Earticle

Since 1978 the world’s first baby was born by IVF, intracytoplasmic sperm injection (ICSI) using single sperm was developed in 1992. The assisted reproductive technology (ART) has been considered ultimate process for infertility caused by male or female factors. Especially, IVF with preimplantation genetic diagnosis (PGD) can prevent disease that may be inherited from the mother or father. Recently, mitochondrial (or nuclear) replacement techniques such as pronuclear transfer (PNT), maternal spindle transfer (MST) and polar body transfer (PBT) were developed between oocytes or zygotes for preventing mitochondrial disease carrying pathogenic mtDNA or improving oocyte quality. However, animal models are mostly non-human primates, such as monkeys and mice, and are not suitable for animal models that can represent the human reproductive system and life style. I hereby introduce ST and PBT as an assisted reproductive technique using micromanipulation, and would like to ask what animal models are appropriate for these research.

Laboratory animals more than 100 million have consumed annually in laboratories for test of chemicals, medical devices, drugs and foods, and also have brought about a social problem in aspect of bioethics. Recently, bio-imaging system that can detect the fluorescence or bioluminescence signals in vivo, has considered as a replacement solution in the use of laboratory animals. Here, we tried to construct a mouse model with BRET(Bioluminescence Resonance Energy Transfer) system that is programmed to cut a recognizable site (DEVD amino acid sequence) by caspase-3 when the drug efficacy is observed. BRET expression vector was composed of a bioluminescent luciferase and a red fluorescent protein (tdTomato) under CAG promoter, and DEVD sequence was inserted between their proteins. Luciferase and tdTomato expressed normally in BRET-PC3 human prostatic cancer cells, and red fluorescence was observed under radiation- free status. During drug inducible apoptosis, bioluminescence rate catalyzed by luciferase rapidly increased whereas red fluorescent intensity decreased slightly. Next, BRET-PC3 cells injected into underskin in the femoral region to produce xenograft mouse model and were identified successfully expectable events occurred after treatment of apoptotic drugs like as BRET-PC3 cells. Finally, we constructed mouse expressing BRET system by microinjection and confirmed strong detectable signals using bio-imaging equipment. In this study, we developed a new mouse model with BRET system that can detect apoptotic reagents, and suggest the possibility as a teat model for drug discovery and development.

Pluripotent stem cells (PSCs) can be differentiated into all three germ layers in vitro and in vivo. Embryonic stem cells (ESCs) can be derived from the inner cell mass of blastocyst in vitro, and the epiblast derived pluripotent stem cells (EpiSCs) can be established from epiblast of post-implantation embryos. The pluripotent ESCs and EpiSCs express Oct4 (Pou5f1) gene. Oct4 gene is essential for maintenance of pluripotent state and levels of Oct4 regulate differentiation of pluripotent stem cells. The Oct4 gene contains two cis-regulatory elements, the distal enhancer (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type-specific and stage-specific manner. We generated double transgenic mice carrying both Oct4-Δ PE-GFP and Oct4-ΔDE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. We could classify pure populations of naive (GFP+RFP−) and primed (GFP−RFP+) PSCs by using double transgenic systems. Next, we established ZHBTc4 ESCs (Oct4 tetracycline conditional knockout ESCs) - derived epiblast stem cells like cells (ZHBTc4-EpiLCs) to determine function of Oct4 in differentiation into germ layers. ZHBTc4-EpiLCs could not be formed colonies in doxycycline (dox) contained EpiSC medium, indicated that Oct4 gene is essential for maintenance of primed PSCs. ZHBTc4-EpiLCs were differentiated into neuroectodermal or mesoendodermal cells in differentiation medium with or without dox. The ZHBTc4-EpiLCs could be efficiently differentiated into neuroectodermal lineage in neuroectodermal differentiation medium with dox, whereas mesoendodermal related genes were down-regulated in mesoendodermal differentiation medium with dox. This results showed that Oct4 gene regulates maintenance and differentiation potential in primed pluripotent stem cells.

Pectolinarigenin, an aglycone of pectolinarin, has more potent　inhibitory activities on melanogenesis than pectolinarin. Pectolinarin and pectolinarigenin have been reported to be major compounds in Cirsium setidens. In the present study, we demonstrated inhibitory effects of pectolinarin and pectolinarigenin from C. setidens on melanogenesis. Melanin synthesis was decreased in both pectolinarin- and pectolinarigenin-treated melan- a cells and in a reconstructed human skin model. However, pectolinarigenin treatment showed more potent inhibitory activity of melanin synthesis than did pectolinarin treatment. The concentrations of pectolinarin and pectolinarigenin in C. setidens water extracts were determined by HPLC. Unfortunately, the amount of pectolinarigenin of C. setidens water extract was lower than that of pectolinarin. To increase the pectolinarigenin content in C. setidens water extract, several component conversion methods were studied. Consequently, we identified that microwave irradiation under 1% acetic acid was an optimum sugar elimination method. Ameliorative effects of Juniperus rigida fruit on atopic dermatitis in mice. The fruits of Juniperus rigida have been used in Korean traditional medicine for the treatment of inflammatory diseases in humans such as rheumatoid arthritis. Aim of the study: This study aimed to investigate the anti-atopic properties of J. rigida fruit in in vivo murine atopic dermatitis (AD) models. BALB/c mouse ears ad SKH-1 hairless mice stimulated with oxazolone (4 weeks) and DNCB (3 weeks), respectively, were treated with the 1% Juniperus rigida fruit EtOH extract (JFE). The JFE improved AD symptoms in both oxazolone- and DNCB-induced AD mice by accelerating skin barrier recovery function and suppressing the overproduction of serum immunoglobulin E (IgE) and interleukin 4 (IL-4). The JFE was found to contain isoscutellarein-7-O-β-xylopyranoside, cupressuflavone, podocarpusflavone A, and hinokiflavone as major components based on phytochemical analysis. Eight flavonoids were isolated from JFE, and of those, cupressuflavone and isoscutellarein-7-O-β-xylopyranoside strongly down-regulated IL-4 expression and β-hexosaminidase release in RBL-2H3 cells. Therapeutic attempts with J. rigida fruit and its active components might be useful in treating AD and related skin inflammatory diseases.

