Earticle

Interferon induced transmembrane protein-1 (IFITM1) is one of transmembrane protein which is differentially expressed in uterus during estrus cycle and pregnancy, that IFITM1 gene is highly expressed in estrus stage by the effect of estrogen, and in parturition by the effect of PGF2 alpha. This genes are also up-regulated in cells with hyperactivation of the WNT/β-catenin signaling pathway. In this study, to identify a function of IFITM1, the binding partner of IFITM1 were determined using immunoprecipitation and LC- MASSMASS methods. 1, 3 and 5 ug of polyclonal anti-IFITM1 antisera were used for immunoprecipitation, and the 75 kDa of specific band was detected in silver stained polyacylamide gel. This band were chracterized using LC-MASS-MASS, and revealed this band is glucose regulated protein 75 (GRP75) which binds to p53 and inhibits the p53 action in nucleus. To identify the localization of GRP75 in cells, immunocytochemical approach has been applied, and GRP75 is expressed in mitochondria of L929 murine connective tissue cells. Co-localization study between IFITM1 and GRP75 in L929 cell identified that these two proteins were closely expressed in mitochondria. Although the role of the interaction of these two protein need to be clarified in various biological phenomena, this data suggest that close interaction of IFITM1 and GRP75 may regulate cellular functions in uterus on sets of estrus cycle and pregnancy.

191개의 아미노산으로 구성된 인간 성장 호르몬(human Growth hormone, hGH)은 성장 촉진뿐만 아니라 근육량 증가, 뼈 강화, 체내 지방 분해 등의 약리적 작용을 가지며, 이와 연관된 여러 질환에 대한 치료 및 기타 치료보조제(미용 및 노화억제 분야 등)로 사용되고 있다. hGH를 비롯한 대부분의 단백질 치료제는 98% 이상이 주사제로 투여되고 있는데 이 러한 투여 방식은 환자의 통증 및 감염 가능성 뿐만 아니라 투여 횟수가 많은 경우에는 시 간적, 비용적, 편리성의 측면에서 환자에게 부담이 가중된다. 본 연구에서는 이러한 문제점 을 해결하기 위하여 감염의 우려도 없으며 주사공포증 없이 복용할 수 있는 hGH와 hTf 단 백질을 융합시킨 형태의 경구 투여용 hGH를 생산하고자 하였다. hGH와 hTf 융합 단백질의 유전자 서열은 HepG2 세포에서 분리한 RNA로부터 RT-PCR 을 수행하여 cloning한 hTf 유전자의 서열과 cDNA로 합성한 hGH 유전자 서열을 cyclo peptide linker나 helical peptide linker로 연결하여 retrovirus vector에 도입하였다. 구축한 각 virus vector는 virus 생산 세포주인 GP2 293과 VSV-G 피막단백질 유전자를 이용하여 retrovirus로 생산한 후, 닭의 배아섬유아세포인 CEF와 CHO 세포에 감염시켰다. 각 세포에서 hGH-hTf 유전자의 발현은 RT-PCR, Western blot, ELISA 실험을 통하여 확인하였다. RT-PCR 실험 결과에서는 virus에 감염된 세포주와 감염되지 않은 세포주에서 GAPDH 유전자에 대한 증폭 단편이 확인된 데 반해, hGH 유전자와 WPRE 서열에 대한 증폭은 virus 에 감염된 세포주에서만 이루어 졌다. Virus에 감염된 세포에서 hGH 단백질과 hTf 단백 질의 발현 양상을 확인하기 위하여 각각의 세포 배양액과 세포에서 분리한 단백질을 이용 하여 Western blot을 실시하였다. 세포 배양액과 세포에서 분리한 단백질에서의 hGH와 hTf 발현을 비교한 결과, 두 단백질 모두 세포 배양액에서의 발현이 강한 것으로 확인되었다. hGH 단백질은 약 20 kD의 크기를 나타내었으며 hTf 단백질은 80 kD의 크기를 가지는 것 으로 확인되었다. 각 virus에 감염된 세포에서는 hGH 단백질이 hTf 단백질과 융합된 형태 로 발현되어 약 100 kD의 크기를 가지는 것으로 확인되었다. ELISA 분석 실험에서도 virus 에 감염된 각 세포에서의 hGH 단백질의 발현 및 분비를 확인하였다. 본 연구에서 확보한 경구 투여용 인간 성장 호르몬인 hGH-hTf는 차후 형질전환 동물의 개발이나 물질 생산 세포주의 확립을 통해서 대량생산함으로써 주사용으로만 개발되어 있 는 바이오의약품의 경구용화 관련 연구에 필요한 핵심 기술을 제공할 수 있을 것이다. * 본 연구는 농촌진흥청 차세대 바이오그린21사업(과제번호: PJ00804101)의 지원에 의해 이루어졌다.

