Pigs may be considered as a suitable organ source for its characteristics in xenotransplantation if significant immunological barriers can be overcome. However, xenograft could be rejected by T cells, especially CD8+ cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenograft by recognizing Swine Leukocyte Antigen (SLA)-I. Human cytomegalovirus (HCMV) encodes unique short (US) 11 gene, which interferes with cellular immune responses by inducing rapid degradation of newly synthesized heavy chains (HC) of MHC class I from endoplasmic reticulum (ER) to the cytosol. In this study we established two US11 clonal cell lines by transfection into minipig fetal fibroblasts and confirmed the integration of US11 gene by PCR and FISH. The reduction of Swine Leukocyte Antigen (SLA)-I which was expressed on the cell surface by US11 was also detected by flow cytometry assay. The level (14.6 % to 21.2%) of SLA-I expression in US11 clonal cell lines was decreased relative to the control. The reconstructed embryos were produced with these clonal cells and transferred to nine surrogate gilts. Ultrasound examination of recipient surrogates on days 35 after embryo transfer confirmed established pregnancies in two recipients. One recipient delivered one piglet with normal birth weight. PCR analysis revealed that transgene vector was integrated in the offspring genome. Transgene-expression analysis and CTL assay are currently underway. The present results show that transgenic pig was produced with US11 cDNA for controlling cell-mediated rejection. This result indicated that grafts of transgenic pigs expressing human cytomegalovirus protein US11 could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival. This research was supported by the BioGreen 21 Program (#20110301-061-541- 001-05-00), Rural Development Administration, Republic of Korea.

Several human leukocyte subsets including natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and polymorphonuclear neutrophils (PMN) participate in cellular immune responses directed against vascularized pig-to-human xenografts. As these leukocytes express the death receptor Fas either constitutively (PMN) or upon activation (NK, CTL), we explored in vitro whether the transgenic expression of membrane-bound human Fas ligand (mFasL) on porcine fetal fibroblasts is a valuable strategy to protect porcine xenografts. cDNA of mFasL carrying the deletion at the cleavage site with metalloproteinase and lacking the death domain in its cytoplasmic tail was subcloned into pCAGGS expression vector driven by the chicken β-actin promoter containing blastidin- resistance cassette. The mFasL expression vector was transfected into mini-pig fetal fibroblasts by lipofection method. Blastidin-resistant cells were screened by PCR and FISH. The expression of mFasL was confirmed by Western blot and FACS with the mouse anti-human FasL antibody. Interaction of two transgenic clonal cell lines with human leukocytes was analyzed using functional assay for cytotoxicity. mFasL expressed on porcine fetal fibroblasts protected porcine fetal fibroblasts against killing mediated by human NK cells. The rate of NK cell mediated cytotoxicity was significantly reduced in transgenic clonal cells (54±10.80%) compared to normal minipig fetal fibroblasts. This result indicated that grafts of transgenic pigs expressing mFasL could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival.

(‐)‐Epigallocatechin 3‐gallate (EGCG) is a potent antioxidant polyphenol in green tea that acts as an anticancer agent via both direct and indirect pathways. Although the relationship between EGCG’s anticancer effects and its antioxidant activity is not fully understood, it is known that EGCG stimulates production of reactive oxygen species (ROS), which induce oxidative stress leading to cell death. In IM9 multiple myeloma cells, EGCG acted in a dose‐ and time‐dependent manner to induce apoptotic cell death. Among the antioxidant enzymes expressed in IM9 cells, levels of peroxiredoxin (Prdx) V were selectively and significantly reduced by EGCG. Moreover, the ROS scavenger NAC completely inhibited EGCG‐induced apoptosis and PrdxV reduction, while overexpression of PrdxV, but not a PrdxVC48S mutant, protected IM9 cells from EGCG‐induced apoptosis. EGCG‐induced reductions in cell viability and PrdxV levels were also observed in primary CD138+ multiple myeloma cells from patients. These results suggest that PrdxV is a key target via which EGCG mediates its anticancer effects.

