as autoprothrombin ⅡA and blood coagulation factor ⅪⅤ, is a zymogenic (inactive) protein, the activated form of which plays an important role in regulating blood clotting, inflammation, cell death and maintaining the permeability of blood vessel walls in humans and other animals. hPC is a 62 KD disulfide- linked heterodimer consisting of a 21 KD light chain and a 41 KD heavy chain which circulates as an inactive zymogen in plasma. In this study, we focus on generation of hPC transgenic mice. hPC transgenic mice were produced by using micro-injection method. The hPC cDNA was cloned into pBC1 vector under goat β-casein promoter. One-cell stage embryos microinjected were transferred to 24 recipient mice on day 1 of the estrus cycle. We screened 61 mice by the PCR. Four line transgenic mice were identified and confirmed expression of protein C gene in mammary gland and several organ. We also analyzed the expression of mRNA and protein through the northern blot and western blot in mammary gland of hPC transgenic mice. hPC was localized in the alveolar epithelial cell by immunohistochemistry. Now, we are collecting the milk from the 2 found lines. After then, we are checking the activity of hPC produced from mice milk.

Phosphoprotein Enriched in Astrocytes (PEA15) is a 15kD-sized intracellular signaling protein, highly expressed in astrocytes and constitutively expressed in peripheral tissues. Recently it was found that PEA15 expression was elevated in patients suffering type 2 diabetes and suggested to be involved in the syndrome of insulin resistance. To investigate the functional role of PEA15 for the control of blood glucose level, we produced a transgenic pig over-expressing mouse PEA15 (mPEA15). As a model animal, pig has many advantages. They have a higher fecundity and a short generation time and are physiologically similar to human. Using the transgenic pig, we carried out a series of experiments to establish a link between PEA15 expression and the insulin resistance. Our results suggested that, compared with control pig, mPEA15 pig has, (1) a higher blood resistin level, (2) a lower cell membrane-embeded GLUT4 level, and (3) a lower glucose clearing ability based on oral glucose tolerance test (OGTT). When our results combined, it can be concluded that mPEA15 over-expressing pig has many symptoms of insulin resistance and these pigs will become a useful disease model to investigate diabetes mellitus in the near future.

Setdb1/Eset, a histone lysine methyltransferase, is recruited by various transcription factors to modify local chromatin. The observation that Setdb1-null blastocysts fail to produce epiblast-lineage cells suggests a role for Setdb1 in generating mouse embryonic stem cells (mESCs). When examined in mouse zygotes, Setdb1 proteins appeared as dots at perinucleolar rims of pronuclei, with the dot-shaped signals more prominent in male pronuclei. Setdb1 signals were observed diffusely in the nucleus from the two-cell stage onward and, by the blastocyst, took a punctate form, away from nucleolus. Such varying expression patterns suggest its involvement in diverse biological processes at preimplantation stage. Setdb1 appeared in Oct4-positive cells of inner-cell-mass origin but not in trophectoderm-lineage cells in blastocyst outgrowths. Setdb1 co- immunoprecipitated with Oct4 in mESCs, and Setdb1 expression was markedly reduced upon retinoic acid- induced differentiation. These observations suggest that Setdb1 has an important role in maintaining the self-renewal of mESCs through collaboration with Oct4.

