The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

The aim of this study was to investigate effect of heat stress on expression levels of plasminogen activators (PAs) related mRNAs and proteins, and changes of PAs activity in porcine endometrial explants. The endometrial explants (200 ± 50 mg) were isolated from middle part of uterine horn at follicular phase (Day 19-21) and were pre-incubated in serum-free culture medium at 38.5oC in 5% CO2 for 18 h. Then, the tissues were transferred into fresh medium and were cultured at different temperature (38.5, 39.5, 40.5 or 41.5oC) for 24 h. The expression level of urokinase-type PA (uPA), type-1 PA inhibitor (PAI-1), type-2 PAI (PAI-2), and heat shock protein-90 (HSP-90) mRNA were analysis by reverse-transcription PCR and proteins were measured by western blotting. The supernatant were used for measurement of PAs activity. In results, mRNA and protein levels of HSP-90 was higher in 41.5oC treatment groups than other treatment groups (p < 0.05). The expression of uPA, PAI-1, and PAI-2 mRNA were slightly increased by heat stress, however, there were no significant difference. Heat stress condition suppressed expression of active uPA and PAI-2 proteins (p < 0.05), whereas PAI-1 protein was increased (p < 0.01). Although PAI-1 protein was increased and active uPA was decreased, PAs activity was greatly enhanced by exposure of heat stress (p < 0.05). These results suggest that heat stress condition could change intrauterine microenvironment through regulation of PAs activity and other factors regarding with activation of PAs might be regulate by heat stress. Therefore, more studies regarding with regulatory mechanism of PAs activation are needed.

The aim of this study was to investigate change of plasminogen activators (PAs) and their inhibitors (PAIs) mRNA and protein expression level by heat stress in porcine endometrial cells. The endometrial epithelial cells were isolated from endometrial epithelium in porcine uterus and cultured in different temperature conditions (38.5 and 41.5℃) for 24 h. Expression of urokinase-type PA (uPA), tissue-type PA (tPA), PA inhibitor-1 (PAI-1) and -2 (PAI-2) mRNA in epithelial cells were analyzed using reverse transcription-PCR and protein levels were measured by immunofluorescence. In result, mRNA expression of uPA, tPA, PAI-1 and PAI-2 were decreased in 41.5℃ than 38.5℃ culture condition, however, significant differences were no detected. uPA, tPA and PAI-2 protein were mainly expressed in nucleus, whereas PAI-1 was distributed in cytoplasm and nucleus. uPA and tPA protein levels were increased by heat stress treatment and significant difference was only detected in tPA level (p<0.05). In contrast, two types of PAIs protein level were decreased in 41.5℃ cultured group compared with 38.5℃ group. In present study, tPA protein expression was upregulated by heat stress in porcine endometrial cells. This result suggest that change of tPA by heat stress may be related to blood flow into uterus and intrauterine microenvironments, and could directly and indirectly influence to reproductive performance in pigs.

This study was to investigate effect of progesterone (P4) on prostaglandin (PG) synthases and plasminogen activators (PAs) system in bovine endometrium during estrous cycle. Endometrium tissues were collected from bovine uterus on follicular and luteal phase and were incubated with culture medium containing 0 (Control), 0.2, 2, 20 and 200 ng/ml P4 for 24 h. The PGF2α synthase (PGFS), PGE2 synthase (PGES), cyclooxygenase-2 (COX-2), urokinase PA (uPA), and PA inhibitors 1 (PAI-1) mRNA in bovine endometrium were analyzed using reverse transcription PCR and PA activity was measured using spectrophotometry. In results, COX-2 was higher at 2 ng/ml P4 group than control group in luteal phase (p<0.05), but, it did not change in follicular phase. Contrastively, PGES was significantly increased in 2 ng/ml P4 group compared to control group in follicular phase, but there were no significant differ among the treatments in luteal phase. uPA was no significant difference between P4 treatment groups and control group in both of different phase. PAI-1 was decreased in 20 ng/ml P4 group compared to control group in follicular phase (p<0.05). PA activity was decreased in 2 ng/ml P4 group compared to other groups in follicular and luteal phase (p<0.05). In conclusion, we suggest that P4 may influence to translation and post-translation process of PG production and PA activation in bovine endometrium.