현재 널리 사용되고 있는 목적 유전자 발현 조절 시스템은 Gossen과 Bujard에 의해 개발 된 tetracycline-inducible gene expression system (Tet system)으로서, 유도체인 tetracycline 계열의 물질의 공급 여부에 따라 외래 유전자의 발현을 가역적이며 유도적으로 조절할 수 있다. 이 시스템은 중금속이나 steroid hormone 등을 이용한 발현 조절 시스템에 비해 발 현 유도율이 높고 비특이적인 발현이 상대적으로 낮으며, 유도물질에 의한 세포 독성이나 다 면 적 영향이 거의 나타나지 않는 장점을 가진다. 그러나 비유도 조건에서 완벽한 발현 억제가 이루어 지지 않은 관계로 background 활성이 미미하게 존재하고 있어서 이를 해결하기 위 한 연구가 진행되고 있으며 유도물질에 대한 transactivator의 감수성을 향상시켜서 낮은 유 도체의 농도에서도 유전자의 발현을 극대화하기 위한 시도도 이루어지고 있다. 본 연구에서는 가장 개선된 형태의 Tet system의 각 요소들을 재조합하여 one vector 형 태의 유전자 발현 조절 시스템을 구축한 후, 일차배양한 세포주에서 이 시스템의 효율성을 증명하고자 하였다. 재조합한 요소는 유전자 발현 조절을 위한 Tet system의 구성에 있어 서 가장 중요한 2가지 요소인 transactivator와 tetracycline response element (TRE)로 각 각 의 일부 서열이 변형된 형태이다. 도입한 transactivator는 유도 조건에서의 외래 유전자의 발현을 극대화시키고 발현 유도물질인 doxycycline에 대한 감수성을 높여서 저농도의 doxycycline 조건에서도 발현 유도가 가능하다. 또한, 변형된 TRE 서열에는 endogenous mammalian transcription factor 결합 부위가 존재하지 않으므로 transactivator가 없는 경우 유전 자의 발현이 turn on되지 않으므로 background 활성이 거의 나타나지 않는다. 실제적으로 기존의 Tet system에서는 비유도 조건에서의 외래 유전자인 GFP의 발현을 미미하게 나타 낸 데에 비해 개선된 Tet system에서는 GFP의 발현이 거의 나타나지 않았다. 또한, 유도 조건에서는 기존의 system에 비해 새롭게 구축한 system에서 강한 발현을 나타내었으며 발현 유도율도 매우 높은 것으로 확인되었다. 구축한 Tet vector system에서 WPRE 서열의 위치와 표적세포주의 종류에 따른 GFP의 발현 양상을 확인한 결과, 소의 태아섬유아세포 에서는 WPRE가 transactivator 서열의 3′ 위치에 존재한 vector에서 발현이 가장 강하게 나 타났으며 닭의 배아섬유아세포에서는 WPRE가 TRE 서열의 3′ 위치에 존재한 vector에서 강한 발현을 보였다. 본 연구에서 구축한 개선된 형태의 Tet system은 완벽한 외래 유전자의 발현 조절을 가 능하게 함으로써 세포 수준에서나 개체 수준에서의 관련 연구에 있어서 유용한 유전자 전 이 수단으로 이용될 수 있을 것이다. * 본 연구는 농촌진흥청 차세대 바이오그린21사업(과제번호: PJ007990042012)의 지원에 의해 이루어졌다.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Palatal development is one of the crucial events in craniofacial morphogenesis, according to the significant signaling pathway including the out growth, elevation, and fusion of palatal shelves. In the fusion of palatal shelves, epithelial to mesenchymal transition (EMT) is a fundamental process to achieve the proper morphogenesis of palate. Mechanisms of EMT have been reported as the processes of migration, apoptosis or general EMT through the modulations through various signalling molecules. Rgs19, known as a regulator of G protein signaling (RGS) family through GTPase activity, showed the interesting epithelial expression patterns in various organogeneses including the significant expression patterns of Rgs19 in palatal development. To evaluate the precise function of Rgs19 in palatogenesis, we employed the gain and loss of function studies using ASODN treatments and gene electroporations while in vitro palate organ cultivations. Knockdown of Rgs19 using treatments of AS-ODN showed the retarded palatal fusion with the decreased patterns of apoptosis in mesial epithelium edge (MEE). In addition, alteration patterns of related genes were examined with the qRT-PCR. And epithelial mesenchyme transition (EMT) process was delayed in medial edge epithelium (MEE) throught immunohistochemistry of pancytokeratin, which known as epithelial cell marker. Morphological changes were observed with the three dimensional reconstruction method. These results show that expression of Rgs19 in MEE has crucial role of EMT, also Rgs19 affects to palatal fusion by regulation of apoptosis through the signalling modulations.

