Hairtails (Trichiurus lepturus) are the most popular marine products in Korea because of their taste and nutritional value, and Koreans consume them in large quantities. Hairtail, ecologically important warm water fish species, belonging to family Trichiuridae, widely distributed on the coast of the West Sea, South Sea and Jeju Island in the Korean Peninsula and the several sea areas in China under the natural ecosystem. However, in spite of their economic and scientific consequences, a little information currently exist regarding the physiological and ecological levels only of hairtail species in Korea (Koo et al., 2004). Simply the biological fisheries feature, distribution and migration of hairtail (T. lepturus) in Korean waters were surveyed (Park et al., 2005). Currently, imported hairtail have been altered into endemic hairtail because of high edge. In the present study, to explicate the genetic distances and differences among geographical hairtail populations, we accomplished a clustering analysis of three hairtail populations collected from Atlantic, Korea and Chinese site. Muscle tissues were obtained separately from individuals from Atlantic hairtail population (AHP), Gunsan hairtail population (GHP) and Chinese hairtail population (CHP), respectively. The muscle was collected in sterile tubes, immediately placed on dry ice, and stored at －40℃ until the genomic DNA extraction. Genomic DNA was extracted and purified under the conditions described previously (Yoon and Kim, 2004). Seven primers (BION-02, BION-03, BION-04, BION-08, BION-09, BION-13 and BION-17) were shown to generate the shared loci, specific loci, unique shared loci to each population and shared loci by the three populations which could be obviously scored. The degree of variability was calculated by use of the Dice coefficient (F), which is given by the formula: F = 2 nab / (na+nb), where nab is the number of bands shared between the samples a and b, na is the total number of bands for sample a and nb is the total number of bands for sample b (Jeffreys and Morton, 1987; Yoke-Kqueen and Radu, 2006). Euclidean genetic distances within- and between-population were also calculated by complete linkage method with the support of the hierarchical dendrogram program Systat version 13 (SPSS Inc., Chicago, IL, USA). Here, the seven decamer primers BION-02, BION-03, BION-04, BION-08, BION-09, BION-13 and BION-17 were used to generate the shared loci, specific, unique shared loci to each population and shared loci by the three populations. In the present study, averagely, a decamer primer generated 64.7 amplified products per primer in the AHP population, 55.7 in GHP population and 56.4 in CHP population. The number of unique shared loci to each population and number of shared loci by the four populations generated by genetic analysis using 7 decamer primers in AHP, GHP and CHP population. 119 unique shared loci to each population, with an average of 17 per primer, were observed in the AHP population, and 28 loci, with an average of 4 per primer, were observed in the CHP population. Many researchers studied the sizes of DNA fragments in the PCR profiles of five species of Eastern Pacific abalone (genus Haliotis) (Muchmore et al., 1998), black tiger shrimp (Penaeus monodon) (Tassanakajon et al., 1998), shrimp populations (Yoon and Kim, 2003) and deep sea lobster (Puerulus sewelli) (Park et al., 2005). The hierarchical dendrogram point out three main branches: cluster 1 (ATLANTIC 01 ~ ATLANTIC 07), cluster 2 (GUNSAN 08 ~ GUNSAN 14) and cluster 3 (CHINESE 15 ~ CHINESE 21). The shortest genetic distance displaying significant molecular difference was between individuals’ CHINESE no. 16 and CHINESE no. 18 (0.045). In the long run, individual no. 01 of the AHP population was most distantly related to CHINESE no. 19 (genetic distance = 0.430). The genetic distance between the Indian Ocean lobster and the Korean Slipper lobster species ranged between 0.040 and 0.612 (Park et al., 2005). Consequently, PCR analysis generated on the genetic data displayed that the geographic AHP population was widely separated from CHP population. From what has been said above, the potential of genetic analysis to identify diagnostic markers for the identification of three hairtail populations has been demonstrated. Generally speaking, using a variety of arbitrary primers, PCR has been applied to identify polymorphic/specific markers particular to line, species and geographical population, as well as genetic diversity/polymorphism in diverse species of organisms (McCormack et al. 2000; Yoon and Kim 2004).

We investigated the effect of light spectra on circadian rhythm by exogenous prolactin (PRL) by using light emitting diodes (LEDs): red, green, and purple. We injected PRL into live fish or treated cultured brain cells with PRL. We measured changes in the expressions of period 2 (Per2), cryptochrome 1 (Cry1), melatonin receptor 1 (MT1) mRNAs, and MT1 proteins, and in the plasma PRL, serotonin, and melatonin levels. After PRL injection and exposure to green LED light, MT1 expression and plasma melatonin levels were significantly lower, but the expressions of Per2 and Cry1 were significantly higher than others. Plasma serotonin after PRL injection and exposure to red LED light was significantly lower than others. These results indicate that injection of high concentration PRL inhibits melatonin, and inhibited melatonin regulates circadian rhythm via clock genes and serotonin. Thus, exogenous PRL regulates the circadian rhythm and light spectra influence the effect of PRL in goldfish.

Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.

Multipotent perivascular stem cells (PVCs) have gained much attention as an alternative source for cell based regenerative medicine in recent years. Due to their rarity in human tissues, developing methods to efficiently isolate and expand PVCs from various fetal and adult tissues is necessary to obtain a clinically relevant number of cells that maintain progenitor potency. Here, we report on a non-enzymatic isolation (NE) method of PVCs from human umbilical cords (HUCs) and compare its efficiency with the conventional collagenase treatment method (CT) in terms of proliferation and immunophenotypes. The cells isolated by NE displyed acceptable surface marker profile of PVCs and showed multilineage (osteogenic, chondrogenic, and adipogenic) differentiation potential. While both methods provided similar levels or patterns of proliferation and immunophenotypes, PVCs by NE retained a higher level of CD146(+) frequency compared to that of CT over passage. Furthermore, we have investigated potentials of various exogenous factors to promote proliferation of HUCPVCs in vitro. Among these factors, supplementation of basic fibroblast growth factor (bFGF) provided the optimal condition to significantly enhance the proliferation rate of HUCPVC and increased a proportion of stage-specific antigen-4 (SSEA-4) positive subset. Collectively, our study suggests that NE method with bFGF supplementation offers an alternative way to obtain sufficient numbers of HUCPVCs with higher number of primitive SSEA-4(+) subpopulation that are applicable in therapeutic doses for regenerative medicine.

Molecular markers are useful for selecting to include superior character genetic like as strong immune system and rapid growth in fish. The marker is also very important part of breeding technology in Olive flounder (Paralichthys olivaceus). Single nucleotide polymorphisms (SNPs) marker is already in use widely for genomic research and breeding. But this SNPs marker hardly has been validated for screening functional genes in Olive flounder. We study identify single nucleotide polymorphisms (SNPs) on Expressed sequence tag (EST) database, develop usable SNP marker and apply to wild sample and cultured of olive flounder. As a result, Out of total 4.327 ESTs, 693contigs and 514 SNP from total contigs were detected while these substitutions include 297 transitions and 217 transversions. 144 developed markers were applied in 16 samples (wild 8, culture 8), Out of total marker, only 32 markers had detected polymorphic in sample. Polymorphism of 32 markers was observed in the variety genes region involved in immunity and protein synthesis. And the 32 marker were identified 21 transitions, 11 transversions, and indel was not detected in polymorphic SNPs. The analysis on heterozygosity by sample showed 0.34 in wild sample and 0.29 in cultured sample. In conclusion, we was identified SNP and Polymorphism by designed new marker, it supports that development marker is suitable for SNP detection and diversity analysis in Olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.

Reactive oxygen species (ROS) are produced in organisms as the natural products of oxidative metabolism by environmental stress and pathogen invasion. ROS, such as superoxide anion and hydrogen peroxide, can be toxic to cells and tissues to cause oxidative stress. Recent study revealed that olive flounder (Paralichthys olivaceus) superoxide dismutase (SOD) has been identified as a partial gene and strongly induced to benzoin[a]pyrene and it was deduced indicator of aquatic oxidative stress responses, but its transcriptional response against viral infection has not been investigated. In the present study, spatial and temporal expression profile was analyzed to investigate the function of Of-SOD in the anti-viral response. Of-SOD transcripts were ubiquitously detected in diverse tissues with variable levels using a real-time PCR. The expression of Of-SOD was significantly higher in the muscle, liver and brain, but extremely low in the stomach and spleen. Following VHSV challenge, the expression of Of-SOD increased within 3 hours and subsequently decreased to the original level at 2 days post-challenge in kidney. Although expression pattern and induction time are slight differences depending on the tissue, the transcript of Of-SOD was consistently increased in acute infection response, but expression is low in the chronic response. Collectively, Of-SOD expressions were inducible after VHSV infection and they were probably involved in the immune response against viral challenge. These results suggest that SODs may play important roles in the immune defense system of P. olivaceus and perhaps contribute to the protective effects against oxidative stress in this flounder.

