In order to investigate the effects of antioxidants on the culture of mouse preantral follicles in vitro, we examined the effects of taurine, glutathione and catalase on their growth and maturation. Addition of taurine was not effective on the survival of preantral follicles. However, metaphase II rates of oocytes within preantral follicles were signifi-cantly higher in 1 mM treated group than in control and 10 mM treated group (p<0.05). Glutathione did not improved the rates of survival and metaphase II oocytes. However, metaphase II rates of oocytes progressively decreased with increasing glutathione concentration. Catalase also showed that the rates of survival and metaphase II oocytes pro-gressively decreased with increasing concentration. Especially, all of preantral follicles cultured in medium containing 100 IU/ml catalase were degenerated. These results suggest that low concentraion of taurine, as an antioxidant, have positive effect on the culture of mouse preantral follicles in vitro.

Toxic heavy metals like mercury and cadmium are known to involve in altering the salivary flow so that can be appeared sialorrhea or ptyalism, the condition of increased salivary flow, or xerostomia (“dry mouth”), the condition related to inhibited or decreased salivary flow. Although many people were exposed to these heavy metal in work environment, dental clinics, the mechanism is rarely discussed in the clinical literature. The present study is to carried out analysis of AQP5 expression that play a key role in saliva fluid secretion and cell membrane water permeability on mercury- or cadmium-exposed mice submandibular gland. To investigate AQP 5 expression, immunohistochemical study and western blot assay were carried out on mercury- or cadmium-exposed mice. Additionally, RT-PCR, real- time PCR with specific primers were carried out. Cadmium or mercury exposure led ductal extension, ductal cell increase, and blood vessel increase in mouse submandibular gland. The mRNA and protein expression of AQP5 were increased in time dependent manners on cadmium or mercury exposed mouse. Also, AQP5 were translocated from basolateral membrane to apical membrane of acini cell. In conclusion, toxic heavy metal such as mercury and cadmium appear to alter the AQP5 expression and distribute to apical membrane of ductal cell and lead to alter salivary secretion.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-34TM cell culture media at 37℃. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin α6 and β1, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

노화란 시간경과에 따라 생체기능의 불가역적인 저하로 치매, 뇌혈관질환, 고혈압, 암 등과 같은 노화관련 질병이 생겨나며, 세포들이 생명력을 잃어가는 일련의 현상이다. 노화로 인한 대표적인 질병은 퇴행성 뇌 질환에 의한 알츠하이머, 치매와 파킨슨병이 있다. 이 병들은 초기의 변화가 두드러지지 않아 조기 진단이 어렵고, 질병의 진행을 막는데 어려움이 있다. 이를 해결할 방법으로 유전자 지표를 개발하는 것이 중요한 과제라 할 수 있는데, 본 연구는 노화 동물모델인 SAMP1 마우스를 대조군인 SAMR1 마우스를 비교하여 노화의 초기단계에 차등 발현하는 유전자를 확인하여 향후 노화와 연관 지표를 발굴하기 위하여 실시되었다. 먼저, 성장단계인 8주령과 노화초기진행단계인 26주령의 뇌 조직에서의 차등발현 유전자를 발굴하고자 cDNA chip 분석을 수행하였고, 신뢰성을 가지고 생물학적 변이를 검출할 수 있는 유전자는 13,939개의 유전자를 발굴하였다. 이들 중 발현이 2-fold 이상 증감하는 유전자들을 조사한 결과, 2,029개의 유전자를 선별할 수 있었다. 또한, 기능별로 분류한 결과 노화가 진행되는 과정에서 총 증가 혹은 감소한 유전자의 개수는 660개였으며, 이 중 노화와 연관성이 높은 유전자는 74개였다. 26주령에 증가양상을 보인 것이 46개, 감소양상을 보인 유전자가 28개로 밝혀졌다. 이를 검증하기 위해 노화와 관련된 유용한 유전자인 Wnt7a, Prkcb, Dgke가 선별되어 real-time qPCR을 수행하였다. 그 결과 Wnt7a 유전자는 노화가 진행됨에 따라 발현양이 급격히 감소하여 노화 초기 단계의 지표로 활용이 가능할 것으로 사료된다. 반면에 Prkcb 유전자는 성장단계의 발현량에는 관계없이 노화초기단계에서 억제됨을 확인하였고, Dgke는 성장단계부터 유전자 발현이 억제되어 노화에 영향을 미칠 것으로 판단된다.