본 연자는 1999년부터 시작하여 청도싸움소 정액보존을 시작으로 전국의 칡소 및 흑우 의 정액을 채취하여 희소성이 높은 유전자원을 확보하여, 현재까지 약 20년간 한우, 칡 소, 흑우정액 및 수정란을 40,000스트로 이상 생산하여 확보 중이다. 전국 최대의 한우유 전자 뱅크로 한우의 체내 ‧ 외수정란, 생체난포란(OPU) 수정란, 개체식별 체외수정란 등 수정란을 생산하여 12개 시, 군에 4,000~5,000두 정도 수정란을 이식하고 있다. 또한, 고 품질의 수정란생산 배양시스템 개발을 하여 국내와 일본, 중국에도 기술을 이전하여 선 업화 발전에 기여하고 있다. 이런 연구와 현장노하우의 기술바탕으로 국내산업적 동물 간의 교배로 실용축 생산을 목적으로 하였다. 국내 한우의 역사는 고구려의 고분벽화에 삼국시대 얼룩소와 흑소, 하얀소가 있었던 것 으로 생각되고 고구려는 한우를 농경 외에도 수레를 끌기 위해 사용했으며, 신라의 민정 문서에 국가에서 소의 숫자를 철저하게 파악하고 관리했음이 확인해준다. 한우는 1399년 에 우리나라 최초의 수의학서 조선우마의방 책에서는 한우를 총 9종으로 분류하여 기록 되어 있고 그 당시에 현재와 마찬가지로 크게 황소와 칡소, 흑소 등이 있었고 털색도 더 세부적으로 구분하여 보다 더 다양한 종이 있었던 것으로 보인다. 현재 한우는 290만두 재주흑우는 1,000여 두, 칡소는 3,500여 두 내륙에 흑우는 약 200여 두 사육되고 있으며 한우가 주류를 이루고 있다. 특히 제주흑우는 천연기념물 제546호로 지정되어 옛 문헌은 나라에서 제사를 지낼 때 쓰는 제향품이나 임금에게 바치는 진상품으로 엄격히 사육 ‧ 관 리됐다. 그러나 현제 흑우는 유전자보존으로만 관리되어 산업적인 측면에서는 미미한 수 준이다. 본 연구소는 발생공학과 유전공학 분야에서 개발되고 있는 수정란이식기술, 성감별, DNA 검사, 유전자분석, 초음파 진단 등을 활용하여 한우, 칡소, 내륙흑우 등 우수한 유전 형질을 이용한 실용축생산과 유전자분석을 통한 육량과 육질의 유전자를 분석하였고, 비 육과정을 통해 도축성적, 고기성분분석을 하여 지방함량과 맛 등급에 중요한 올레인산, 불포화지방산등 분석을 하였다. 그리고 10일 간 숙성 후 시식회를 하여 가칭 “지리산흑 우”의 고기 품평을 하였다. 앞으로 내륙의 실용축 흑우의 품종고정과 개량, 증식에 새로운 전기를 마련해줄 수 있 고 축산농가의 소득과 우리들의 입맛에 맛 나는 소고기를 먹을 수 있을 것으로 생각된다.

BPA is an endocrine disruptor, has been investigated for its impact on male fertility in several species of animals and human. However, the underlining molecular mechanisms and possible health hazards of BPA exposure are still far from being well understood. Considering in vitro experimental model, we investigated the effects of BPA (0.0001 to 100 μM) exposure on spermatozoa. We demonstrated that BPA is capable of affecting several sperm functions by upregulating the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase-A (PKA) activity. High concentrations of BPA was also responsible for the activation of protein tyrosine phosphorylation in a PKA-dependent signaling subsequently induced a precocious acrosome reaction. Simultaneously, BPA has been found to compromise the rate of fertilization and early embryonic development. Proteomics investigation of spermatozoa reveled that BPA induced differential expressions of major sperm proteins that were responsible for the pathogenesis of many diseases. Considering in vivo experimental model, we studied the effects of gestational BPA exposure (environmental relevant doses) on non-capacitated/ capacitated spermatozoa in F1 adult mice. We demonstrated that BPA inhibited several sperm function in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased PKA activity and tyrosine phosphorylation (non-capacitated) in spermatozoa. We also noticed that BPA decreased average litter size as well as compromise the rates of cleavage and blastocyst formation. Proteins differentially expressed in capacitated/ non-capacitated spermatozoa play a critical role in energy metabolism, stress responses, and fertility finally predispose to the development of several diseases. On the basis of these results, both direct and gestational exposure to BPA alter spermatozoa function and the proteomic profile, ultimately affecting their fertility potential. Therefore, it is of critical public health significance to reevaluate the levels of BPA exposure that are currently deemed to be acceptable.