한우 암소를 사육하는 농가 현장에서 번식효율은 농가의 수입에 지대한 영향을 미치는 요인으로써 국내 번식 간격은 15.9개월로써 일본의 번식 간격 13.6개월에 비하여 2개월 이 상이나 차이가 나고 있고, 한우가 번식 간격이 길어진 주된 요인은 우선 분만후 발정재귀일 의 지연으로 인한 무발정 기간이 길어진 영향과 인공수정시 수태율이 떨어지고 있는데서 공태기간이 길어지는 요인으로 작용하는 것을 꾭을 수 있으며 따라서 다양한 발정동기화 방법중에서 한우에게 가장 높은 수태율을 제공할 수 있는 방법을 개발하고자 수행하였으며 아울러 발정동기화 처리가 혈중 P4 수준에 미치는 영향을 조사하였다. 발정동기화와 배란동기화를 혼합한 복합 프로그램 적용시 발정발현율에서 대조구는 PGF ₂α (25 mg; i.m.)를 1차 투여하고 11일이 경과한 후 PGF₂α(25 mg; i.m.)를 2차 투여하고 72시 간에 인공수정하였고, 처리 1구는 CIDR을 삽입하고 7일후 CIDR 제거와 동시에 PGF₂α(25 mg; i.m.)를 투여하고 72시간 경과후 인공수정하였으며, 처리 2구는 CIDR을 삽입하면서 GnRH 100 μg을 투여하고 7일후 CIDR를 제거함과 동시에 PGF₂α(25 mg; i.m.)를 투여하고 2일후 GnRH 100 μg을 재투여하고 24시간후 인공수정을 실시한 처리구로써 발정발현율은 대조구 85%, 처리 1구 88.3% 및 처리 2구는 93.3%로 나타냈다. 발정동기화와 배란동기화 복합 프로그램을 적용했을 때 P4 수준은 0일차에 1.64～6.56 ng/ml이였고 3일이 경과한 후 7.18～9.9 ng/ml로 최고 수준으로 증가하였고, PGF₂α 투여 시 점인 7일이 경과한 후 6.59～9.58 ng/ml 수준으로 분포하였으며, 10일이 경과한 인공수정 시점에는 0.7～1 ng/ml 수준으로 하강하여 대조구, 처리 1구, 처리 2구 공히 발정이 적절히 유도되었으나 인공수정후 발정 한주기가 지나간 재발정 시기에는 처리 1구 11.02 ng/ml, 처 리 2구 13.65 ng/ml 대비 대조구에서는 낮은 수태율로 인하여 혈중 P4 수준이 5.73 ng/ml 수준으로 낮게 나타났다. 발정동기화와 배란동기화 복합 프로그램 병용처리시 수태율 개선 효과에서 발정동기화와 배란동기화 복합 프로그램 사용에 따른 1회 수정 수태율은 대조구 51.6% 대비 처리 1구 58.3%, 처리 2구 66.6%로써 15% 개선되는 경향이였고 수태율은 각각 83.3%, 88.3% 및 93.3%였다.

today's dairy production management systems, reproductive efficiency affects the income of any farm by influencing overall milk production, genetic gain, amount of replacement heifers, and wise cullling decisions. Nutrition, management, and genetics play a major role achieving this maximum herd-reproductive performance. Several reports using trace minerals on the diet of cattle have shown reproductive effects. The main justification for using blocks, to provide deficient nutrients is, therefore, their convenience for packaging, storage, transport and ease of feeding. Dairy cattle are annually exposed to prolonged periods of elevated humidity and heat which reduce feed intake and reproductive performance. The objectives of the present study were to evaluate the effects of mineral on reproductive performance of dairy cows during Summer. The experimental group of cows had access ad libitum to the mineral lick in the following composition : Zn, Fe, Mn, Cu, I, Co, Se. Environmental heat load was described using the THI (temperature humidity index) because thermoregulation in cows is affected by both Temp and RH. The THI was calculated: THI=(0.8×Temp)+[RH×(Temp— 14.4)]+ 46.4. All cows were monitored for estrus twice daily in the morning and late afternoon. And any animal found in estrus was artificially inseminated. Pregnancy diagnosis was performed via ultrasonography at 45 to 70 d after insemination. The visitation and intake of mineral were higher with the maximum THI. The mean intake of mineral block was 23.1 kg in Summer and 17.7 kg in other seasons. The reproductive performance was considerably improved by mineral supplementation. The factors influencing consumption of mineral mixtures include: soil fertility and forage type, available energy and protein, individual requirements, salt content of the water, palatability of mineral mixture, availability of fresh minerals and physical form of minerals. These results show that minerals have a great impact on animal's reproductive physiology.