Immunological rejection of the organ grafted onto a primate arises from two antibody mediated processes, hyperacute rejection (HAR) and acute humoral rejection (AHR). Functional ablation of α1,3-galactosyltransferase (GalT) and concurrently overexpression of complement regulatory proteins are known to inhibit HAR and AHR. In previous study, we reported that production of porcine male fibroblasts harboring a MCP expression cassette targeted to GalT locus. In this study, we constructed a different MCP expression cassette, in which the EF1α promoter regulates MCP expression and internal ribosome entry site-mediated neomycin resistance gene expression. Subsequently, this cassette was inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Female fibroblasts were isolated from ear skin of 10 days old miniature pig, and used for nucelofection of the the construct for MCP expression at GalT locus. PCR analysis showed that four clones of forty neomycin resistant clones carry MCP expression cassette at exon 9 of the GalT gene. Two clones analyzed downregulated GalT expression, as determined by quantitative reverse transcriptase polymerase chain reaction. Flow cytometry analysis showed that MCP was efficiently expressed at the cell surface.

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber’s hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH–quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes. The NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results shown that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, I will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

Successful pregnancy requires suppression of maternal immune response to the implanting conceptus, which acts as a semiallograft. During the implantation period in humans and rodents, various immune modulators are produced at the maternal-fetal interface and regulate functions of cytotoxic T cells and NK cells for protection of conceptuses from the maternal immune system. However, maternal immune responses to the conceptuses during the establishment and maintenance of pregnancy are not much understood in pigs which show true epitheliochorial type placentation. Previously, we reported that SLA-DQ molecule, a type of MHC class II molecules, is expressed in the uterine endometrium during pregnancy in a stage- and cell type specific manner, and that SLA-DQ expression is essential for the maintenance of pregnancy. Thus, to understand the role of SLA-DQ and maternal-fetal immune interaction, we examined expression of CD80 and CD86, co-stimulators for T cell activation, in the uterine endometrium during pregnancy. We also measured levels of CD80 and CD86 mRNAs in the uterine endometrium of pigs carrying conceptuses derived from somatic cell nuclear transfer (SCNT) and those from natural mating on Day 12 of pregnancy. Expression of endometrial CD80 mRNA was affected by day of pregnancy, and levels of CD80 mRNA were significantly higher on Day 15 of pregnancy than those of the estrous cycle. Expression of CD86 mRNA did not change during pregnancy. Levels of CD80 and CD86 mRNAs were not different in the uterine endometrium of pigs carrying SCNT derived conceptuses on D12 of pregnancy compared to those with conceptuses derived from natural mating. These findings suggest that CD80 and CD86 are involved in immune interactions at the maternal-fetal interface during pregnancy for the establishment and maintenance of pregnancy in pigs.

Prostaglandins (PGs) are critical lipid mediators involved in many reproductive processes including luteolysis, maternal recognition of pregnancy, and implantation in domestic animals. In pigs, PGs, especially PGE2 and PGF2α, are produced in the uterine endometrium. The actions of PGE2 and PGF2α are mediated by signaling receptors, PTGERs and PTGFR, respectively, but their expression in the uterine endometrium is not well elucidated. In this study, we determined expression of PTGERs and PTGFR 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. Temporal expression of all genes studied was analyzed by real-time RT-PCR. PTGERs except for PTGER1 were expressed in the uterine endometrium during the estrous cycle and pregnancy. Levels of PTGER2 and PTGER3 mRNA increased during early pregnancy and late pregnancy, respectively, and levels of PTGER4 mRNA were not changed during pregnancy. Levels of PTGFR mRNA were highest on D90 of pregnancy. Results of this study showed that expression of PG receptors was dynamically regulated in the uterine endometrium during pregnancy in pigs. These results indicate that actions of PGs are dependent on types of receptors and is critical to support the establishment and maintenance of pregnancy at the maternal-fetal interface in pigs.