The purpose of this study was attempted to new methods in mammalian embryos vitrification. This method was affected to increase of the embryo vitrification efficiency and it would be applied to the field of embryo transfer to recipient by modified loading method of embryo into 0.25 ml plastic straw. The frozen mouse embryos were carried out warmed from two different cell stages (8-cell and blastocyst, respectively) by attachment of an embryo in the vitrification straw (aV) method. All groups were cultured in M-16 medium to determine the development and survivability for 24 h, respectively. Results shown that, the survivability of two different groups were significantly different (94.8% vs. 70.9%). Total cell number was not significantly different the non-frozen blastocyst (99.7 ± 12.4) compared to the post-thaw blastocyst (94.8 ± 15.1). From the 8-cell embryo, total cell number of frozen blastocysts were significantly lower than others groups (74.7 ± 14.6, p<0.05). In the case of cell death analysis, the blastocysts from non-frozen and frozen-thawed 8-cell group were not different (0.0 ± 0.0 vs. 1.9 ± 3.1, p>0.05). However, the apoptotic nuclei of blastocyst were significantly observed the frozen-thawed group (5.4 ± 4.4) compared to non-frozen group (p<0.05). Therefore, this new method of embryos using in-straw dilution and direct transfer into other species would be more simple procedure of embryo transfer rather than step-wise dilution method and cryopreservation vessels, so we can be applied in animal as well as human embryo cryopreservation in further.

Matrix metalloproteinases (MMPs) have been known to affect to cell migration, proliferation, morphogenesis and apoptosis by degrading the extracellular matrix. In the previous studies, undifferentiated mouse embryonic stem cells (ESCs) were successfully proliferated inside the extracellular matrix (ECM) analog-conjugated three-dimensional (3D) poly ethylene glycol (PEG)-based hydrogel. However, there is no report about MMP secretion in ESCs, which makes it difficult to understand and explain how ESCs enlarge space and proliferate inside 3D PEG-based hydrogel constructed by crosslinkers containing MMP-specific cleavage peptide sequence. Therefore, we investigated what types of MMPs are released from undifferentiated ESCs and how extracellular signals derived from various niche conditions affect MMP expression of ESCs at the transcriptional level. Results showed that undifferentiated ESCs expressed specifically MMP2 and MMP3 mRNAs. Transcriptional up-regulation of MMP2 was caused by the 3D scaffold, and activation of integrin inside the 3D scaffold upregulated MMP2 mRNAs synergistically. Moreover, mouse embryonic fibroblasts (MEFs) on 2D matrix and 3D scaffold induced upregulation of MMP3 mRNAs, and activation of integrins through conjugation of extracellular matrix (ECM) analogs with 3D scaffold upregulated MMP3 mRNAs synergistically. These results suggest that successful proliferation of ESCs inside the 3D PEG-based hydrogel may be caused by increase of MMP2 and MMP3 expression resulting from 3D scaffold itself as well as activation of integrins inside the 3D PEG-based scaffold.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the causative bacteria that can induce chronic enzootic pneumonia, resulting in low production in the swine industry. Potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia by M. hyopneumoniae has also been recognized. Although some available vaccines have been developed for prevention of M. hyopneumoniae infection, protective immunity is still poor. In this study, in order to provide valuable information on vaccine antigen, we investigated the immunogenicity of M. hyopneumoniae on mouse spleen cells. Concanavalin A (ConA) and lipopolysaccharide (LPS) were used for generation of activated T and B lymphocytes. M. hyopneumoniae made clusters of spleen cells and also affected the cellular activity and viability of spleen cells by alone or with mitogens. Of particular interest, it induced a significant increase in production of TNF-alpha in ConA- treated spleen cells, meaning T helper 1 response. In addition, cell size and mitochondrial membrane potential of M. hyopneumoniae–treated spleen cells were measured by flow cytometric analysis. M. hyopneumoniae did not affect the cell size by alone, whereas ConA or LPS profoundly increased the cell size. Taken together, M. hyopneumoniae significantly affect the cellular activity and cytokine production of spleen cells by alone or in a combination of ConA. This study provides valuable information for production of the vaccine against M. hyopneumoniae.