젖소의 고능력화와 동반하여 전 세계적으로 번식효율은 매년 저하되고 있다. 소의 번식효 율에 영향하는 요인은 매우 다양하며 농가의 사양관리 수준에 따라 여러 가지 번식 문제가 발생하고 있다. 대부분의 농가들이 번식현황을 제대로 파악하지 못하고 있으며, 문제점 또 한 파악하지 못하고 적절한 대응을 위한 자가진단이 이루어지지 않고 있는 실정으로 무엇 보다 번식현황 및 경영진단이 필요한 실정이다. 본 연구는 국립축산과학원 낙농과에서 개 발 된 젖소 번식상황 자가진단 프로그램을 이용해 6개 농가 450두를 공시하였으며, 번식상황 자가진단 프로그램 기술 투입 후 2년 동안 번식성적을 비교 분석하였다. 기술 투입 전 6개 농가의 평균 분만간격은 438일, 평균 공태일수는 156.8일, 분만 후 첫 수정일수는 98.6일, 평균 수정횟수는 2.1회, 발정발견율은 32.5% 및 수태율은 41.2% 였다. 번식상황 자가진단 프로그램 기술 투입 후 6개 농가의 평균 분만간격은 409.8일로 약 28일 단축 되었으며, 평 균 공태일수는 129.3일로 약 27일, 분만 후 첫 수정일수는 77.2일로 약 21일로 발정 한 주 기가 단축되는 것을 확인할 수 있었다. 수정횟수는 2.0회로 기술투입 전후 비슷한 수치를 보였으며, 발정발견율은 47.5%로 기술투입 전보다 약 15%, 수태율에서도 50.5%로 약 9% 정도 높았으며, 모든 번식성정이 향상되는 것을 확인할 수 있었다. 번식상황 진단 프로그램 은 다수 있지만 농가에서 쉽게 활용할 수 있는 프로그램이 없는 상태에서 누구나 손쉽게 활용할 수 있는 엑셀로 된 번식상황 자가진단 프로그램을 활용하여 농가의 문제점을 확인 하고 목표를 세워 노력한 결과라고 생각된다. 추후 번식상황 자가진단 프로그램을 적극적 으 로 활용한다면 농가의 번식효율 향상에 크게 기여 할 것으로 생각되며, 국내 낙농가들도 분 명한 목표설정, 농가의 번식현황에 대한 정확한 문제점 파악을 통해 번식효율 개선에 최선의 노력을 기울여야 할 것이다. ○ 기술 투입 전 · 후 6개 농가 번식성적 비교 구 분 평균분만간격 평균공태일수 분만 후 첫수정일수 평균수정횟수 발정발견율 수태율 기술투입 전(평균) 438일 156.8일 98.6일 2.1회 32.5% 41.2% 기술투입 후(A) 408 118.7 72.6 2.0 48.3 53.0 기술투입 후(B) 402 115.5 68.9 1.8 52.0 56.8 기술투입 후(C) 412 132.4 81.5 2.0 43.2 49.2 기술투입 후(D) 417 141.8 81.3 2.0 45.8 45.3 기술투입 후(E) 405 128.6 76.8 1.9 50.6 52.6 기술투입 후(F) 415 138.6 81.9 2.0 45.3 46.2 기술투입 후(평균) 409.8 129.3 77.2 2.0 47.5 50.5