Proteases and their inhibitors are involved in the process of pregnancy by remodeling uterine endometrium and placenta in many mammals. During placentation, proteases and their inhibitors contribute to formation of epitheliochorial type placentation in pigs. Our previous study showed that LGMN and CST6 were expressed in the uterine endometrium and localized mainly to glandular epithelial cells (GE) and chorionic membrane (CM) during mid to late pregnancy. In this study, we investigated expression of LGMN and CST6 in the uterine endometrium and fetal membrane during pregnancy in pigs. Uterine endometrial tissue samples and fetal membrane samples were collected from D30, D60, D90, and D114 of pregnancy. Real-time RT-PCR analysis showed that both LGMN and CST6 mRNAs were detected in the uterine endometrium and fetal membrane in all samples with higher levels during mid to late stage of pregnancy. Analysis by immunoblotting revealed that LGMN protein was present in the porcine uterine endometrium and fetal membrane. Based on the placental and endometrial distribution of proteases and their inhibitors, we examined LGMN mRNA and LGMN protein expression in the neonatal pigs. In situ hybridization analysis using the intestine from D90 of piglet revealed that LGMN mRNA was highly expressed in the absorptive epithelium of the intestinal villi. Immunohistochemical experiments demonstrated that LGMN protein was localized to epithelial villi. These results suggest a possible role of LGMN in modification of proteins that are transported through the fetal membrane from the uterine for successful transport and utilization in the fetus.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane. Na+/K+-ATPase consists of α, β, and γ subunits, but only α and β subunits are needed for basic functions. Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca2+ exchanger involved in intracellular calcium extrusion. Our previous study showed that calcium regulatory molecules including Na+/Ca2+ exchanger are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs, however, expression of Na+/K+-ATPase in the uterine endometrium has not been determined. Thus, we examined expression of α1 (ATP1A1) and β1 (ATP1- B1) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of ATP1A1 m- RNA in the uterine endometrium during the estrous cycle and early pregnancy were higher than those during mid and term pregnancy, and that levels of ATP1B1 mRNA were highest on day (D) 12 of the estrous cycle. In situ hybridization analysis revealed that ATP1A1 and ATP1B1 mRNAs were localized to luminal (LE) and glandular epithelia (GE) in the endometrium. During mid to term pregnancy, localization of ATP1A1 mRNA was confined to LE, GE, and chorionic membrane (CM) of areolae and ATP1- B1 mRNA was localized to LE, GE and CM with the strongest intensity in LE of areolae. Signal intensity of ATP1B1 mRNA in LE was slightly stronger than that in GE. RT-PCR analysis showed that ATP1A1 and ATP1B1 mRNAs were expressed in conceptuses on D12 and D15 of pregnancy. These results showed that ATP1A1 and ATP1B1 were expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase may play a key role in the establishment and maintenance of pregnancy by regulating intracellular concentrations of various ions including calcium at the maternal-fetal interface in pigs.