The aim of present study was to confirm changes of results in in vitro fertilization (IVF) by two types of exogenous plasminogen activators (urokinase-type, uPA; tissue- type, tPA), and their inhibitor (PAI-1) and investigate the regulatory mechanism. the cumulus-oocyte complexes (COCs) were aspirated from 3~6 mm antral follicles and matured for 44 hours. Then, the cumulus cells were removed for fertilization and denuded oocytes were co-incubated with spermatozoa for 18~20 hours in IVF medium containing 100 pg/mL uPA, tPA or PAI-1. The number of sperm that bound to zona pellucida (ZP) and ZP solubility were measured using hoechst 33342 and 0.5% (w/v) pronase, respectively. Aceto-orcein stain was used to assess sperm penetration and sperm characteristics were analyzed by flow cytometry. In result, sperm penetration was increased by uPA and PAI-1 treatment than other groups (p<0.05). Interestingly, treatment of tPA enhanced ratio of monopsermic fertilization in penetrated zygotes population and reduced polyspermy than uPA groups (p<0.05). Similar to sperm penetration, number of penetrated sperm per zygote were higher PAI-1 treatment than tPA group (p<0.05). The number of ZP bounded sperm and ZP digestion time were lower in uPA and tPA-treated zygotes than other treatment groups (p<0.05) and ZP resistance was increased by treatment of PAI-1 (p<0.05). In results in sperm assay, acrosome reaction did not affected by all treatment in live and all sperm population. However, tPA treatment increased ratio of damaged sperm (p<0.05). These findings shown that exogenous PAs could affect to result of IVF via regulation ZP resistance, ZP binding, and sperm viability and two-types PAs differently regulated the fertility of boar spermatozoa.

The transcription factor OCT4 has a critical role in central regulator of maintaining pluripotency and self-renewal. Also, it is an important marker of undifferentiating status in early mammalian embryonic development and embryonic stem cells. There are four conserved regions (CR1, CR2, CR3 and CR4) in the 5’ upstream regulatory region of various species. It contains core promoter and two conserved enhancers, distal enhancer (DE) and proximal enhancer (PE). Even though study related dual-fluorescence reporter was reported, there is still a dearth of sufficient information about porcine OCT4 upstream reporter system until now. So, the purpose of this study is investigation to find conserved regions among other species using comparative sequence analysis of porcine OCT4, and conduct functional analysis of porcine OCT4 promoter region. And we designed four constructs containing the different elements of the OCT4 promoter and enhancers in luciferase vectors, and they were transfected into mouse embryonic stem cells and embryonic carcinoma cells. Our results indicate that porcine OCT4 promoter and regulatory regions have a high similarity with other mammalian species. Four porcine OCT4-luciferase constructs showed different expression patterns in mouse embryonic stem cells, mouse embryonic carcinoma cells, and mouse embryonic fibroblast. And we designed four constructs containing the different elements of the OCT4 promoter and enhancers in GFP, RFP vectors and they were transfected into mouse embryonic stem cells. Those constructs can work in mouse embryonic stem cells and mouse embryonic carcinoma cells. This work could provide basic information for porcine OCT4 upstream region and suggest the various type of porcine OCT4-Fluoresence reporter constructs, which can use for reporter system of studying species-specific pluripotency in early embryo developments and establishment of embryonic stem cells.

In mammals, the uterus is the organ of pregnancy. In most mammals, conceptus implantation to the uterine endometrium consists of blastocyst hatching, migration, apposition/ attachment, invasion and subsequent placental formation. It is known that close to 50% of fertilized pre-implantation embryos in mammals, including humans, fail to implant. We hypothesized that there was the embryo quality-dependent expression of embryo development associated genes on Endometrial Epithelial Cells (EECs) of superovulated Hanwoo. Therefore examined to study the mode of EECs expression during pre-implantation. Additionally, we investigated how EECs respond to the presence of embryos by FSH and LH in bovine EECs in vitro(The EECs were divided into three different groups as follows good (n=4), fair (n=4), bad (n=2)). Our results found that the different expression of EST mRNA to each the group. In addition, it was found that the expression of genes related to MMP-9 and development showed different appearance in each group, and the result of embryo development was different. In particular, the expression of genes associated with the growth of cells from the Good group was very high, and the expression of genes affecting cell activity was also very high. However, Bad group had a high expression of Casp-3 protein and expression of metabolism-related genes of other cells was very low. Also, it is interesting that the expression of FSH and LH in the EEC of Good group is very high, and even in the evaluation of the development rate of in vitro fertilized egg using co-curture, the expression of genes related to cellular metabolic processes increases It was confirmed. These results suggest that FSH and LH and cellular action increase the metabolism of EEC and also increase the development of Embryo.

Porcine spermatogonial stem cells (SSCs) are a useful model for a successful gene modification to generate transgenic sperm, and have been considered as an crucial tool for biomedical research. However, there have been no report presenting transfection methods suitable for porcine SSCs. Accordingly, we report optimized condition using an electroporation for gene modification of SSCs isolated from porcine testes. As the results. the highest transfection efficiency was observed in porcine SSCs transfected with 1 μg transgenic vector with a single electric pulse from an electroporator at a voltage of 100 V and a capacitor setting of 250 μF with minimal cell death. Moreover, the transfection efficiency and cell viability were constant without consideration of plasmid size. Furthermore, we didn’t observe loss of spermatozoa differentiation potential in the transfected porcine SSCs. From these results, we customize electroporation condition to porcine SSCs for establishment of successful gene delivery system effectively introducing foreign DNA into the genome of porcine SSCs without any loss of the original porcine SSCs characteristics.