Embryo transfer (ET) is the final procedure for getting pregnancy through assisted reproductive technology such as IVF (in vitro fertilization), SCNT (somatic cell nuclear transfer). In our laboratory, the porcine cloned embryos loaded in ET medium are carried for 3 hours by portable incubator because of the great distance from the laboratory to the experimental farm. Thus, before transferring into recipient, porcine cloned embryos are exposed in vitro condition for long time. Medium which is used in this process is the TALP (Tyrode’s medium supplemented with 10 mM HEPES), but it includes little nutrients for embryo. Thus, the aim of this study is to determine whether ET media containing nutrients affect the in vitro development of embryos compared to TALP. For the experiment, porcine zygote medium (PZM)-5 which has amino acids for developing embryo was chosen as ET medium containing nutrients, added 10 mM Hepes as PZM-5 does not contain buffering system. For experiment, we carried out parthenogenesis through a chemical method using Thi/DTT. Parthenogenetic embryos were cultured in PZM-5 for 2 days, and then they were randomly divided into two group; loaded in a straw with TALP or PZM-5-Hepes, respectively. They were stored in a portable incubator for 3 hours to simulate the time consumed in ET, thereafter embryos in both TALP and PZM-5-Hepes groups were respectively cultured in PZM-5 for additional 5 days. All experiments were repeated 5 times. In result, blastocyst formation rate were 22.46%±1.47 and 23.17%± 2.13, respectively and total cell number were 32.9±2.22 and 37.09±2.18, respectively. There is no significant difference between TALP and PZM-5-Hepes groups. * Further study will investigate effect of PZM-5-Hepes on in vivo development of porcine cloned embryo. This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program and TS Corporation.

수정란 이식기술은 가축의 효율적인 증대와 조기번식에 유용한 기술로 알려져 있으며, 단 기간에 다량의 수정란을 확보할 수 있다. 수정란 이식기술은 한우의 개량 및 육종과 희소한 우의 조기증식 및 유전자 다양성 확보, 유전자 보존 등의 목적에 접근하는 데 매우 유용한 방법으로 인식되고 있다. 본 연구는 일반한우와 희소한우간의 비외과적 채란 성적과 이식 가 능 수정란의 비율을 비교 검토하였다. 본 실험은 국립축산과학원 가축유전자원시험장에서 보유중인 일반한우 13두, 희소한우 11두를 공시하였다. 발정주기에 관계없이 CIDR를 질 내에 삽입하고, 4일 후부터 4일간 FSH (Antorin) 28AU를 12시간 간격으로 점감 주사하였다. FSH(Antorin) 투여 3일째 CIDR를 제 거하고, PGF2α(Lutalyse) 3 ml을 근육 주사하여 과배란을 유기하였다. 인공수정은 PGF2α (Lutalyse) 주사 후 발정을 확인하고 12시간 간격으로 3회 실시하였으며, 1차 인공수정 전 GnRH 1.0 ml을 주사하였다. 수정란 회수는 1차 인공수정 후 8일째에 3-Way Catheter를 이용 비외과적 방법으로 채란하였다. 일반한우와 희소한우에서 회수 된 수정란 총 개수는 각각 87개, 77개로 그 중 이식 가능 한 수정란 수는 51개, 53개였다. 평균 회수란 수는 각각 6.69±1.54개, 7.00±1.13개로 차이 가 없었으며, 평균 이식가능 수정란 수는 각각 3.92±1.11개, 4.82±1.09로 유의적인 차이는 없는 것 같다. 이 같은 결과들로부터, 일반한우와 희소한우 간 수정란 회수율과 이식가능 수정란 간의 비율의 차이는 소의 종 특이적인 차이보다 같은 종 내의 개체 차이로 인식해야 할 것으로 사료된다.