Human tissue-type plasminogen activator (t-PA) is responsible for fibrin-specific plasminogen activation and plays a key role in fibrinolysis thereby aiding breakdown of blood clots in the vasculature. In the present study, in order to develop a system for production of recombinant st-PA and t- PAHis6 proteins in transgenic rice seeds, a DNA fragment encoding t-PA gene was selected and cloned to a plant binary vector (pMJ21) harboring a rice GluB1 promoter, an N-terminal signal peptide of the rice glutelin B1 protein and a Pin II terminator. The constructed plasmid was transformed into Agrobacterium tumefaciens LBA4404 (pSB1) to facilitate introduction into rice callus. The insertion of the st-PA and t-PAHis6 genes into the genome of transgenic rice seeds and their transcripts were confirmed using PCR, and Southern blot as well as RT-PCR, respectively. The highest level of recombinant st-PA expression as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 2,916 ng/total soluble protein (mg) in transgenic rice seeds. The amount of recombinant proteins expressed in transgenic plants was estimated to range from 634 ~ 2,916 ng/TSP mg (st-PA) and 925 ~ 2,640 ng/TSP mg(t- PAHis6), respectively. Immuno-blot analysis of transgenic rice seeds revealed single bands of approximately 68-kDa representing recombinant st-PA and t-PAHis6 proteins. These results demonstrate the expression and in vivo activity of recombinant st-PA and t-PAHis6 in transgenic rice seeds. This study is a promising endeavor for production of recombinant pharmaceutical proteins using rice seed system.

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. Two type of PAs are urokinase-type PA (uPA) and tissue-type PA (tPA). Plasminogen is present in most extracellular fluids. PAs play in various reproductive processes including implantation, ovulation and fertilization. In the spermatozoa, PAs and PAIs play a role in sperm motility and fertilization. PAs in the sertoli cell are stimulated spermatozoa maturation and sperm activation through the phospholipase A2. The oocyte maturation is the process for fertilization and implantation. PAs in cumulus-oocyte complexes (COCs) are related to oocyte maturation by protein kinase A and C. In the ovulatory process, PAs activity are changed and it are related to reducing the tensile strength of ovarian follicle wall. The uterine environment is important for reproduction and the uterus undergo tissue remodeling. In the uterus and oviduct of mammals, expression and activity of PAs are changed during estrous cycle. Thus, expression and activity of PAs are concerned to many reproductive functions. Therefore, PAs seem to important factor of regulator in reproductive events.

The aim of this study was to establish a three dimensional (3D) culture system of endometrial cells and to examine the plasminogen activators (PAs) activity in porcine uterine. The 3D culture system in porcine endometrial cells was composed to mixture 3D gel, stromal cells and epithelial cells. The 3D culture system was used to identify normal structure search as uterine tissue and PAs expression in this study. In results, porcine endometrium epithelial cells forming a top monolayer and endometrium stromal cells developed as fibroblast-like within 3D matrix scaffold. Expression of urokinase-type PA (uPA) and tissue-type PA (tPA) were observed during the 3D culture using immunofluorescence. PA activity in 3D-cultured endometrial cells was no significant difference between the tissue type, but 2D culture system were significantly lower than in 3D-cultured endometrial cells (P<0.05). Therefore, basic system and functional aspect of 3D culture could be established with similar system of endometrium tissue. We suggest that this study was assumed applicable as baseline data to investigate mechanism between porcine uterus cells in vitro.