The present research was carried out to evaluate the possibility of female offspring production using artificial insemination buffer (AIB) before artificial insemination (AI). To do it, we carried out the optimization of AIB, making of AIB gun and analysis of affecting AI rate after AIB treatment. AIB made with the base of Tris‐buffer supplemented with L‐arginine and several materials that could be reduced the motility of male sperm compared with female one. This mean that female sperm could be increased the possibility of fertilization with ovum compared with male one. AIB must be deposited into 2nd to 4th cervix by the guide of AIB gun. After 15 min of AIB insertion, frozen semen was deposited into same place after. Total 352 cattle were inseminated with AIB insemination and was not significant difference between AIB and traditional AI rate (56.8 vs. 55.7%). However, AIB AI rate was significantly differs among 12 different farms. The parturition number of cows did not effect on AIB AI rate among 1st to 7th parturition number of cows. The proportion of AIB AI success rates in hanwoo cows was significantly higher than in dairy cows (61.0% vs. 48.7%), but the average AI success rate was not different between AIB and conventional AI (56.8% vs. 55.7%). The female offspring production rate in 2nd to 4th cervix deposition place was significantly higher than in uterus body (77.7% vs. 59.6%, p<0.05). The injection volume of AIB in 5 and 10 ml was significantly higher than in 2 ml (77.7, 78.7 vs. 51.8%, p<0.05), but not different between 5 and 10 ml ABI volume. The best exposure time of AIB in the cervix was 10 and 15 min rather than that of 5 min (79.2%, 77.2% vs. 63.2%, p< 0.05), and so AIB have to expose at least 10 min to get higher female offspring. In conclusion, AIB could be used in AI industry to produce female offspring and also AIB AI can be increased the AI success rate compared with traditional AI rate.

The present study was performed to identify the role of plasminogen activator (PA) and the location of PA expression in porcine uterus tissues during the estrous cycle. Porcine uterus tissues were obtained from ovary in pre-ovulatory (Pre-Ov), post-ovulatory stage (Post-Ov) and early to mid-luteal stage (Early-mid L). The uterus tissue was immediately fixed by PBS with 10% formalin. There were fixed porcine uterus tissue for 24 hours at room temperature and porcine uterus tissue dehydrate for 12 hour in sucrose solution. For immunohistochemical staining, porcine uterus tissues were cut to 4 μm by micro frozen section microtome. The nucleus and cytoplasm of porcine uterus tissues were stained by Hematoxin and Eosin. Porcine uterus tissues were evaluated by Immunofluorescence using anti-tissue type PA (tPA) and urokinase type PA (uPA). The location of PA expression was identified by observing the PA fluorescence using fluorescent microscope and optical telescopes. As a results, when Pre-Ov and Post-Ov were identified endometrial blood vessel in an inner layer that were observed tPA and uPA. Especially, expression of PA was observed around secretory gland. But the expression of PA were not confirm in Early-mid L. Also, The expression of PA were higher in Post-Ov than Early-mid L. In conclusion, during the estrous cycle, the expression of PA were increased from Pre-Ov to Post-Ov and was decreased from Post-Ov to Early-mid L.

현재 경북지역에는 약 700여두의 칡소가 사육되고 있으나, 대부분이 자연종부에 의한 번식에 의존하고 있어 사육농가에 따른 근친의 위험도가 증가하고 있고 품조의 퇴화가 의심되고 있다. 따라서 본 연구에서는 경북지역에 사육하고 있는 칡소의 유전적 근연관 계를 분석하여 향후 교배계획 등에 활용할 자료를 확보하고자 실시하였다. 칡소 개체에 따른 모색발현은 전체 칠소무늬, 부분 무늬, 황색 및 흑색으로 구분하여 조사하였다. 경북지역 사육 칡소 암소와 수소 총 67두로부터 혈액을 채취하여 DNA를 분 리하였다. 근연관계분석은 ISAG에서 추천하는 27개 초위성체 마커를 활용하여 PCR 분 석을 하였다. 결과는 POPGENE32 및 UPGMA 등의 클러스터링 프로그램을 활용하여 유 전적 거리 및 다양성을 분석하였다. 칡소의 모색은 전체 칡소 무늬, 9.6%, 부분 칡소 무늬, 56.0%, 황색 20.8% 및 흑색 13.2%로 조사되었다. 칡소 혈액으로부터 DNA를 분리하여 genetic distance(GD)를 조사 하여 모색 발현과 비교한 결과 전체 칡소무늬는 GD 값이 0.92, 부분 칡소무늬는 1.32, 황색은 0.79 및 흑색은 0.77로 분석되었다. GD 값이 큰 개체들 간에 교배한 경우 칡소무 늬가 발현된 송아지가 많았다. 본 연구결과 경북지역 칡소의 기초적인 유전적 근연관계를 확인할 수있었으며, GD값이 클 수록 모색이 정확하게 발현되는 경향이었다. 하지만 근연관계 분석에 더 많은 칡소의 분석과 모색유전자에 대한 추가적인 분석이 필요할 것으로 판단된다.