소에서 BCS는 영양관리를 평가하는 방법으로 이용되고 있으며, BCS 조건에 따라 번식 성적에 영향을 주며 난소활동 재개 지연 등의 현상을 초래하는 것으로 알려져 있다. 우리나 라에서 수정란 이식기술은 우수종축의 우수한 유전능력을 단기간에 확대 보급할 수 있는 방법으로 이용하고 있다. 수정란 이식 기술은 단기간에 다수의 수정란을 확보 할 수 있고, 우수한 종축의 능력을 증식 · 보존할 수 있는 기술로 전 세계적으로 이용하고 있는 기술이 다. 본 실험은 한우의 BCS별 체내수정란 회수율과 이식가능 수정란의 비율을 비교하였고, BCS별 채란일로부터 발정재귀까지의 기간을 비교 조사하였다. 본 실험은 국립 축산과학원 가축유전자원시험장에서 보유중인 한우 55마리를 공시하고, BCS 2.5 이하인 개체 8두, BCS 3.0인 개체 36두, BCS 3.5이상인 개체 11두, 세 그룹으로 구분하였다. 발정주기에 관계없이 CIDR를 질 내에 삽입하고, 4일 후부터 FSH(안토린) 28 AU를 12시간 간격으로 주사하고, FSH(안토린) 주사 3일째 CIDR을 제거함과 동시에 PGF2α (Lutalyse) 3 ml을 주사해 과배란을 유기하였다. 인공수정은 PGF2α(Lutalyse) 주사한 뒤 발 정 확인 후 12시간 간격으로 3회 실시하였다. 1차 인공수정 전 GnRH 1.0 ml을 주사하고, 수정란 회수는 1차 인공수정 후 8일째에 3-Way Catheter를 이용해 실시하였다. 발정재귀 확인은 채란 후 발정확인일까지의 날짜 수로 계산하였다. BCS 2.5 이하, 3.0, 3.5 이상 그룹에서 회수된 총 수정란 개수는 각각 44개, 363개, 87개 였으며, 평균 회수란의 개수는 각각 6.29±1.91개, 12.96±1.50개, 7.91±1.68개였다. 평균 이 식가능한 수정란의 개수는 각각 5.43±1.67개, 8.50±1.41개, 4.82±1.79개로 BCS 3.0인 집단 이 다른 그룹에 비해 높은 수정란 회수율과 이식가능 수정란 회수율을 보였다. 발정재귀일 까지 걸리는 기간은 각각 평균 28.40±1.94일, 22.38±4.02일, 19.22±6.76일로 나타나 유의 적 차이는 없는 것으로 보인다. 본 실험의 결과로부터 적절한 BCS는 체내수정란 회수시 수정란 회수율과 이식가능한 수 정란의 비율에 좋은 영향을 미치는 것을 알 수 있었으며, BCS와 채란 후 발정재귀일의 관 계는 집단 내 개체간의 차이가 큰 것으로 사료된다.

한우 고급육 생산을 위해 종모우의 개량과 선발이 이루어지고 있으나 좀 더 효율적인 개 량을 위해서는 고급육을 생산할 수 있는 한우 암소의 개량이 필수적이다. 따라서 본 연구는 경기도 안성지역 한우의 육질 개량을 위해 지역 한우농가의 소를 대상으로 육질 초음파 측 정과 육질관련 유전자와의 비교 분석을 하기 위해 실시하였다. 육질분석을 위해 3산 이상 한우 번식우 86두를 대상으로 초음파로 등심단면적, 등지방두께, 근내지방을 측정하였다. 각 개체별로 혈액을 채취하여 유전자를 분석하였으며, 유전자 분석은 육질관련 유전자로 알 려진 FABP4와 FABP5-1, FABP5-2를 이용하였다. 개체별로 고급육 생산을 위한 사양관리 전과 사양관리 후 표현형과 유전자와의 상관 관계를 분석하였다. 번식우의 일반 사양관리 조건(비육전)에서 FABP4는 육질초음파 측정 결과와 유전자 분석과의 상관 관계가 0.006으 로 매우 높은 것으로 나타났다. 그러나 FABP5는 상관 관계가 0.084로 비교적 낮은 결과를 보였다. 그러나, 비육후 도축하였을 때 FABP4는 육질초음파 측정 결과와 유전자 분석과의 상관 관계가 0.0054로 매우 높은 것으로 나타났다. 그러나 FABP5는 상관 관계가 0.0899로 비교적 낮은 결과를 보였다. FABP4 유전자에서 비육전 초음파 측정 결과와 비교하였을 때 GG type은 7.18, AG type은 8.50, AA type은 10.50이었으나(n=50), 비육후 도축한 결과 GG type에서 4.88, AG type은 2.33, AA type은 나타나지 않았다(n=20). FABP5 유전자에 서 비육전 초음파 측정 결과와 비교하였을 때, GG type은 9.30, AG type은 7.95, AA type 은 7.40이었으나(n=50), 비육후 도 축결과는 GG type에서 2.67, AG type은 3.50, AA type 5.00으로 나타났다(n=20). 두 가지 유전자에서 비육전과 비육후 유전자와 표현형과의 관계 가 반대로 나타났음을 알 수 있었다. 즉, 이 결과를 통해 육질관련 유전자를 보유하고 있더 라도 비육을 하지 않고 일반 번식우 사양관리를 하였을 때 육질이 떨어진다는 것을 알 수 있었다.