본 연구에서는 뽕잎차와 Monascus pilosus로 발효시킨 뽕잎차가 ICR mouse의 체중과 간 조직 항산화계 효소류의 활성에 미치는 영향을 조사하기 위하여 정상대조군(NC), 고지방식이 대조군(HC), 뽕잎차 분말을 2% 혼합한 고지방 식이군(UM), 발효 뽕잎차 분말을 2% 혼합한 고지방 식이군(FM)으로 나누어 8주간 사육하였다. 체중증가량에 있어서는 HC군의 주당 체중증가량은 NC군에 비해 2.13배로 높았으나, UM군과 FM군은 NC군의 1.80 및 1.67배로 체중증가량이 크게 완화되었다. 장기 중량은 고지방 식이군에서 증가되었던 간의 중량은 UM과 FM군에서 회복되는 경향을 보였으며, 신장의 경우에도 NC군 수준으로 회복되는 경향을 나타내었다. 부고환 주변 지방의 함량은 UM과 FM군의 경우 모두 고지방식이로 현저하게 증가된 지방함량을 유의적으로 감소시켰으며, 그 효과는 FM군에서 현저하였다. ALT 활성은 HC군이 NC군에 비하여 2.56배나 높은 수치를 나타내었으며, UM군과 HC 간의 유의차는 보이지 않으나 FM군은 HC군에 비하여 유의적으로 낮았다. TG, TC, LDL-cholesterol의 함량 및 동맥경화지수(AI)는 HC군은 NC군에 비하여 각각 18.42%, 39.59%, 212.56% 및 230.70%가 증가하였으며, HDLcholesterol 함량은 17.84%가 감소하였다. UM군의 TG, TC, HDL- 및 LDL-cholesterol 함량은 HC군과 유의적인 차이가 없으나, FM군에서는 유의적인 차이를 보였으며, 동맥경화지수도 HC군 2.63, UM군 2.09, FM군 1.70으로 FM군에서 양호한 결과를 나타내었다. 간 조직 TG 함량은 UM 및 FM군이 HC군에 비하여 25.18∼25.72%가 감소되었으며, 총 콜레스테롤의 함량에서도 TG와 유사한 경향을 보였다. Glutathione(GSH)의 함량은 FM군이 UM군에 비하여 유의적으로 증가하였으며, lipid peroxide(LPO)의 함량은 UM군과 FM군은 HC군에 비하여 각각 49.82% 및 44.52% 감소하였다. Xanthine oxidase(XO) 활성은 UM군과 FM군에서는 HC군에 비하여 각각 38.41% 및 53.62%가 감소하였다. SOD 활성은 UM과 FM군 모두 NC군과 유사한 활성을 나타내었다. Glutathione S-transferase(GST)활성은 UM군과 FM군은 고지방 식이에 의해 감소된 HC군의 활성을 14.81∼23.46% 증가시켰다. Glutathione peroxidase(GPX) 활성(NADPH nmole/min/mg protein)은 고지방 식이 대조군인 HC군에서의 활성은 NC군의 65.59%에 불과하였으나, UM군과 FM군은 HC군에 비하여 각각 22.95% 및 38.52% 증가하였다. 간 조직을 검경한 결과, 고지방 식이 대조군인 HF군에서는 간 조직 세포가 지방 방울의 축적에 의해 확장되어 간굴모세혈관(hepatic sinusoids)이 폐쇄된 상태였다. 하지만, UM군과 FM군에서는 중심정맥 주위의 세포들이 정상적으로 잘 보존되어 있으며, 간 소엽의 간세포삭과 간 조직 세포 내에 지방방울의 축적이 보이긴 하나 구분이 가능하며, UM군에 비하여 FM군 쪽의 간 손상이 보다 경미한 것으로 나타났다. 이상의 실험결과, M. pilosus로 발효시킨 뽕잎차는 정상식 이군에서는 간 조직 손상과 관련된 LPO의 함량을 경감시키는 효과와 함께 경미한 체중 감소 효과가 있는 것으로 사료되며, 고지방 식이에 의한 비정상 생리 상태에서는 비만 해소와 ROS 생성계효소의 저해와 소거계 항산화 효소의 활성화를 통한 간 기능 증진과 조직 손상을 예방 또는 경감시키는 효과가 있는 것으로 생각된다.