The present study was performed to identify the role of plasminogen activator (PA) and the location of PA expression in porcine uterus tissues during the estrous cycle. Porcine uterus tissues were obtained from ovary in pre-ovulatory (Pre-Ov), post-ovulatory stage (Post-Ov) and early to mid-luteal stage (Early-mid L). The uterus tissue was immediately fixed by PBS with 10% formalin. There were fixed porcine uterus tissue for 24 hours at room temperature and porcine uterus tissue dehydrate for 12 hour in sucrose solution. For immunohistochemical staining, porcine uterus tissues were cut to 4 μm by micro frozen section microtome. The nucleus and cytoplasm of porcine uterus tissues were stained by Hematoxin and Eosin. Porcine uterus tissues were evaluated by Immunofluorescence using anti-tissue type PA (tPA) and urokinase type PA (uPA). The location of PA expression was identified by observing the PA fluorescence using fluorescent microscope and optical telescopes. As a results, when Pre-Ov and Post-Ov were identified endometrial blood vessel in an inner layer that were observed tPA and uPA. Especially, expression of PA was observed around secretory gland. But the expression of PA were not confirm in Early-mid L. Also, The expression of PA were higher in Post-Ov than Early-mid L. In conclusion, during the estrous cycle, the expression of PA were increased from Pre-Ov to Post-Ov and was decreased from Post-Ov to Early-mid L.

In the last few decades with the industrial revolution many environmental contaminants have estrogenic activity (endocrine disruptors, EDs) are released into the environment affecting the male reproductive system and male fertility. Sperm motility is one of the initial tests performed to assess sperm function; only motile sperm can achieve fertilization in vivo. The present study aimed to investigate the possible effects of a group of EDs that represent a widespread chemicals in the environment genistein (Gen), is a naturally occurring isoflavone (100 μM), bisphenol A (BPA), that is used in the manufacture of plastics and other products and released largely into the environment (100 μM), nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical (10 μg/ml), TCDD, that is formed as an unwanted by-product in the manufacture of chlorinated hydrocarbons (2.5 μg/ml), atrazine (Atraz) is a herbicides (500 μM), dibromochloropropane (DBCP) is a pesticide (10 μg/ml), and diazinone (Diaz) is a insecticide (500 μM) on human sperm motility and kinematic characteristics. Human spermatozoa were incubated in Ham's F10 media with/without the tested chemicals or DMSO as positive control for 6 hr at 37℃ in 5% CO2. Then, sperm motility was assessed using computer assisted semen analyzer. Interestingly, all the chemicals tested significantly decreased sperm motility as compared to the control groups. However, only Diaz significantly decreased sperm kinematic characteristics namely, VCL, VSL, STR, VAP, and ALH. We suggest that the environmental chemicals may have an effect on male fertility via decreasing sperm motility.