The success of in vitro embryo production (IVP) demonstrates that it’s possible to bypass the oviduct throughout early development. However, several studies show that embryos developed in vivo are superior to embryos developed in vitro. Using an ex vivo model of porcine uterus is one of the strategies to investigate fertilization within the oviductal environment. During this study, in vitro matured porcine oocytes (MII) were fertilized with 7.5×107, 15×107 and 30×107 sperm cell for 20 min in porcine uterine ex vivo model. The oocytes were then flushed and performed in vitro culture (IVC) at 39°C for 168 hours under 5% O2, 5% CO2. MII oocytes used for in vitro fertilization (IVF) served as control-1. Before IVF, MII oocytes cultured in porcine uterine ex vivo model for 20 min served as control-2. Within the results, penetration rate, MPN formation, monospermy, polyspermy, and efficiency of fertilization had not shown significant difference between control-1 and control-2 group, respectively. However, penetration rate (treatments: 29.7±4.4, 34.3±3.2, 44.3±7.4 vs. 80.0±1.7), polyspermy (treatments: 5.7±5.7, 9.7±5.8, 8.0±4.0 vs. 33.7±9.5) and efficiency of fertilization (treatments: 23.7±2.3, 29.0±3.6, 35.0±4.6 vs. 43.0±5.8) were significantly decreased in treatment groups compared to control-1 (p<0.05). GSH accumulated levels were significantly decreased in 30×107 sperm cell treated group compared to control-1 (p<0.05) and there was no significant difference in ROS accumulated levels among the groups. For embryo development, the cleavage rate and blastocyst rate had not shown significant difference between control-1 and control-2 group. However, the cleavage rate (treatments: 16.3±2.6, 20.1±2.7, 40.7±13.4 vs. 69.5±6.3, 74.2±3.4) was significantly decreased in treatment groups compared to control-1 and control-2 (p<0.05). And the cleavage rate in the treatment group of 30×107 (40.7±13.4) was significantly higher than the treatment group of 7.5×107 (16.3±2.6) (p<0.05). The blastocyst rate (treatments: 31.7±4.0, 25.7±4.0, 26.7±6.5 vs. 7.2±2.4, 9.9±3.0) was significantly increased in control-1, control-2 and the treatment group of 30×107 compared to 7.5×107 and 15×107 (p<0.05). Therefore, these results suggest that ex vivo model may decrease the penetration rate and efficiency of fertilization by reducing GSH accumulated levels. Cleavage rate and blastocyst rate can be promoted by increasing sperm number during ex vivo fertilization.

For the establishment of successful pregnancy the maternal immune system must tolerate the implanting semi-allogenic conceptus, but the mechanism underlying this process is not fully understood in pigs. Among many factors, members of the tumor necrosis factor superfamily (TNFSF) have been considered as important immune regulators in cell-mediated immunity. TNFSF members bind to their responsive receptor containing cytoplasmic death domain to induce apoptosis in immune cells. Action of TNFSF members at the during pregnancy has been studied in some species including humans, mice and cows, suggesting that the TNFSF-induced apoptosis of activated maternal immune cells at the maternal-conceptus interface may be one of the mechanisms against rejection of semi-allogenic fetus. However, the expression and function of TNFSF members at the maternal-conceptus interface have not fully understood in pigs. Thus, to initiate the study on the role of TNFSF members for the establishment of pregnancy, we determined the expression of the TNFSF members, CD40 ligand (CD40LG), FAS ligand (FASLG), TNFα and TNF-related apoptosis inducing ligand (TRAIL; TNFSF10) in the endometrium and conceptus tissues during pregnancy in pigs. Real-time RT-PCR analysis showed that CD40LG, FASLG, TNFα and TNFSF10 mRNAs were expressed in the uterine endometrium during the estrous cycle and pregnancy. Levels of CD40LG, FASLG, TNF-α and TNFSF10 mRNA in endometrial tissues significantly increased on Day 15 of pregnancy, and levels of FASLG, TNF-α and TNFSF10 were also high on Day 60 of pregnancy and decreased thereafter. Immunohistochemical analysis showed that CD40LG and TNFSF10 proteins were localized mainly to luminal epithelial (LE) cells on Day 15 of pregnancy and amniotic membrane during late pregnancy, while FASLG protein was localized to LE cells during Day 30 to term and glandular epithelial cells during the estrous cycle and pregnancy. To understand the regulatory mechanism of endometrial CD40LG, FASLG and TNFSF10 expression by conceptus-derived cytokines, we treated endometrial tissues with increasing doses of interferon-γ (IFNG) and interferon-δ (IFND) and found that IFNG increased the expression of CD40L, TNFα and TNFSF10 mRNA, but not FASLG mRNA, and IFND increased TNFSF10 mRNA. To further understand the role of TNFSF10 on apoptosis of immune cells at the maternal-conceptus interface, we measured cell death of peripheral blood mononuclear cells by uterine epithelial cells expressing TNFSF10, and found that TNFSF10-expressing epithelial cells decreased survival of immune cells, especially of myeloid lineage. These results showed that CD40LG, FASLG, TNFα and TNFSF10 were expressed in a cell-type and stage-specific fashion in the endometrium during pregnancy, suggesting that CD40LG, FASLG, TNF and TNFSF10 may play an important role in the establishment of pregnancy by regulating the maternal immune response at the maternal-conceptus interface in pigs.