The endometrium undergoes a cyclic growth and tissue remodeling as changes of epithelial cells, and plasminogen activators (PAs) are related to endometrium tissue remodeling. This study was to evulate expression of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in porcine uterine epithelial cells. In results, the uPA and tPA were expressed in uterine tissue, epithelium and secretory glands in porcine endometrial cell. In addition, the uPA and tPA were expressed in cultured epithelial cells, and it were mainly expressed in cytoplasm. In porcine uterine tissue and epithelial cells, uPA activity was higher than activity in tPA. In PAs mRNA expression levels, uPA mRNA level was significantly higher than tPA mRNA level (P<0.05). The fluorescence intensity of uPA protein was also higher than fluorescence intensity of tPA protein, and uPA protein expression was significantly higher than in tPA protein expression (P<0.05). Therefore, we suggest that a physiological function in porcine uterine epithelial cells should be more influenced by uPA than in tPA during pre-ovulatory phase. The endometrium undergoes a cyclic growth and tissue remodeling as changes of epithelial cells, and plasminogen activators (PAs) are related to endometrium tissue remodeling. This study was to evulate expression of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in porcine uterine epithelial cells. In results, the uPA and tPA were expressed in uterine tissue, epithelium and secretory glands in porcine endometrial cell. In addition, the uPA and tPA were expressed in cultured epithelial cells, and it were mainly expressed in cytoplasm. In porcine uterine tissue and epithelial cells, uPA activity was higher than activity in tPA. In PAs mRNA expression levels, uPA mRNA level was significantly higher than tPA mRNA level (P<0.05). The fluorescence intensity of uPA protein was also higher than fluorescence intensity of tPA protein, and uPA protein expression was significantly higher than in tPA protein expression (P<0.05). Therefore, we suggest that a physiological function in porcine uterine epithelial cells should be more influenced by uPA than in tPA during pre-ovulatory phase.

New therapeutic measure are needed to improve the outcome for patients with oral squamous cell carcinoma(OSCC) because OSCC continues to portend a relatively unfavorable prognosis. Recently RNA interference(RNAi) has emerged as an effective method to target specific genes for silencing. Although overexpression of urokinase-type plasminogen activator receptor(uPAR) has been implicated in progression and metastasis of OSCC, the transfection effect of RNAi- uPAR on OSCC has been rarely reported. The purpose of this study were to examine the efficient and specific inhibition of uPAR mRNA and protein expression by siRNA targeting of uPAR through RT-PCR and immunoslot blotting, and to study cell proliferation activity, adhesion, invasion and migration in vitro compared to the controls. In MTT assay, siRNA-uPAR transfected cells showed about 70-80% cell proliferation compared to OSCC cell lines after 2 days. In adhesion assay, siRNA-uPAR transfected cells showed about 20-30% adhesion activity compared to OSCC cell lines, but similar features to those of BSA coated wells. In migration assay, siRNA-uPAR transfected cells showed about 60% migration activity compared to OSCC cell lines, but higher 3.5 folds to those of BSA coated wells. In invasion assay, siRNA-uPAR transfected cells showed about 55% invasive activity compared to parental cell lines. mRNA expression of siRNA-uPAR transfected cells showed about 10-15 % compared to parental cell lines by RT-PCR. Protein expression of siRNA-uPAR transfected cells showed about 25% compared to parental cell lines by ELISA assay. It suggested that RNAi-uPAR tranfection might be used as a potent and specific therapeutic tool for the treatment of oral squamous cell carcinoma, especially in inhibiting invasion and metastasis.