Cathepsins (CTSs), a family of lysosomal cysteine proteases, and their inhibitors (CSTs) play a critical role in remodeling of the uterine endometrium and placenta for the establishment and maintenance of pregnancy in many animal species including rodents, sheep, cow and pigs. It has been shown that the high rate of pregnancy failure by somatic cell nuclear transfer (SCNT) is associated with abnormal placental development. Our previous study has shown that CST6 is highly expressed in the uterine endometrium from mid to late pregnancy in pigs. In this study, to understand whether appropriate endometrial and placental tissue remodeling occurs in the uterine endometrium from gilts with conceptuses derived from SCNT during pregnancy in pigs, we investigated expression of CST6 in the uterine endometrium. Uterine endometrial tissues were obtained from gilts that carried SCNT-derived normal conceptuses (NT-No) and abnormal conceptuses (NT-Ab), and from gilts carrying conceptuses from natural mating (Non-NT) on D114 of pregnancy. Immunoblot analysis showed that CST6 protein levels in the endometrial tissues of gilts carrying NT-No were lower than those of gilts carrying Non-NT. The levels of CST6 protein in the endometrial tissues of gilts carrying NT-Ab decreased even more than those of gilts carrying NT-No. These results indicate that decreased expression of CST6 in the endometrium with NT-No and NT-Ab reflects inappropriate endometrial tissue remodeling and pregnancy failure of pigs with SCNT derived conceptuses and that CST6 plays an important role for the maintenance of pregnancy in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA, Republic of Korea.

Prostaglandins (PGs), especially PGE2 and PGF2α, are critical local mediators that play important role in luteolysis and maternal recognition of pregnancy in pigs. Luteolysis during the estrous cycle in pigs is induced by PGF2α synthesized and secreted by the uterine endometrium. In pregnant pigs, PG synthesis is changed in favor of PGE2 synthesis. However, molecular and cellular mechanisms by which PGE2 and PGF2α are produced in the uterine endometrium during pregnancy are poorly understood. Therefore, we determined immunolocalization of PTGES, AKR1B1, CBR1, and HPGD that are involved in synthesis and catabolism of PGE2 and PGF2α in the uterine endometrium during the estrous cycle and pregnancy in pigs. Uterine endometrial tissue samples were collected from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90, and D114 of pregnancy. Spatial expression of all proteins studied was analyzed by immunohistochemistry. PTGES were localized primarily to luminal and glandular epithelial cells. AKR1B1 were localized to luminal epithelial cells during early pregnancy and chorionic membrane during mid- to late pregnancy. CBR1 and HPGD were localized to luminal epithelial cells. Our results showed that expression of proteins responsible for synthesis and catabolism of PGE2 and PGF2α were dynamically regulated in the uterine endometrium during the estrous cycle and pregnancy in pigs. These results indicate that PGs play critical roles to support the establishment and maintenance of pregnancy at the maternal-fetal interface in pigs. This research was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA, Republic of Korea.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN or corresponding configurations to stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the follicle size could determine chromatin configuration in porcine oocyte. For this experiment, follicles was divided into three groups (<1 mm follicle, 1～3 mm follicle and 3～6 follicle). Using DAPI staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN, SN-NSN and NSN configurations. MⅠ and M Ⅱ of three groups's Mature oocytes by staining was confirmed the configuration of chromatin. The maturation rate and parthenogenetic development potential were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of porcine embryos.

