Lipopolysaccharide (LPS)는 염증유발 cytokine 분비를 자극하고 염증을 유발하는 그람음성균의 내독소이다. 본 연구에서는 LPS가 신경아교세포 활성과 해마에 있는 nuclear factor kappa B (NF-κB) 매개 염증유발 요소를 조절하는지를 조사하였다. 성체 수컷 쥐를 대조군과 LPS를 투여한 실험군으로 무작위로 나누어 vehicle 또는 LPS (250 μg/kg)를 5일 동안 복강투여하고 무게를 측정했다. 해마의 활성산소와 과산화지방질 수준을 분석하고, 형태학적 연구를 위해 Hematoxylin and eosin 염색을 시행하였다. 또한, 해마에서 ionized calcium-binding adapter molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), NF-κB, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α)의 발현을 확인하기 위해 Western blot 분석과 immunofluorescence 염색을 시행하였다. 그 결과, LPS를 투여한 쥐들의 체중이 감소하였다. LPS 투여는 활성산소와 과산화지방질 수준의 증가를 유발하였고 LPS를 투여한 쥐의 해마에서 심각한 조직병리학적 변화를 확인했다. 또한 LPS 투여는 신경교세포와 별아교세포의 표시물인 Iba-1과 GFAP의 발현을 증가시켰고, NF-κB의 발현과 IL-1β와 TNF-α와 같은 염증성인자의 발현을 증가시켰다. 이러한 결과들을 통해 LPS 투여는 해마손상과 염증반응을 유도한다는 것을 알 수 있고 LPS 투여가 해마조직에서 신경아교세포와 NF-κB에 매개된 염증인자들을 활성화시킨다는 것을 확인하였 다. 따라서, 본 연구는 LPS 투여는 해마조직에서 산화적 스트레스 증가와 염증인자 활성을 증가시켜 신경손상을 유도함을 보여준다.

Our study has analyzed whether inappropriate gonadotropin secretion affects the morphological changes due to the activation of intrauterine MMP. Methods A total of each 6 mice were injected with PMSG, Progesterone, and Androgen in 5 IU of intraperitoneal injection every 2 days after estrus synchronization, and morphological and MMPs expression patterns were compared after inducing hormone secretion. Also, cell survival and death related genes were compared and analyzed. The endometrium was highly developed in the PMSG, and the androgen was not developed at all. In particular, the diameter of the uterus of the Androgen group was also very narrow. MMPs activity assay in the case of PMSG was confirmed that showed low activity, whereas, progesterone and androgen In showed high activity and, in particular, very high activity of MMPs in the case of androgen in glandular cell. The expression of VEGF in the tissues of each group was different from that of MMPs. In the PMSG group, the activity of VEGF was increased in both the Myo-metrium and the endo-metrium, whereas the progesterone group showed low overall expression in the endo-metrium. Therefore, the present study showed that the activities of the endo-metrial cells and the restructuring of the endometrial cells differed according to the type of the abnormal secretory hormone. In particular, the secretion of androgen increased the activity of MMPs throughout the uterus, The endo-metrial epithelial cells are affected by the progesterone group. In conclusion, this study suggests that inappropriate gonadotropin secretion increases the functional changes of the uterus and this reconstruction may be caused by increased activity of MMPs

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitromatured pig oocytes and treated for 4 h after electric activation with 0.5 μM latrunculin A (LA), 10.4 μM cytochalasins B (CB), and 4.9 μM cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8～88.6%), blastocyst formation (30.7～ 32.4%), and mean cell number of blastocysts (33.5～33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5～78.0%) and mean blastocyst cell number (33.6～35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5～85.7%) and blastocyst cell number (41.1～43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21～22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05<P<0.1) nuclear maturation when compared with oocytes matured in M199 (76.9% vs. 83.8%) but no significant differences were found in embryo cleavage, blastocyst formation, and mean number of cells in blastocyst (73.8% vs. 74.6%, 11.1% vs. 12.1%, and 28.4 cells vs. 30.1 cells, respectively). The oocyte degeneration was not reduced by CB treatment during ICSI (11.9%) when compared with no treatment control (11.3%) while the treatment showed detrimental effects (P<0.05) on embryonic cleavage (40.0%) and blastocyst formation (1.8%) rates when compared with control (60.0% and 11.5%, respectively). For activation of ICSI oocytes, additional electric stimulus has no positive or negative effect on in vitro development of preimplantation stage ICSI porcine embryos. Our results demonstrate that CB treatment during ICSI inhibits embryonic development of ICSI oocytes and additional electric activation after ICSI has no effect in improving ICSI embryonic development in pigs. Further studies are needed to improve ICSI efficiency by investigating factors influencing embryonic development after ICSI in pigs.