The present study was performed to identify the relationship between plasminogen activator (PA) and Heat Shock Protein-90 (HSP-90) in porcine uterus tissues during the estrous cycle. Porcine uterus tissues were obtained from preovulatory (Pre-Ov), post-ovulatory (Post-Ov) and early to mid-luteal (Early-mid L) stages. The protein was extracted from uterus tissue by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by RIPA Buffer and quantified by BCA methods. As results, t-PA expression was significantly (p<0.05) higher from pre-ovulatory(Epithelium tissue: 29,067 μg/μl, Myometrium tissue: 30,797 μg/μl) compared to the post-ovulatory stage(Epithelium tissue: 54,357 μg/μl, Myometrium tissue: 53,270 μg/μl) and early to mid-luteal stage(Epithelium tissue: 42,380 μg/μl, Myometrium tissue: 43,139 μg/μl). On the other hand, the uPA expression indicated higher from early to mid-luteal stage (Epithelium tissue: 0.02198 μg/μl, Myometrium tissue: 0.02412 μg/μl) than pre-ovulatory stage (Epithelium tissue: 0.01577 μg/μl, Myometrium tissue: 0.01531 μg/μl) and post-ovulatory stage(Epithelium tissue: 0.01414 μg/μl, Myometrium tissue: 0.01429 μg/μl). However, expression of u-PA did not differ from each estrous cycle in the epithelium tissue and myometrium tissue(p<0.05). Expression of HSP-90 was differ t-PA and u-PA from pre-ovulatory in Epithelium tissue(25,423 μg/μl) and early to mid-luteal stage in epithelium tissue(177,922 μg/μl) and myometrium tissue(26,664 μg/μl). These results suggest that HSP-90 and u-PA were related with change of uterus cycle according to the reformation of the tissues in porcine uterus.

선행 연구에서 형질전환 모상근 대량생산 최적배지 선발을 통해 WPM(Woody Plant basal salt Mixture) 배지가 형질전환 모상근 대량생산의 최적배지임을 확인할 수 있었다(Kim et al., 2012). 이에 당, 탄소원, pH, 무기염, 식물 호르몬 등이 형질전환 모상근의 수량성에 미치는 연구를 통해 최적의 형질 전환 모상근 대량생산 조건을 확립한 결과는 다음과 같다. 1. 형질전환 모상근 대량생산에 적합한 pH는 7.0이었으며, 최적의 탄소원과 농도는 1%의 sucrose임을 확인하였다. 2. 형질전환 모상근 대량생산에 적합한 적정 무기염들 (KH2PO4,NH4NO3, KNO3)의 농도는 KH2PO4 경우 MS배지와같은 농도인 0.94 mM에서 수량성이 303 g으로 가장 높았고, NH4NO3 및 KNO3 농도는 MS배지 농도의 1/4배인 3.87 mM, 3.52 mM 처리가 345 g과 358 g으로 수량성이 가장 높았다. 3. 형질전환 모상근 대량생산에 있어 식물생장조절물질 NAA, IBA의 최적 농도는 무처리 경우가 각각 595 g과 402 g으로 수량성이 가장 높아 대량생산에는 식물생장조절물질 처리가 필요 없을 것으로 판단되었다.

Urokinase-type plasminogen activator(uPA) bound to urokinase plasminogen activator receptor(uPAR) expression is strongly correlated with the metastatic potential of various tumors by enhancing ECM degradation through plasminogen and matrix metallopreotease activation. But expression of uPA/uPAR in human malignant salivary gland tumors has been rarely reported. The purpose of this study were to investigate mRNA expression and cytologic concentration of uPAR in SGT cell line compared to various cancer cell lines by RT-PCR and ELISA method, and to study migration and adhesion assay. These results would be to apply the pathogenesis and prognosis of malignant salivary gland tumors. All the cell lines(SGT, HN 4, SCC 25, and HeLA) were cultured under DMEM with 10% FBS at 37℃ in a 5% CO2 incubator. We studied a possible association between mRNA expression and cytosolic concentrations of uPAR in SGT cell line compared to various cancer cell lines using RT-PCR and an enzyme-linked immunoassay( ELISA) method. And also cell adhesion and migration assay were done in all the cell lines. In migration assay SGT cell line was about 2.5-4 folds higher than another cell lines. In adhesion assay SGT cell line was about 1.1-2 folds higher than another cell lines. uPAR cytosolic concentrations of SGT cell line was about 3.4-10 folds by ELISA, while mRNA expression was about 2.5-5 folds by RT-PCR. Oral Scc cell lines showed the lowest value. uPAR protein and mRNA expression were correlated with migration and adhesion assay. From the aboving results, high cytosolic concentrations and mRNA expression of uPAR were correlated with migration and adhesion assay. It suggested that this might be a specific marker for malignant potential of SGT cell line and would be contributed to pathogenesis and prognosis of human salivary gland adenocarcinoma