The efficiency of the in vitro production of oocytes is significantly lower than that of in vivo culture. Allicin (AL) is generally known to regulate the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study performed to reveal the effects of allicin treatment on porcine oocyte maturation and developmental competence. Cumulus oocyte complexes were cultured in IVM medium, containing 0, 0.01, 0.1, 1, 10, and 100 μM AL for 44 h. The rate of polar body emission was tended to be higher in the 0.1 AL-treated group than in the control group (p<0.1). The rates of cleavage and blastocyst formation significantly increased in the 0.1 AL-treated group (p<0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. The expression of maternal marker (BMP15 and CCNB1) and anti-apoptotic genes significantly increased in the 0.1 AL-treated group at MII stage, respectively (p<0.05). The rate of phosphorylated p44/42 MAPK to p44/42 MAPK was ~4-fold higher in the 0.1 AL-treated group than in the control group (p<0.05). Consequently, these results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes. In conclusion, allicin enhances the efficiency of IVM to establish a novel IVC system, and we suggested that it will be helpful reagent as assisted reproductive technology (ART) in the future.

Xenotransplantation has been presented as a strategy to meet the imbalance between organ donors and recipients. However, the immune suitability of organs between the animal organs and human bodies do not match, therefore, hyperacute-rejection (HAR) and antibody-dependent cellular cytotoxicity (ADCC), cell-mediated immune rejection arises from transplanting animal organ becomes necrosis appeared in a short time. Recently, various genetic engineering has produced GTKO pigs that knockout alpha- 1,3-gal genes known to be the cause of hyperacute rejection, In addition, various immunosuppressive studies are underway. The ADAM10 and 17 genes are proteins belonging to a disintegrin metalloprotease (ADAM) family and mainly function as sheddase. In normal cells, ADAM10 and ADAM17 has cut off EGH ligands to produce growth hormone, or TGFa is activated to inhibit viral infection. But, in tumor cells, MHC Class I-related proteins A, B and other ligands expressed on the cell surface are converted into soluble forms by ADAM10, 17 proteins and immunosuppressive reactions are performed for survival of tumor cells. The aim of this study was to investigate whether ADAM10 and 17 genes could be used to mimic immune-evasion mechanisms of tumor cells into normal cells to reduce cell-mediated rejection. In this experiment, the ADAM 10, 17 gene was obtained miniature pigs and amplified by PCR, inserted into pcDNA3.1 vector, and introduced Porcine Fetal fibroblast (PFF). Transgene cells selected by geneticin (500 ug/mL) treatment for 2 weeks were used target cell for cytotoxicity experiments. NK92MI cells (ATCC, CR2408) were used for NK cell cytotoxicity assay, and CD8+ T cells were separated by Ficoll gradient and stimulated with PMA (25 ng/mL) and IL-2 (50 U/mL) for 2 weeks. The results showed that the cytotoxic inhibitory effect of ADAM10, 17 on CD8+ T cells was greater than NK cells. Further studies will need to evaluate the soluble forms of NKG2DL that are involved in the immune response of NK cells and CD8+ T cells.

We examined the effect of endoplasmic reticulum (ER) stress inhibitor treatment during somatic cell nuclear transfer (SCNT) process on the reprogramming efficiency of porcine embryos. ER stress inhibitors such as Salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, 100 μM) were added to the manipulation medium and holding medium. Porcine SCNT embryos were sampled at the 1-cell stage for mRNA extraction. The expression of x-box binding protein 1 (Xbp1), ER stress-associated genes and apoptotic genes were confirmed by RT-PCR or RT-qPCR. The levels of X-box binding protein 1 (Xbp1) splicing and the expression of ER stress-associated genes were significantly decreased by TUDCA treatment at the 1-cell stage of SCNT embryos (p<0.05). The expression of ER stress-associated genes was also slightly decreased by a combination treatment of salubrinal and TUDCA (Sal+TUD). The expression levels of Caspase-3 and Bcl2-associated X protein (Bax) mRNA were also significantly lower in TUDCA and Sal+TUD groups (p<0.05). The blastocyst formation rate (20.2%, 41/203) and the mean cells number in blastocysts (63.0±7.2, p<0.05) were significantly increased in the TUDCA treatment group compared with the control group (12.6% and 41.7±3.1, p<0.05). These results show that the treatment of ER stress inhibitor, especially TUDCA, during SCNT process can inhibit cellular damages and enhance in vitro development of SCNT embryos by reducing ER stress.