국내의 말 산업 확대와 승마인구 저변확대에 따른 승용말 생산의 필요성이 대두되고 있으나, 현재 대부분의 승용말은 경주 퇴역마 또는 고가의 수입말을 활용하고 있다. 따라 서 우수하고 경제적인 승용말의 국내 생산이 필요하며, 이에 따른 인공수정 및 수정란이 식 연구가 필요하다. 본 연구에서는 말의 발정동기화 처리법에 따른 난소와 혈중 호르몬 농도의 변화를 조사하였다. 공란말 및 수란말의 발정동기화는 CIDR-Plus, 경구용 프로제 스테론(Regumate) 및 PGF2a를 이용하였다. CIDR-Plus 및 Regumate는 투여 시작부터 3 일간격으로 채혈 및 초음파 검사를 하였고, PGF2a는 투여전 및 투여 후 3일간격으로 채 혈과 호름파 검사를 하였다. CIDR-Plus 삽입 전 프로제스테론 농도가 평균 1.02 ng/ml, 3 일, 6일 및 9일째 각각 1.10 ng/ml, 4.21 ng/ml 및 8.90 ng/ml이었고, 9일째 PGF2a를 투 여하여 배란유도한 후 3일째에 0.04 ng.ml로 급격히 감소하였다. 한편 Regumate는 투여 전, 투여 후 3일, 6일 9일 및 PGF2a 투여 후 3일째에 각각 평균 0.04 ng/ml. 0.03 ng/ ml, 1.59 ng/ml, 8/84 ng/ml 및 0.93 ng/ml이었다. 발정 증상이 없는 번식말에 PGF2a 투 여전 혈중 프로제스테론 농도는 평균 2.45 ng/ml였으며, PGF2a 투여로 평균 0.03 ng/ml 미만으로 감소하였다. CIDR-plus는 발정 증상이 대체로 미약하였고, 기구 삽입에 따른 질 의 발적과 염증소견이 관찰되었다. Regumate는 급여 후 황체의 크기가 6일째에 평균 3.2 cm로 성장하였고, PGF2a 투여 후 황체 소실 및 난포의 성장이 관찰되었다. 본 연구 를 통하여 말의 발정동기화는 경구용 프로제스테론이 기구에 비하여 효과적인 것으로 판 단된다.

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum during the estrus cycle in bovine ovary by proteomics ^techniques. Our study was devided into five steps for follicular, ovulatory, early-lteal, midluteal and late-luteal. The protein was extracted from glanulosa cell and corpus luteum proteins by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 700 μg. Immobilized pH gradient (IPG) strip was used 18 cm and 3 11 NL. SDS-PAGE was used 10% acrylamide gel. The protein spots were visualized by Coomassie Brilliant Blue (CBB) staining, analyzed by MALDI mass spectrometry and searched on NCIBlnr. As the result, 61 spots of total 85 spots were repeated on follicular stage and 51 spots of total 114 spots were repeated on ovulatory stage. 40 spots of total 129 were repeated on early-luteal and 49 spots of total 104 spots were repeated on mid-luteal stage. Also 41 spots of total 60 spots were repeated on last-luteal stage. There were differences in the ovulation (follicular∼ovultory stage) in which the spots of follicular stage 19 was only and in ovulation stage was 10 spots. The difference between the luteinization (ovultory∼mid-luteal stage) was the spots counted in each stage. The spots of ovulatory stage was 1, early-luteal stage was 1 and in mid-luteal stage was 2. Eleven spots were found in mid-luteal stage and 2 spots were found in last-luteal stage. In conclusion, we confirmed that there were 7 spots in ovulation, 4 spots in luteinization and 2 spots in luteolysis. Spot No. 89-93 from ovulation were transferrin, and spot No.94 and 95 were HSP60. Spot No. 103 were Dusty PK, spot No. 135 were OGDC-E2, and spot No. 175, 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 from luteolysis were vimentin.

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase- mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinization to predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally.