An understanding of oocyte gene expression is a necessary for the study of early female gamete development. Recently, oocyte has been used in many techniques such as somatic cell nuclear transfer, intracytoplasmic sperm injection and embryonic stem cell derivation. The purpose of this study was to investigate in the proteomes of pig oocytes and identification of differential proteins between using DIGE technique. In this experiment to overcome of limitation of 2D gel method like a low reproducibility and low sensitivity for proteome analysis of very small quantities, 2D fluorescence difference gel electrophoresis (DIGE), which enables co-detection of up to three samples on the same 2DE gels with CyDyes was used for analysis of oocyte proteins. Proteins within an isoelectric point (pI) range of 3 to 10 and a molecular weight (Mw) range of 20～100 kDa were primarily analyzed in DIGE with 2 replications of each sample. Approximately 1000 spots were detected in 2-D gel. Then, image analysis of DeCyder was performed to detect variations in protein spots between mature oocyte and parthenogenesis embryo. In the comparison of mature oocyte and parthenogenesis embryo, 11 spots were identified to be up-regulated proteins and 2 spots to be down-regulated proteins in parthenogenesis embryo, among which proteins were zona pellucida glycoprotein 4, transferrin receptor, apolipoprotein B, L-3-Hydroxyacyl Coa Dehydrogenase Revisited, cytochrome P450 2C33, similar to Monocarboxylate transporter 2, 2'-5' oligoadenylate synthetase 3, interferon alpha/ beta receptor-1, Chloride channel protein 6, pyruvate carboxylase as well as2'-5' oligoadenylate synthetase 3 using MALDI-TOF-MS. These results suggested that differential proteins were present between mature oocyte and parthenogenesis embryo.

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). In our previous study has results showed that the donor cells treated with 5-aza-2’- deoxyctidine (5-aza-dC, DNA methylation inhibitors) and Trichostatin A (TSA, histone deacetylase inhibitors) could improve the development of porcine nuclear transfer embryos in vitro. In this study we want to investigate why these two drugs treatment with the donor cell can improve the cloning efficiency, whether they can alter the epigenetic status of the genome of the donor nucleus. This study included 6 groups: control group, the donor cell (porcine fetal fibroblast cell) with no treatment; 2.5 nM 5-aza-dC group, the donor cells treated with 2.5 nM 5-aza-dC for 1h; 5-aza-dC group, the donor cells treated with 5 nM 5-aza-dC for 1h; TSA group, the donor cells treated with 50 nM TSA for 1h; 2.5 nM 5-aza-dC+TSA group, the donor cells treated with 2.5 nM 5-aza-dC for 1h and subsequently treated with 50 nM TSA for another 1h; 5-aza-dC+TSA group, the donor cells treated with 5 nM 5-aza-dC and 50 nM TSA together for 1h. The first experiment detected the DNA methylation status in the different groups. After treatment with these two drugs, the DNA methylation level of the donor cells decreased, however there is no significant difference among the groups. This result indicated that the donor cell treatment with 5-aza-dC and TSA can partially alter the DNA methylation status of the donor cells. The second experiment checked the histone acetylation level of the donor cells treated with these two drugs by western blot. TSA, 2.5 nM 5-aza-dC+TSA, 5 nM 5-aza-aC+TSA, these three groups can significantly improve the hisone acetylation level compared with control and 5-aza-dC groups, there is no significant difference among these three groups. The results of this study suggest that the donor cells treated with 5-aza-dC and TSA can partially decrease DNA methylation and can significantly improve the histone acetylation level of the donor cells, these alterations of the epigenetic modification maybe can improve the clonging efficiency.

