Morphometric changes in the Ussurian bullhead, Leiocassis ussuriensis, and the Korean bullhead, Pseudobagrus fulvidraco, were observed during the early period of growth. Yolk length, yolk height, and yolk volume in the two species decreased within 9 days post-hatching (DPH) (p<0.05). The body lengths and body heights of both species increased gradually to 150 and 130 DPH, respectively (p<0.05). The horizontal distance between the anteriormost extension of the head and the anterior insertion of the pectoral fin, the anteriormost extension of the head × the verticality position of the anterior insertion of the primary dorsal fin rays, and the anterior insertion of the primary dorsal fin × the anterior insertion of the pectoral fin were greater in the Korean bullhead than in the Ussurian bullhead (p<0.05). However, the relative sizes of the head region, pectoral fin, ventral fin, and anal fin were greater in the Ussurian bullhead than in the Korean bullhead (p<0.05), and relative body depth and the size of the outer-mandible barbel were greater in the Korean bullhead than in the Ussurian bullhead (p<0.05). The growth curves of the morphometric characteristics of both species were divided into three types.

To effects of sex maturation in olive flounder by regulating long photoperiod, gonadal development and GTH mRNA expression in the pituitary were investigated. Photoperiod was treated natural photoperiod and long photoperiod (15L:9D) conditions from September 2011 to March 2012. The results showed that natural photoperiodic group showed a higher gonadosomatic index (GSI) than long photoperiodic group during the spawning season (March 2012). The histological analysis of ovarian tissue showed that natural photoperiod group of ovaries contained vitellogenic oocytes, but long photoperiod group of ovaries mainly contained perinucleolus staged oocyte and oil-drop staged oocytes. The FSH mRNA of olive flounder, under natural photoperiod group, showed a significantly higher expression but no significant difference under long photoperiod group. The LHβ mRNA showed a significantly higher expression only under natural photoperiod group. These results may suggest that long photoperiodic information regulates secretion of pituitary FSH and LH and maintain early growing stage of gonadal development in this species.

Olive flounder (Paralichthys olivaceus) is one of the commercial important flatfish species in Korea. The ocular signal transduction pathway is important in newly hatched flounders because it is closely involved in the initial feeding phase thus essential for survival during the juvenile period. However, the study of gene expression during ocular development is incomplete in olive flounder. Therefore we examined the expression analysis of specifically induced genes during the development of the visual system in newly hatched flounders. We searched ocular development-involved gene in the database of expressed sequence tags (ESTs) from olive flounder eye and this gene similar to arrestin with a partial sequence homology. Microscopic observation of retinal formation corresponded with the time of expression of the arrestin gene in the developmental stage. These results suggest that arrestin plays a vital role in the visual signal transduction pathway of the retina during ocular development. The expression of arrestin was strong in the ocular system during the entirety of the development stages. Our findings regarding arrestin have important implications with respect to its biological role and evolution of G-protein coupled receptor (GPCR) signaling in olive flounder. Further studies are required on the GPCR-mediated signaling pathway and to decipher the functional role of arrestin.

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87－98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

Ionosphere is one of the largest error sources when the navigational signals produced by Global Positioning System (GPS) satellites are transmitted. Therefore it is very important to estimate total electron contents (TEC) in ionosphere precisely for navigation, precise positioning and some other applications. When we provide ionospheric TEC values in real-time, its application can be expanded to other areas. In this study we have used data obtained from nine Global Navigation Satellite System (GNSS) reference stations which have been operated by Korea Astronomy and Space Science Institute (KASI) to detect ionospheric TEC over South Korea in real-time. We performed data processing that covers converting 1Hz raw data delivered from GNSS reference stations to Receiver INdependent Exchange (RINEX) format files at intervals of 5 minutes. We also analyzed the elevation angles of GPS satellites, vertical TEC (VTEC) values and their changes.

Photometric observation is one of the most effective techniques for determining the physical characteristics of unknown space objects and space debris. In this research, we examine the change in brightness of the Communication, Ocean, Meteorological Satellite-1 (COMS-1) Geostationary Orbit Satellite (GEO), and compare it to our estimate model. First, we calculate the maximum brightness time using our calculation method and then derive the light curve shape using our rendering model. The maximum brightness is then calculated using the induced equation from Pogson's formula. For a comparison with our estimation, we carried out photometric observation using an optical telescope. The variation in brightness and the shape of the light curve are similar to the calculations achieved using our model, but the maximum brightness shows a slightly different value from our calculation result depending on the input parameters. This paper examines the photometric phenomenon of the variation in brightness of a GEO satellite, and the implementation of our approach to understanding the characteristics of space objects.