산소소비량은 수정란이식 수태율에 영향을 미치는 여러 요인들 중 하나인 수정란의 품 질을 판정할 수 있는 기준으로 알려져 왔다. 최 등(2010)은 한우 체내수정란의 형태학적 인 등급에 따른 산소 소비량을 비교, 체외수정란의 산소 소비량과 총세포수의 상관관계 를 확인한 결과, 국제수정란이식학회(International Embryo Transfer Society, IETS)에서 규정하고 있는 수정란의 등급 판정 기준에 준하여 형태학적 등급이 좋을수록 수정란의 산소 소비량도 높아지고, 체외수정란의 산소 소비량에 따른 총 세포수와 산소 소비량 사 이에도 정의 관계가 있다고 보고 했다. 따라서, 본 연구는 수정란의 산소 소비량을 측정 하여 그 품질을 확인하고, 이를 바탕으로 체내수정란의 산소 소비량이 수태율에 미치는 영향을 구명하고자 수행하였다. 한우 체내수정란의 생산은 난소 및 자궁질환이 없는 건 강한 한우 공란우를 과배란을 유기하여 3 way Foley catheter를 이용하여 수정란을 채란 하였다. 체외수정란은 국립축산과학원 가축유전자원시험장 관행방법을 기준으로 생산하 여 시험에 사용하였다. 수정란의 산소 소비량 측정은 수정란 호흡장치(FHK, HV-405, Japan)를 이용하였고, 수정란 호흡 장치는 주사형 전기화학현미경(Scanning Electrochemical Microscopy, SECM)을 이용하여 산소 농도 분포를 각각 측정하였다. 한우 체내수정 란의 산소 소비량(10¹⁵/mol s— 1)을 측정하고 이를 수란우에 이식한 후 수태율을 조사한 결 과 산소 소비량이 가장 높은 12.0 이상에서 85.7%의 높은 수태율을 나타내었다. 이에 반 하여, 10.0 미만의 낮은 산소 소비량에서는 흥미롭게도 수태율이 0.00%를 나타내었다. 이런 결과들은 형태학적 등급이 좋을수록 수정란의 산소 소비량도 높아진다는 보고를 근 거하여, 산소 소비량이 높은 수정란을 이식하면 수정란이식 수태율이 높아진다는 것을 확인하였다.

Sperm examination is an important tool in estimating the fertilizing capacity of an ejaculate. The number of spermatozoa in a semen dose, morphology and motility are important for the fertilization process. By evaluation of semen, artificial insemination (AI) using high quality of semen can increase fertilization rate. Boar semen is subject to contamination by various pathogens that can result in fertility disorders in sows. Among these pathogens, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus-2 (PCV-2) are of particular importance and accurate monitoring prior to and during the presence of boars in AI stations is essential. Because of the high risk of dissemination of disease via AI, The absolute goal is to provide pathogen-free semen and this is feasible with the adequate measures. The disease affects boars semen causes a significant reduction quality. In this study we investigated the characterization boar semen in Jeju, interaction of pathogenic virus infection with characterization of boar semen. Forty-two boar semen from 13 farms were investigated. The semen were stored during 5 days at 17℃ and the sperm qualities in the stored semen were analysed. Visual-motility assessment is a tool (Computer- Assisted Semen Analysis) used to determine the quality of boar semen. Percentage of morphologically normal spermatozoa were assessed. PRRS ,PPV and PCV-2 were detected in boar semen using PCR. The motion characteristics in boar semen was showed 68.4±9.1% for motility, 48.6±7.1 μm/s for VAP, 45.3±7.0 μm/s for VSL, 79.1±8.7 μm/s for VCL, 1.3±0.2 μm/s for ALH, 8.3±0.4 Hz for BCF, 93.6±3.5% for STR, 57.9±6.4 % for LIN. The percentage of sperm with abnormal head, midepeice and tail were 0.3±0.7%, 14.4±12.5%, 4.9±6.6%, respectively. Based on the PCR method, PPV was detected in 20 samples (48%). However, PCV-2 and PRRSV were not detected in any cases. Marked differences in motility and morphology between PPV negative and PPV positive semen were not observed. Sperm cell production was not affected by PPV infection. However, slight increases in detached head, coiled tail after infection were observed (p<0.05). The motility of semen in Jeju is similar to case comparing with other regions in Korea. Although PPV in semen was not affected in semen quality, there is the high risk of virus excretion in the semen of Jeju boars. Therefore continuous screening tests for some particular pathogens in boar semen would be warranted.