Chemoresistance is one of the main problems to treat different kinds of cancers or cancer cells. Therefore, it is necessary to find out the strategies to make the cancer cells sensitive to chemotherapy along with optimal dosage of drugs. We examined sensitivity of MCF7 cells through pretreating with an epigenetic modulator, azacytidine (AzaC) to doxorubicin (Dox). The cells were treated with 5 and 10 mM of AzaC for a week, subsequently with 50, 100 and 500 nM of doxorubicin for 24 and 48h. It was found that pretreatment of AzaC significantly enhance the sensitivity of MCF7 cells to Dox, inducing cell death. After 24h 15% cells underwent apoptosis in 500 nM dox treatment group while 23.4% cells death occurred in AzaC pre treatment group. After 48h MCF7 cells treated with Dox showed 19.0% cell death while AzaC sensitized cells showed 50.0% cells death when exposed to 500 nM of Dox for 48h. Western blot analysis showed the upregulations in the expression of bax, caspase-3, caspase-9 and p53 in AzaC-sensitized MCF7 cells treated with Dox as compared to those treated with only Dox. There was no clear indication for pro-apoptosis genes in the cells treated with individual drugs. These results showed that pretreatment with the epigenetic modulator significantly increased the sensitivity of MCF7 cells to Dox. Therefore it is concluded that demethylation event might enhance the activity of DNA intercalating agents to induce DNA damage in breast cancer cells.

Development of the central nervous system (CNS) occurs normally in mammalian fetus despite lower temperature in the brain region than in the heart. To investigate the effects of temperature niche on the neural differentiation of stem cells in vitro, P19 embryonic carcinoma (EC) stem cells and N2a neuroblastoma stem cells were induced to undergo neural differentiation by retinoic acid and LiCl, respectively. The cells were analyzed for the expression of neural marker genes during 12 days differentiation. Although there were Map2 and NCAM expressions in both groups, no clear difference was found. Similarly, expression patterns of Tuj1 and NF-M were not different in both groups, showing more intensive staining patterns at day 12 than those at days 4 and 8, respectively. However, more cells expressed GFAP markedly at day 12 in 37℃ group. There was little expression of the above markers in N2a cells during differentiation except for Ngn2 and Tuj1. It was found that Ngn2 was expressed more intensely at days 6 and 9 in 33℃ group. Tuj1 expression showed a similar pattern to those of P19 EC cells. RT-PCR analysis also showed that the expressed transcripts did not quite different in both groups, although they were different among the days of differentiation. Thus, it appears that neural differentiation occurs normally with a slight delay and probably less cell death in the cells at 33℃ than that at 37℃.

Skeletal deformities are important factor of evaluation of fish value commercially. Deformities of opercular are commonly observed type of fish deformation. Although these malformations in fish can be caused by culture conditions, the environmental factors are unknown. This study examined the effect of water temperature on the opercular deformity of the red spotted grouper, Epinephelus akaara. Experimental fish (TL; 7.49±0.10 cm) were respectively divided into 3 groups that were reared at 20, 24, 28℃ for 6 weeks. All specimens were photographed from the left lateral view using a Canon EOS 70D. We placed 11 landmark points for visualization the shape differences of operculum in the whole body. In order to measure the reduction of opercular, we estimated total length (TL) and shortening of the distal part (distance between landmarks 10 and 11). After 6 weeks, both growth rate and incidence of opercular shortening were high in 24 and 28℃. At 28℃, the distance of distal part of operculum was the highest as 0.36 cm and exposure to the 24℃ induced the highest growth rate during this experiment. On the other hand, both growth rate and opercular deformity were low at the lower temperature (20℃). This study shows opercular malformation as well as the growth rate of E. akaara are influenced by the high water temperature.

일주기 리듬(Circadian rhythm)은 대부분의 생물에서 나타나며, 생화학적, 생리학적 또는 행동학적인 패턴이 거의 24시간의 주기성을 가진다. 포유류에서도 호르몬 분비의 일주기적 패턴이 보고된 바 있는데, 이러한 내분비의 일주기 리듬은 생식기능의 조절에 중요한 역할을 한다. 본 연구에서는 섭식조절에 따른 일주기 패턴의 교란이 생체 리듬의 에너지 균형을 교란하고 생식기능의 변화를 유도할 것이라는 가설을 세웠다. 생후 6주의 준 성체 수컷 흰쥐를 사용하였으며, 섭식을 조절하기 위해 대조군(CTL)은 19:00시부터 익일 08:00시까지 먹이를 제공하였고, 실험군(RF 군)은 08:00시 부터 19:00시까지 먹이를 제공하였다. 4주간 체중과 섭식량을 조사하고, 4주 후 희생하여 조직의 무게를 측정하였다. 실험 결과 두 군에서 섭식량과 체중, 정소와 부정소의 무게는 유의한 차이가 없었다. 그러나 저정낭(control : RF group = 0.233 ± 0.014 g : 0.188±0.009 g, p<0.01)과 전립선(control : RF group = 0.358 ± 0.015 g : 0.259 ± 0.015 g, p<0.001)의 무게는 RF 군에서 유의하게 감소하였다. 뇌하수체에서 PCR을 통해 mRNA의 발현을 조사 한 결과, Cgα(control : RF group = 1.0 ± 0.0699 AU : 0.1923 ± 0.0270 AU, p<0.001)와 FSHβ (control : RF group = 1.0 ± 0.1489 AU : 0.5237 ± 0.1088 AU, p<0.05)는 RF 군에서 유의하게 감소하였다. 그러나 ACTH에서 유의한 차이가 나타나지 않았다. 조직학적 조사 결과 부정소의 미세소관에서는 차이가 나타나지 않았으나, 저정낭의 경우 RF 군에서 다소 형태적 차이가 나타났다. 본 연구는 섭식의 제한 및 역전이 뇌하수체의 성선자극 호르몬의 유전자 발현에 영향을 미치며, 저정낭과 전립선의 발달을 저해함을 확인하였다. 이와 같은 결과는 뇌하수체에서의 성선자극 호르몬 신호의 감소가 정소의 안드로젠 분비를 감소시켜 저정낭과 전립선과 같은 안드로젠 의존적인 조직의 기능 저하를 초래한 것으로 추정된다.