최근 들어 암소의 수태율와 관련된 수많은 생리활성물질들에 대한 연구결과이 보고되 어지고 있다. 이들 중 저농도의 resveratrol 처리에 따른 효과 관련 연구결과들에서 임신 호르몬인 progesterone (P4), estradiol-17β (E2) 등의 생리적인 변화에 따라 sirtuin (SIRT1)의 발현에 유의적인 차이가 나타난다고 보고하고 있다. 따라서 본 연구목적은 한 우 암소 자궁 내 resveratrol을 주입하여 암소의 수태율을 관찰하고 이를 바탕으로 P4, E2, SIRT1의 변화양상을 조사하고자 한다. 공시축은 생후 14개월령 미경산 암소 20두를 대상으로 체중, 십자부고, 흉위, BCS 등 을 측정한 후 대조군 5두, 0.5, 1.0, 2.0 μM resveratrol 처리군 각 5두씩 설정하여 주입하 였다. Resveratrol 주입에 대한 수태율 효과를 정확하게 분석하기 위해서 전체 실험축군 을 배란동기화 처리하였고 10일째 일괄 인공수정 하였다. Resveratrol은 7일째 자궁 내 두당 20 mL 주입하였다. 배란동기화로 예상되는 주요 발정일은 9일째이며, 9일째 발정이 확인된 개체는 0.5 μM Resveratrol 주입군에서 100%로 가장 높게 나타났으며 대조군은 40%, 1.0, 2.0 μM Resveratrol 주입군에서는 20%로 각각 확인되었다. 임신감정은 수정 후 40일 경과 후 초음파장비를 통해 수행하였으며 그 결과 발정확인률과 유사한 경향치 를 보이며 0.5 μM Resveratrol 주입군이 60%, 대조군은 40%, 1.0, 2.0 μM Resveratrol 주입군은 20%로 나타났다. 따라서 선행된 in vitro 수준의 연구결과와 동일하게 in vivo 수준에서도 저농도의 Resveratrol이 암소 수태율에 긍정적인 영향을 미친다는 것을 확인했으며, 추후 사전에 확보 한 혈액으로부터 serum을 추출하여 P4, E2 등의 주요 호르몬 수치변화와 SIRT1의 발현 여부를 분석하여 resveratrol이 어떠한 상호작용을 통하여 oocyte maturation과 embryonic development에 관여하는지 추가로 검증할 예정이다. 본 연구결과는 농가에서 쉽고 간편 하게 저비용으로 수태율을 향상시킬 수 있는 기술이기 때문에 한우 사육농가의 수익향상 에 기여할 수 있다고 기대된다.

Tissue-type plasminogen activator, a protein used to treat heart attacks, converts plasminogen into plasmine, which digests fibrin and induces the dissolution of fibrin clots. The purpose of this study was to produce a tPA transgenic pig by somatic cell nuclear transfer. The human tPA (htPA) genes were transfected into porcine ear fibroblast cells derived from a female piglet and established two transgenic cell lines (Tg) with normal karyotypes. Tg line #3 and #4, which expressed high levels of the transgenes. Blastocyst rates of SCNT embryos derived from Tg lines were significantly lower than those of Non-Tg. A total of 2799 SCNT embryos were transferred to 9 surrogates. Seven offspring were delivered, and the production efficiency was 1.0~1.3% (3/324 and 4/304). All offspring exhibited htPA genes. These piglets revealed by microsatellite testing to be genetically identical to Tg cell lines, but not to the surrogate. Our goal is to establish a htPA transgenic pig for studying biopharmaceutical production.

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1- specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

Somatic cell nuclear transfer(SCNT) using micromanipulator have been used as a traditional cloning technique and applied various researches during the last decades. However the micromanipulator is expensive and relatively longer training period is required to operate it efficiently. Handmade cloning(HMC) is an alternative cloning method in a simplified way compared to the traditional cloning. Here, we suggested modified porcine handmade cloning(mHMC) as a new approach to clone porcine embryos as a substitute of the traditional SCNT. In mHMC, a nucleus was removed by an aspiration method by using a glass pipette, instead of bisection method. In this study, we investigated the efficiency of the enucleation method in mHMC by assessing the developmental competence of embryos in comparison with the traditional SCNT. The efficiency of enucleation was evaluated based on the rate of the accuracy and oocyte survivability. The accuracy of enucleation was lower in mHMC compared to those in SCNT(98.01±0.57 vs. 83.83±2.47), and the rate of survived oocytes was also lower in mHMC(96.50±0.84 vs. 90.10±2.11, respectively). And the developmental competence was assessed. The blastocyst rate was significantly higher in mHMC group(13.53±2.08 vs. 20.48±0.99). The levels of apoptosis and ROS were investigated to evaluate embryo quality. The expression of ROS and apoptosis-related genes showed no difference between groups. And the relative expressions of mRNA of pluripotency genes and reprogramming genes were evaluated. Although DNMT1 and DNMT3α were not differently expressed in two groups, the expression of the one of pluripotent gene, Oct4 was significantly higher in mHMC. In conclusion, based on the comparable results of mMHC and SCNT, the mHMC could be a suitable alternative technique to clone embryos in cost effective way compared to traditional SCNT.

The purpose of this study was to observe the changes in motility after frozen-thawing of black goat epididymal sperm. The black goat testis was transported to the laboratory within 30 minutes from the slaughterhouse and the temperature was set at 36.5℃. Sperm were collected by pressing the sliced epididymis and used the Semen Washing Media. The egg yolk-triladyl frozen solution was used for the freezing of black goat sperm and the freezing concentration was set to 1×108 sperm/mL. The frozen sperm were thawed in water bath at 37.5℃ for 45 seconds and motility was measured after preservation for 0, 30, 60, 90 and 120 min at 4℃, 17℃ and 37℃, respectively. In results, sperm motility at 30 min after thawing was significantly higher in 17℃ than 37℃ (50.44% vs. 26.60%; p<0.05). Likewise, sperm motility at storage time 60, 90, and 120 min after thawing was significantly lower in frozen-thawed sperm stored at 37℃ groups than frozen-thawed sperm stored at 4℃ and 17℃ (p<0.05). Although there was no significant difference between frozen-thawed sperm stored at 4℃ and 17℃, motility in the frozen-thawed sperm stored at 17℃ was slightly higher than stored at 4℃. In conclusion, this results suggested that 4℃ and 17℃ temperature are more efficient for storage of frozen-thawed spermatozoa in black goat than 37℃. However, more researches are needed to establish the optimal conditions or proper supplementation for sperm preservation.