This study investigated the changes of plasminogen activators (PAs) activity, expression and localization of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) during the estrous cycle in pigs. Estrous cycle was sorted into three group by pre-ovulation (Pre-Ov), post-ovulation (Post-Ov) and early to mid-luteal stages (Early to mid-L). Analysis for immunohistochemistry was confirmed by location of tPA and uPA. Porcine uterus tissue was cut into 1 × 1 cm squares, and were incubated in DMEM/F-12 medium for 1 h at 38℃, 5% CO2 for measurement of PA activity. Western blotting was implemented for measurement of PA quantity. In results, the blood vessels and secretory glands were increased in Post-Ov stage than Pre-Ov and Early to mid-L stages. The tPA and uPA was located mainly within lumen of blood vessels and secretory glands. The PA activity in Post-Ov (0.99±0.03) stage were significantly (p<0.01) higher than Pre-Ov stage (0.51±0.03) and Early to mid-L stage (0.21±0.04). Expression of PAs were significantly (p<0.05) higher in Early to mid-L stage than other stages. These results indicate that PAs activity and expression may change in uterus tissue during the estrous cycle in pigs.

Plasminogen activator(PA) system such as urokinase plasminogen activator(uPA), urokinase PA Receptor(uPAR), tissue, tissue PA, and PA inhibitor-1&2(PAI-1&2) play a role in tumor invasion, metastasis, and proliferation. It is interested that these factors in patients with primary oral squamous cell carcinoma(Oral SCC) will be evaluated and correlated with clinicopathologic variables. Recently, these expression of primary oral SCC has been restricted to clinical or immunohistochemical study such in vivo study. The purpose of this study were to investigate the mRNA expression and cytologic concentration of uPA, uPAr, tPA, and PAI-1,2 in oral SCC cell lines compared to NHOK and to apply these results to evaluate early detection biomarkers of oral SCC in future. All the cell lines(NHOK, HN 4 and SCC 25) were cultured under KBM bullet kit at 37℃ in a 5% CO2 incubator. We studied a possible association between mRNA expression and cytosolic concentrations of uPA, uPAR, tPA, and PAI-1,2 in oral SCC cell lines compared to NHOK using RT-PCR and an enzyme-linked immunoassay(ELISA) method. uPA mRNA expression was about 5-6 folds, while uPAR was a bout 3 f olds, and PAI-1 was about 1 .5-1.6 f olds. PAI-2 was a bout1.2 -1.3 f olds t han that o f NHOK, w hile t PA w as l ower t han that of NHOK. uPA cytosolic concentrations was about 15-19 folds, while uPAR was about 8 folds, and PAI-1 was about 3-4.5 folds. PAI-2 was about 2 folds than that of NHOK, while tPA was lower than that of NHOK. Both uPA, uPAR, and PAI-1,2 cytologic concentrations were correlated with mRNA expression of oral SCC cell lines. From the aboving results, high cytosolic concentrations of uPA, uPAR, and PAI-1 & 2 were correlated with mRNA expression. It suggested that these might be specific markers for oral SCC cell lines and these results would be contributed to evaluate early detection biomarkers for human oral squamous cell carcinoma.

Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.

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.

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.

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide (H2O2), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decreased with addition of H2O2, and were significantly (p<0.05) lower in medium with 0.1 mM H2O2 than control group. Also, the rate of degenerated oocytes was increased in as H2O2 concentration increased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When H2O2 concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM H2O2 and (3) control medium with 1.0 mM H2O2 along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. H2O2 decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity against oxidative stress caused by H2O2. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM H2O2 alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.