The transcription factor, early growth response protein 1 (EGR1), act as immediate early response genes to control various cellular and reproductive events. Egr1-deficient female mice show infertility by anovulation resulting from luteinizing hormone-β (LH-β) subunit deficiency. While ovulation, fertilization and embryo development normally occur in Egr1-deficient mice treated with a superovulation regime to rescue LH deficiency, embryo implantation was completely failed. The morphology and ultrastructure of uterine tissues were observed by light and transmission electron microscopy during the peri-implantation period in Egr1-deficient mice. To examine alterations in cellular organelles, the uterine horns were fixed with 2.5% glutaraldehyde and postfixed with 1% osmium tetroxide in PBS. After dehydration and infiltration, the samples were embedded in Epon 812. Semi-thin sections 0.5 μm thick were cut with an ultramicrotome and stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife of the ultramicrotome and placed on copper grids. The sections were double stained and examined under a transmission electron microscope. The height of luminal epithelial cells was decreased and the polarity was poorly differentiated in the Egr1-deficient comparing to the wild mice. The abundant mucinous materials were observed in the surface of luminal epithelial cells of the Egr1-deficient. It was confirmed the microarray and real time qPCR data. The luminal epithelial cells of wild mice had many dense lipophilic granules and healthy mitochondria, but not in the Egr1-deficient. It may related to production and secretion of steroid hormones and prostaglandins in the luminal epithelial cells for successful implantation. These results show that Egr1 is a critical transcription factor to fine-tune subcellular morphological and functional changes for the receptive phase of peri-implantation period of uterine tissue in mice.

Estrogen is an important regulator of reproduction in both male and female. The two forms of estrogen receptor (ER) are known, ERα and ERβ. To understand the role of ERα in the testis, we investigated the expression of ERα in the mouse Leydig cells during postnatal development and the effects of estrogen on steroidogenesis and proliferation in progenitor Leydig cells (PLCs). In the testis, ERα mRNA and protein levels were markedly increased from postnatal day (PND) 1 to 14 and decreased thereafter until PND 56. During postnatal development ERα immunoreactivity was strong in the nucleus of Leydig cells at PND 14 when PLCs were abundant in the interstitium and low in the mature adult Leydig cells (ALCs). In fetal Leydig cells (FLCs), ERα immunoreactivity was negligible at birth and became increased at PND 14. This suggests an important role of ERα in Leydig cells during neonatal period. In isolated PLCs, 17β-estradiol (E2) and ERα-selective agonist, PPT suppressed the hCG-induced progesterone production and steroidogenic pathway genes expression. The hCG-induced PLCs proliferation was significantly inhibited by E2 and PPT. In conclusion, estrogen - ERα signaling may negatively regulate functional differentiation and proliferation of PLCs.

Guppy has become a model organism for studying behavioral traits such as courtship and mate choice, as well as for understanding ecogeographic adaptation. Unfortunately, studying the early development of live bearers is more complicated than that of oviparous species, due to the inaccessibility of developing embryos for experimental manipulation. Ulrike et al. showed that the embryos could not be cultured for the entire period of their embryonic development. To optimize conditions embryo in vitro culture we established system for varying the concentration of fetal bovine serum in the medium impact on the embryonic development of in vitro embryos. For in vitro culture, embryos were incubated in 8 ml of sterile embryo medium (L-15 [Leibovitz] medium, supplemented with 5, 10, 15, and 20% fetal bovine serum respectively, 20 units/ml penicillin, and 200 mg/ml streptomycin) in a dark incubator at 25℃. Our study found that in 5% of FBS of the medium, embryos can be maintained until the middle-eyed. In 10% and 15% embryos can be maintained constant development; some of them can be fed; however, in 15% it is faster than 10%. And although in 20% of FBS can sustain rapid development of early stage, but ultimately died. According to our experimental data, both 10% and 15% FBS in medium can be used for in vitro culture, the slowly development in 10% FBS appears to be more conducive to observation.