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6- dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with 50 μM β-mercaptoethanol (β-ME), 100 μM L-ascorbic acid (Vit. C) or 100 μM L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The H2O2 levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups (26.9±1.6～29.1±1.3 pixels/embryo, p<0.05) compared to control (33.2±1.7 pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0～32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

These study was carried out to investigate the effects of the collection time, culture time and activation of canine oocytes on in vitro maturation rates. The activated oocytes were cultured in 10% FCS+TCM-199 media containing hormonal supplements (10 IU/ml HCG, 10 IU/ml PMSG, 10 ug/ml gonadotropin) at 5% , 95% air, . 1. IVM rate of in vitro cultured cumulus-attached oocytes recovered from ovaries that collected at follicular and luteal stages of the reproductive cycles were 11.4% and 5.7%, respectively. IVM rate of oocytes recovered from ovaries that collected at follicular stages of the reproductive cycles was significantly higher than that of luteal stage (p<0.05). 2. When IVM was carried out at different periods of 40, 48, and 70 hrs, the IVM rates of oocytes matured in vitro were 2.9%, 8.6%, 5.7%, respectively. These results indicate that the IVM time between hrs gives the highest maturation rate for the oocytes matured at the different stages. 3. IVM rate of oocytes matured in vitro for 10 hrs after single and combined activation treatment by ET, IP and CH and Ca+DMAP, CH+DMAP, ET+CH were respectively. This was higher than that in both single and combined stimulated groups compared to control group ().

Electrical treatment has been widely used for porcine oocytes activation. However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide (10μgml) for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to 6.25μM calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to 10μgml cycloheximide and electrical pulse treatment was significantly increased (P<0.05) than in the control group. And exposure to 6.25μM calcium ionophore for 2 min with 10μgml cycloheximide for 10min and electrical pulse treatment significantly increased (P<0.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) E+10 μM Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, 100 μM) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

본 연구의 목적은 1) 사회적 평가, 경쟁, 금전적 보상을 통한 스트레스 유발 처치가 시공간과제 수행에 미치는 영향과 2) 스트레스와 수행간의 관계를 매개하는 신경효율성의 변화를 조사하는 것이다. 본 연구의 피험자는 평균나이 23.4세의 대학생 15명으로 구성되었다. 피험자들은 스트레스 유발 처치조건과 무 처치조건에서 시각-모터 포인팅 과제를 수행하였고, 스트레스 유발 처치의 효과를 검증하기 위해 과제수행 시 각성측정의 지표인 심박수와 visual analogue scale(VAS)을 통해 경쟁심, 스트레스가 측정되었다. 또 스트레스 유발 처치가 수행력과 뇌 활성화에 미치는 효과를 검증하기 위해 과제수행 정확성과 뇌파(EEG)가 분석되었다. 먼저 스트레스 유발처치가 유의한 각성의 증가를 유발했는지를 검증하기 위해 스트레스 유발조건과 무 조건간 심박수, 경쟁심, 스트레스 척도에 대한 t-검증을 실시하였다. 또 과제수행시 뇌파의 차이를 비교하기 위해 조건(스트레스 유, 무), 뇌반구(좌, 우), 영역(전두, 중심, 두정, 측두, 후두엽)이 반복측정된 분산분석을 실시하였다. 이때 종속변인은 델타, 세타, 알파, 베타, 감마파의 파워값이었다. 분석결과, 스트레스 무 처치조건과 비교해서 유발조건에서 심박수, 경쟁심, 스트레스가 유의하게 증가하였다. 그러나 과제수행정확성은 무 처치조건보다 스트레스 조건에서 더 높은 것으로 나타났다. EEG 분석결과 스트레스 유발조건에서 시공간 정보처리를 담당하는 우뇌의 활성화가 스트레스 무 처치조건보다 높게 나타났고, 특히 이러한 조건별 차이는 과제 관련 뇌 영역인 측두엽에서 나타났다. 이 결과는 스트레스 유발 처치로 인한 과제 수행력 향상의 기전이 과제관련 뇌 영역의 활성화를 유도하는 신경효율성의 증가일 수 있다는 가능성을 제시한다.

Pueraria lobata (Willd.) Ohwi was investigated to check its modulatory effects on the activation of macrophages upon inflammatory conditions treatment. For this purpose, we examined several inflammatory responses such as nitric oxide (NO) production, reactive oxygen species (ROS) generation, cytoprotection and phagocytosis under the treatment of methanol extract from P. lobata (Pl-ME). Pl-ME dose-dependently blocked NO production in lipopolysaccharide (LPS)- stimulated RAW264.7 cells but not sodium prusside (SNP)-generated NO release. The NO inhibition seemed to be due to blocking inducible NO synthase (iNOS), since Pl-ME suppressed its expression in a NF-kB-independent manner. Similarly, this extract also effectively protected RAW264.7 cells from LPS-induced cytotoxicity. However, Pl-ME did not block ROS generation and rather it enhanced. Finally, this extract negatively modulated FITC-dextran uptake. Therefore, our data suggested that Pl-ME may be involved in negatively regulating some macrophage-mediated inflammatory responses such as NO production and phagocytic uptake.