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

The purpose of this study was to assess follicular viability and competence through in vitro culture of preantral follicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified-warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro matured oocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution of whole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposure groups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups (69.4±2.8 and 67.8±3.1) when compared to that of control group (71.7±2.1). There was no statistical significant difference in blastocyst development rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cells per blastocyst was significantly lower (p<0.05) in the vitrification group (41.9±20.2) than in the fresh control group (55.1±22.5). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovaries can achieve full maturation, normal fertilization and embryo development.

Interferon induced transmembrane protein-1 (Ifitm-1) has been reported to have an important role in primordial germ cell formation, and it has expressed in female reproductive organ. In the present study, Ifitm-1 gene expression was identified in testes and all part of epididymis using western immunoblot and immunohistochemistry. Interestingly, Ifitm-1 expression was observed on the head of spermatozoa. To investigate the role of Ifitm-1 gene expression in behavior of spermatozoa after acrosome reaction, fresh sperm was incubated with calcium ionophore to induce acrosome reaction, whereas the expression of Ifitm-1 was not altered after the acrosome reaction. Then to identify the effect of Ifitm-1 in sperm motility and other seminal parameters, different concentration of Ifitm-1 antibody was incubated with spermatozoa, and seminal parameters were assessed using computer-assisted semen analysis (CASA). Interestingly, motility, progressive, and VAP were increased in the sperm with Ifitm-1 antibody treated compared to rabbit serum, however other parameters such as straightness were not changed. In order to identify the functional significance of Ifitm-1 in fertilization, capacitated spermatozoa were pre-incubated with anti- Ifitm-1 antibody and subsequently examined the ability to adhere to mouse oocytes. However, any defection or alteration in sperm-egg fusion was not found, Ifitm-1 antibody treated or non-treated spermatozoa showed a normal penetration. Although the precise role of Ifitm-1 in sperm motility and following fertilization need to be elucidated, this study suggests that the activation of Ifitm-1 on the sperm may enhance the motility of spermatozoa in mice.

Human natural killer (NK) cells are major players in innate immune response. The functions of these cells as a scavenger of cancer cells are enhanced by cytokines such as interleukin-2 (IL-2), which play an important role in immune response in both tumors and virally infected cells. Liver cancer has a high incidence rate and is a major cause of death in Korea. We provide evidence that human NK cells inhibit tumor growth of the hepatocellular carcinoma cell line SNU-354. NK cells were cultured with human IL-2 for 14 days, yielding an enriched NK cell population containing 35% CD8+ cells, 6% CD4+ cells, and 51% CD16+ /CD56+ cells. Intravenous injection of NK cells at doses from 2.5 to 10 million cells/mouse was administered once per week in a nude mouse model that retains human liver tumor induced by implantation of SNU-354 cells. The results showed that human NK cells were recruited within tumor tissue and inhibited SNU-354 tumor growth by 32%, 58%, and 65%. The current data suggest the potential for use of NK cell-based immunotherapy for treatment of human liver cancer.