The objective of the present study was to investigate the effects of different concentrations of sorbitol supplementation for in vitro maturation medium and in vitro culture medium, on porcine cumulus oocyte complexe(COC) maturation and subsequent developmental capacity after parthenogenetic activation. Porcine COC were cultured for 44 h(0～ 22 h termed MI stage and 22～44 h termed MII stage) in TCM199 without(— ) or with(+) sorbitol (20 μM, 100 μM, 200 μM), and the resultant metaphase II oocytes cultured in PZM-3 for 7 days following activation. Our results showed that supplementation with appropriate concentrations of sorbitol (20 μM) during full term maturation culture(MI+/MII+) significantly(p<0.05) improved blastocyst formation rates and total cell number. When the concentration of sorbitol were increased to 100 μM and 200 μM during maturation culture, the maturation rate of COC were significantly reduced compared with 20 μΜ or control groups. Also blastocyst formation rates significantly(p<0.05) reduced with increasing concentration of sorbitol(200 μM). Supplementation with sorbitol(20 μM, 50 μM, 100 μM) into PZM-3 for in vitro culture significantly(p<0.05) inhibited blastocyst formation compared with control group. However, the blastocyst formation rates start to rise again when 50 μ M sorbitol was used for the first 48 hours and then cultured in PZM-3 without sorbitol. There was no significant difference in cell number between control and sorbitol treated groups. When the activated oocytes were cultured in PZM-3 for 48h and then cultured in PZM-3 with sorbitol, interestingly, the blastocyst formation rate was similar to that of PZM-3 with sorbitol for in vitro culture and significantly lower than control group. These results suggest that addition of low concentrations of sorbitol(20 μM) during oocyte maturation is beneficial for subsequent blastocyst development and improved embryo quality. However, treatment with sorbitol supplementation during in vitro culture medium is negative effect to blastocyst formation.

The present study examined the expression of porcine sirtuin 1–3 (Sirt1–3) genes in immature (germinal vesicle; GV stage), mature (metaphase II; MII stage) oocytes, preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the role of sirtuins in oocyte nuclear and cytoplasmic maturation, and embryonic development of PA and IVF embryos using sirtuin inhibitor [5 mM nicotinamide (NAM) and 100 μM sirtinol]. The expression of Sirt1–3 mRNA was significantly (p<0.05) up-regulated during IVM. The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF and SCNT were gradually (p<0.05) decreased from MII stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1 and Sirt3 in SCNT blastocysts were significantly lower than IVF blastocysts. Treatment with nicotinamide (NAM) during IVM resulted in significantly decreased nuclear maturation but it was restored when NAM treated with 2 μM resveratrol (RES; known as antioxidant and sirtuin activator) compared to the control (control: 88.9%, NAM: 67.9% and NAM+RES: 86.4% respectively). Intracellular reactive oxygen species (ROS) level of oocytes matured with NAM was significantly increased and with NAM+RES was significantly decreased compared to the control. Treatment with sirtuin inhibitors during IVC resulted in significantly decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate both PA and IVF embryos. Oocytes treated with NAM during IVM showed significantly lower expression of PCNA, Bax, Bcl-2, POU5F1 and Sirt1–3 compared to the control. Oocytes treated with NAM+RES during IVM restored gene expression except POU5F1. Similarly, PA derived blastocysts treated with NAM during IVM showed down-regulation of PCNA, Bax, Bcl–2, POU5F1 and Sirt1–2. The blastocysts derived from PA embryos treated with sirtuin inhibitors during IVC showed lower (p<0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly de -creased in both NAM and sirtinol treated groups compared to the control. These findings indicate that Sirt1–3 which are transcribed and stored during oocyte maturation may have physiological and important roles in porcine oocyte maturation and preimplantation embryonic development by regulating gene expressions. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.