The objective of this study was to investigate the influence of the addition of cryoprotectants to freezing extenders before semen cryopreservation on boar sperm quality as well as investigate their optimum concentrations. To evaluate the effect of pentoxifylline (0, 5, 10, 20 mM) added in egg yolk extender freezing medium on motion characteristics, motility, viability and acrosomal morphology of spermatozoa at post thawing of cryopreservation. Semen were collected from 12 boars and frozen on the same day. Qualified semen samples (motility>80%) from each boar were subdivided four groups, 0 (control), 5 mM (T1), 10 mM (T2) and 20 mM (T3). Motility was assessed for % motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). Frozen sperms were thawed in Beltsville Thawing Solution (BTS) then incubated at 38℃ for 20 minutes. Progressive motility, average path velocity, straight line velocity, curvilinear velocity and total motility were higher (p<0.05) in 0 mM (control group) as compared with pentoxifylline treated group. Moreover, pentoxifylline addition did not damage acrosomal morphogenic effect of spermatozoa. Nevertheless, pentoxifylline concentration treatment did not affect the viability of the spermatozoa as the concentration increased. The data showed that pentoxifylline as an additive cryoprotectant was not able to improve sperm motility but also quality in cryopreservation.

This study was performed to evaluate effects of Leucosporidium ice-binding protein (LeIBP) supplementation on boar (Duroc) sperm freezing. The collected semen was diluted (1.5×10⁸ /mL) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×10⁸ /mL) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05, and 0.1 mg/mL of LeIBP). The straws were stayed above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, sperm parameters such as motility, viability, and acrosome integrity, intracellular reactive oxygen species (ROS), apoptosis, and undetected lipid peroxidation were determined. Computer assisted sperm analysis was used for sperm parameters and flowcytometry was performed to acrosome integrity (FITC-PSA/PI), ROS (DCFHDA/ PI), lipid peroxidation (BODIPY C11/PI), and apoptosis (Annexin V/PI), respectively. The differences were not in sperm motility, but in viability of 0.05 and 0.1 mg/mL groups compared to control (p<0.05). Higher acrosome integrity was observed in LeIBP groups compared to control (p<0.05), except for 0.001 group. Both ROS and lipid peroxidation level were lower in all LeIBP groups than that of control (p<0.05). Furthermore, lower apoptosis rate was observed in both 0.005 and 0.1 LeIBP groups compared to control (p<0.05). It can be postulated that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

본 연구는 칡소의 동결정액 생산 시 항동해제 첨가 후 저온 저장 시간이 정자 생존성 및 운동성 등에 미치는 영향을 조사하고자 수행하였다. 공시축의 사양관리는 한우사양관 리(국립축산과학원, 2007) 기준에 준하여 사육하였다. 공시축은 정액채취를 위해 승가훈 련이 완료된 칡소 3두를 공시하였고 정액채취 빈도는 주 1회로 하였다. 정액 채취는 공시축의 승가 유도 후 인공질을 활용 채정을 실시하였고 위상차 현미경 하에서 생존율이 80∼90%로 검경된 원정액으로 동결정액 생산에 활용하였다. 동결보존 희석액의 기본조성은 Tris(hydroxymethyl)amino-methane 250 mM, Citric-acid monohydrate 80 mM, Fructose 60 mM, Penicillin G Streptomycin 1%, Egg-Yolk 20%, Glycerol 7% 농도로 하여 본 실험에 공시하였다. 동결정액의 생산은 채취된 원정액을 1차 희석 한 다음 저온 저장실로 이동하여 2차 희석 및 Glycerol 평형을 1시간 유도한 다음 0.5 mL Straw에 충진시켜 액체질소를 활용한 간이 급속 동결을 실시하였다. 융해 후 정자 특 성분석은 위상차 현미경과 컴퓨터정자분석기(Computer Assisted Sperm Analysis)를 활용 하여 생존율, 운동성, 선형성, 직진도 등을 Glycerol 평형직후, 4, 8, 12, 24시간 경과 후 각각 검사한 결과 생존율은 평형직후, 4, 8, 12, 24시간(75, 75, 70, 70, 55%)였고 운동성 은 12, 8시간, 평형직후, 24, 4시간(78.8, 78.5, 77.5, 76, 74.6%) 순이었으며, 선형성은 4 시간, 평형직후, 12, 8, 24시간(47.1, 49.5, 51.6, 51.8, 53%) 순으로 낮은 경향을 보였다. 직진도는 평형직후, 4, 8, 12, 24시간(61.2, 66.3, 68, 70.8, 70.8%) 순으로 낮은 경향을 보였다. 금번 연구 결과는 동결보존 희석액에 첨가되는 항동해제인 Glycerol을 첨가한 이 후 저온 저장 조건을 유지함으로써 정자의 가사상태 유지 및 정자의 운동성 등을 억제시 킨 결과로 항동해제 평형시간이 경과함에 따라 생존율 등은 다소 낮아지는 것으로 조사 되었다. 이는 정자의 체외 배출 이후 온도, 삼투압충격 및 대사에너지 소모에 따른 자연 사멸 등의 요인으로 저장시간이 경과함에 따라 생존율에 영향을 미쳤을 것으로 판단된 다. 위와 같은 결과는 동결정액 생산 기반 시설이 없는 농가 또는 타기관 보유 칡소 동 결정액 공유를 위해 사육 현장에서 채정 및 동결보존 희석 처리 시킨 다음 저온 조건을 유지시켜 생산이 가능한 장소로 이동 동결정액을 생산함으로써 시·공간적인 제약 해결이 가능하여 가축유전자원 보존·증식에 기여할 것으로 사료된다.