Atopic dermatitis (AD) is a chronically relapsing, non‐contagious pruritic skin disease with two phases: acute and chronic. Previous studies have shown that cathepsin S (CTSS) is a cysteine protease linked to inflammatory processes, including atherosclerosis and asthma. The possibility that this or other cysteine proteases might evoke itch or be part of a classical ligand‐receptor signaling cascade has not been considered previously. Recently, CTSS was known as a ligand for proteinase‐activated receptor 2 (PAR2) associated with itching. In the present study, we showed that CTSS‐overexpressing transgenic (TG) mice spontaneously developed a skin disorder similar to chronic AD under conventional conditions. This study suggest that CTSS overexpression triggers PAR2 activation in dendritic cells (DCs), resulting in promotion of CD4+ differentiation involved in MHC class II expression. In addition, we investigated mast cells and macrophages and found significantly higher mean levels of T‐helper type 1 (Th1) cell‐associated cytokines than of T‐helper type 2 (Th2) cell‐associated cytokines in CTSS‐overexpressing TG mice. These results suggest that increasing of PAR2 expression in DCs mediated by CTSS overexpression induces scratching behavior and Th 1 cell‐associated cytokines, and can trigger chronic AD symtoms.

Microtubule-associated protein 1B (MAP1B), a member of MAP1 family, plays a key role in the brain development. MAP1B binds to many kinds of proteins directly or indirectly. In our previous studies, MAP1B and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were down-regulated in bovine follicular cystic follicles (FCF). This study was performed to examine interaction between MAP1B and GAPDH in bovine follicles using immunoprecipitation (IP) with western blot analysis and immunohistochemisty. MAP1B and GAPDH mRNA expression levels were down-regulated in bovine FCFs. Consistent with the semi-quantitative PCR data, their protein expressions were also down-regulated in FCFs. IP data showed that MAP1B bound to GAPDH in normal follicles, but their binding was absent in FCFs, suggesting that these data might be resulted from a low level of MAP1B and/or GAPDH expression. These results suggest that GAPDH does not as always function as a loading control in bovine follicles.

The objective of this study was to investigate the proteome composition in pretermand term‐derived human umbilical cord. Umbilical cord samples were collected from 6 preterm infants with gestational age less than 36 weeks or 4 full terms together with medical information during prenatal period. Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Protein profiles were performed on samples from both preterm‐ and termderived human umbilical cord by using Two‐dimensional gel electrophoresis (2‐DE). Approximately 200 different proteins were identified between preterm‐ and term‐delivered umbilical cords. Among them, differentially expressed 34 proteins were identified in 48 protein spots. In the preterm‐derived human umbilical cords, 15 proteins were present at higher levels (2.0‐ to 9.28‐fold increases) and 19 were present at lower levels (2.0‐ to 11.8‐ fold decreases or not detectable) compared to the control term‐derived umbilical cords. Proteomics approaches such as 2‐DE could greatly facilitate the discovery of new and better biomarkers. The high sensitivity and specificity achieved by the combined use of the selected biomarkers show great potential for the early detection of Adverse pregnancy outcomes such as pre‐eclampsia, small for gestational age infants, preterm delivery and placental abruption are associated with higher mortality. Increased amount of HIF‐1 α, GAPDH and HSP27 were observed in preterm‐derived umbilical cords was due to hypoxia‐ dependant and oxidative stress‐independent manner. Moreover, we isolated HUVEC (Human umbilical vascular endothelial cells) from preterm‐ and term‐derived umbilical cords and examined LDH activity. The results of the current study may provide important insights into the molecular mechanisms underlying umbilical cord development and also these data will contribute to a better understanding of the composition of preterm‐ and term ‐ derived human umbilical cord and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities

Tumor cells express altered metabolic activities often linked to mitochondrial dysfunction. Such mitochondrial defects can inhibit oxidative phosphorylation, change the cellular redox status (NAD+/NADH), increase production of reactive oxygen species (ROS), and cause DNA damage that further supports tumorigenesis and a metastatic phenotype1,2. Mitochondrial Complex I (NADH dehydrogenase) is a major site of ROS production in mitochondria and regulator of the NAD+/NADH ratio. This study is focused on mitochondrial complex I as a possible modulator of tumorigenesis and progression in breast cancer. We used NADH dehydrogenase from yeast, called NDI1, to augment complexI activity in metastatic human breast cancer cells. We followed NDI1 functionality and impact on tumor cell behavior in vitro and tumor progression in vivo. Augmentation of NADH dehydrogenase activity through NDI1 resulted in an enhanced NAD+/NADH ratio and slight inhibition of ROS production. Importantly, NDI1 expression inhibited metastasis and tumor growth in the mammary fad pad of immune deficient mice, as seen by non-invasive bioluminescence imaging and histology. The mechanisms involve NDI1-induced inhibition of the AKT/mTOR survival pathway and autophagy stimulation. Knock-down of ATG5 partially reversed the anti-metastatic effect of NDI1, demonstrating that enhancement of autophagy is responsible for NDI1-mediated inhibition of breast cancer spreading. The results indicate that mitochondrial complex I activity can drastically impact tumorigenesis and metastasis in breast cancer, and that augmentation of complex I function through NDI1 can inhibit tumor formation and cancer progression through NAD+/NADH ratio modulation.

The purpose of this thesis is to examine the effect of hormone treatment in blastocyst development of in vitro cultured porcine oocyte. Oocytes used in the study was matured in vitro in the presence of 10% FBS or 10% pFF, and treated with FSH, LH or FSH+ LH, and the rate of blastocyst development was assessed based on the expression of autophagic genes. There was no significant differences in blastocyst development between oocytes maturaed in 10% FBS or 10% pFF. In vitro matured oocytes treated with FSH+LH showed blastocyst development rate as high as that of untreated oocytes, while groups treated with LH only showed a decrease in blastocyst development. About the expression of cell death assosiated factors, mRNA levels of autophagy and apoptosis genes were increased in oocytes matured in 10% FBS and treated with LH. Oocytes that did not receive hormone treatment showed low expression of most cell death genes except ATG5. When oocytes were matured in 10% pFF, ATG5 expression was the highest in FSH treated group, while LC3 showed strong expression in all hormone treated groups. On the other hand, the expression level of mTOR and caspase-3 did not show significant differences between groups. We also examined the protein level of apoptotic genes in the blastocyst. The amount of caspase-3 protein was similar between groups matured in 10% FBS and 10% pFF, but was the highest when treated with LH. Blastocysts treated with FSH and FSH+LH showed similar level of caspase-3 protein, while the level was the lowest when hormone treatment was not given. Within the blastocyst, caspase-3 was mostly expressed in trophoblast cells when matured in 10% FBS, while maturation in 10% pFF caused expression of this protein in the inner cell mass (ICM). Expression of MAP1LC3A was higher in groups matured in 10% pFF than groups matured in 10% FBS in all types of hormone treatment. Among the blastocysts matured in 10% pFF, MAP1LC3A level increased in the order of untreated < FSH < FSH+ LH. Expression of MAP1LC3A within the FBS-matured blastocyst was concentrated to the trophoblast, while pFF-matured blastocyst showed expression in both trophoblast and ICM. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in 10% FBS group without hormone treatment. In both FBS and pFF group, and in all three combinations of hormone treatment, mTOR expression was ovserved mostly in ICM. Together, these results indicated that hormone treatments tend to induce expression of genes associated with programmed cell death. We suggest that proper induction of programmed cell death by FSH and LH treatment would increase the rate of blastocyst development. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

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.