The reproductive system is very complex and diverse. Despite numerous studies, the exact mechanism of reproduction remains a subject to be explored. We have recently confirmed that the novel regulator CREBZF, which has been reported to play an important role in determining the fate of cells, is expressed specifically in the uterus, ovaries and testes, respectively. In the endometrium of uterus, CREBZF was found to be sensitively regulated by estrogen. In addition, the immunostaining showed that CREBZF is specifically expressed in the meiosis stage of spermatocyte during spermatogenesis in seminiferous tubules of testis. Finally, we discovered the strong expression of CREBZF in abnormally activated ovarian follicles by chemotherapeutic drug. These results suggest that CREBZF may play an important role in the activation of reproductive organs. In future study, we will perform the functional study and searching regulatory mechanism of CREBZF in these tissues.

황체는 배란 후 난포 내에 발생하는 일시적인 내분비 기관으로, estrous cycle에 따라 형성과 퇴행을 반복한다. 황체의 발생과정에는 새로운 혈관생성이 수반되는데, 이러한 현 상을 혈관신생(angiogenesis)이라고 한다. 황체의 발달은 초기, 중기, 후기로 구분하고, 황 체기마다 그 기능은 다양하게 작용한다. 또한, 황체 내 미세환경에 의한 angiogenesis의 과정도 다이내믹하게 변화한다. 따라서, 본 연구는 황체기의 변화에 따른 angiogenesis의 매커니즘을 명확하게 밝혀내기 위해서 실시하였다. 실험을 위한 황체조직 시료는 난소로 부터 회수하였고, 황체의 형태학적 구분에 따라 황체기를 구분하였다. 황체조직의 mRNA 와 protein은 Trizol (RNAiso Plus)과 protein lysis buffer 등을 처리하여 각각 추출하였다. 황체기별(초기, 중기, 후기) 황체조직에서 주요 angiogenesis 인자인 VEGF, Ang1과 그 수용체의 mRNA 및 protein 발현을 분석하였다. 그 결과, VEGF와 Ang1의 mRNA은 황체 초기, 중기에서 강하게 발현하고, 황체후기에서는 감소하는 양상을 보였다. 또한, 그들의 수용체인 VEGFR2와 Tie2의 mRNA도 초기, 중기에서는 강하게 발현하였으나, 후기에서 는 감소하였다. 그러나, 황체초기, 중기에서 두 수용체의 protein은 VEGFR2가 Tie2보다 강하게 발현하였고, 황체후기에서는 모든 수용체의 protein이 발현되지 않았다. 본 결과를 토대로, 황체 내 혈관신생 과정에서 VEGFR2와 Tie2는 중요한 역할을 할 것이다. 따라 서, 추가적인 세포실험을 통하여 황체 내 혈관세포와 그 외세포의 명확한 세포기능을 알 아볼 필요가 있다.

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon (IFNT) genes exist in cattle, However, molecular mechanisms of these bIFNT genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through caudal-relatedhomeobox-2 (CDX2) and ETS2 and AP1(JUN) and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine earderived fibroblast (EF) cells, were co-transfected with luciferase reporter constructs carrying upstream (positions −1000 to +51) regions of bIFNT1 and other new type I (bIFNT c1/c2/c3) gene and various transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, AP1 and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, RNA-seq analyses also revealed that the expression levels of bIFNT1/ c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and Madin-Darby bovine kidney (MDBK), Bovine ear fibroblast (EF) and (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

Chicken embryo fibroblast (CEF) cells are widely used in the study of embryology and differentiation. CEF and mammalian fibroblast cells differ in their requirements for defined nutrients in cell growth conditions. However, the molecular mechanism of the induced CEF proliferation is poorly understood. Our previous research has demonstrated that chicken GRP78 deficient promotes chicken cell apoptosis. In additionally, these studies identified the chicken Peroxiredoxin 3 protein (chPRDX3) in chicken cells by chicken serum-induced proliferation following two-dimensional (2D) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analyses. We had confirmed a relation between chPRDX3 upregulaion and cell proliferation in chicken embryo fibroblast cells. Our data demonstrated that the chPRDX3 significantly enhanced fibroblast proliferation. Moreover, knockdown of the chPRDX3 using siRNA decreased fibroblast proliferation and increased apoptosis. Therefore, the chPRDX3 is required for the cell proliferation in chicken fibroblast cells. Based on these results, we suggest that the chPRDX3 signaling pathway plays a critical role in chicken fibroblast survival. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells.

Apoptosis occurs under various developmental, physiological, and pathological conditions, including normal chicken cell death. We have previously shown that chGRP78 is typically expressed in chicken cells and plays a prominent role in the maintenance of cell homeostasis and prevention of apoptosis. In addition, chicken serum-mediated proliferation regulates chGRP78 to prevent apoptosis in chicken cells via chGRP78- mediated anti-apoptosis. However, the precise molecular mechanisms underlying the chGRP78-mediated protection against apoptosis remain undefined. Here, we performed a comparative proteomic analysis of control and chGRP78-overexpressing DF1 cells to elucidate the cellular events that are directly or indirectly regulated by chGRP78. Using 2D gel based proteomics and bioinformatics prediction analysis, we detected chGRP78 binding sites in AKT1-regulated proteins. chGRP78 promoted AKT1 activation, and chGRP78 silencing decreased AKT1 levels. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells via the inhibition of apoptosis.