Recently, two instruments of cosmic ray are operating in South Korea. One is Seoul muon detector after October 1999 and the other is Daejeon neutron monitor (Kang et al. 2012) after October 2011. The former consists of four small plastic scintillators and the latter is the standard 18 NM 64 type. In this report, we introduce the characteristics of both instruments. We also analyze the flux variations of cosmic ray such as diurnal variation and Forbush decrease. As the result, the muon flux shows the typical seasonal and diurnal variations. The neutron flux also shows the diurnal variation. The phase which shows the maximum flux in the diurnal variation is around 13-14 local time. We found a Forbush decrease on 7 March 2012 by both instruments. It is also identified by Nagoya multi-direction muon telescope and Oulu neutron monitor. The observation of cosmic ray at Jangbogo station as well as in Korean peninsula can support the important information on space weather in local area. It can also enhance the status of Korea in the international community of cosmic ray experiments.

Cleft palates with or without cleft lip is one of the most common congenital craniofacial defects in dogs. It has been reported that monogenic autosomal recessive inheritance caused this defect in this species. However, here, we aimed to report cleft palate in a cloned dog. A fibroblast cell line was established from skin tissues of an eight-year-old German shepherd dog. Blood was collected from oocyte donor dogs, and serum progesterone concentration was measured by chemiluminescence enzyme immunoassay method. Ovulation was determined when serum progesterone results reached 5-10 ng/ml, and in vivo matured oocytes were collected surgically about 72 hr after ovulation. Donor cells were cultured with Dulbecco’s modified Eagle medium supplemented with 10% (v/v) fetal bovine serum until confluence. An in vivo matured oocyte was enucleated, and a donor cell was injected into the perivitelline space. The oocyte-cell couplet was electrically fused, and chemically activated. Reconstructed embryos were transferred to an oviduct of a recipient. Pregnancy diagnosis was performed 27 days after the embryo transfer, and ultrasonography of fetal heart beat, and rectal temperature and serum progesterone value of recipient was monitored until the day of delivery. Microsatellite analysis was performed using genomic DNA of cell donor, clones, and oocyte donors. As results, a total of 74 cloned embryos were transferred to five recipients, and one recipient diagnosed as pregnant with two fetuses by ultrasonography and radiology. Caesarean section was performed on day 58 after embryo transfer due to a decreased heart beat of a fetus, which was lower than 180. Two cloned puppies with 640g and 320g of birth weight were delivered safety, but the small one was born with a cleft palate. Microsatellite analysis results of both clones were identical with the cell donor. Cleft palate of the clone was surgically corrected on day 40 after birth. To our knowledge, there has been no report about cleft palate in cloned dogs, and also, no report about clones with different phenotype of cleft palate in dogs. Therefore, this study can give a clue of cleft palate in dogs, which might not be a genetic cause. Further studies about aberrant epigenetic reprogramming in those clones are needed.

The implantation process in pigs is initiated when the conceptus begins secretion of estrogen, the signal for maternal recognition of pregnancy, and cytokines including interleukin-1β(IL1B), interferon delta (IFND) and interferon gamma (IFNG). Our previous study showed that IFNG receptors, IFNGR1 and IFNGR2, were expressed in the uterine endometrium during the estrous cycle and early pregnancy. However, the molecular and cellular mechanism of IFNG in the uterine endometrium in pigs is poorly understood. To determine the role of IFNG on the uterine endometrium during the implantation period, we took advantage of RNA-Seq analysis using explant tissues treated with IFNG in the presence of estrogen and progesterone, and found that many genes including CXCL9, CXCL10, CXCL11, IDO1, IL15, IL15RA, TNFSF10 (TRAIL), and WARS were up-regulated by IFNG. Additional analysis in the uterine endometrial tissues from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy determined the expression of these IFNG-regulated genes in pigs by quantitative real-time PCR Results showed that expression of CXCL9, CXCL10, and IDO1 dramatically increased on D15 of pregnancy, and expression of CXCL11 and TNFSF10 was high during mid- to term pregnancy. These results indicate that IFNG regulates immune-associated genes in the uterine endometrium in a stage-specific fashion during pregnancy, and may play a critical role to support the establishment and maintenance of pregnancy at the fetomaternal interface in pigs.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids, and ions. In addition, Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca+ exchanger expressed at the maternal-fetal interface in pigs. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are required for primary functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. However, it has not been determined that subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we determined expression of alpha (ATP1A1-4), beta (ATP1B1-3), and FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that all alpha, beta, and FXYD subunits, except ATP1A3, were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that ATP1A1, ATP1A4, and ATP1B1 were localized to luminal (LE) and glandular epithelium (GE) during the estrous cycle and early pregnancy, and during mid to term pregnancy. ATP1A1 mRNA was localized to LE, GE, and areolae of the chorioallantois, especially at high levels to LE in areolae regions. ATP1B3 mRNA was detected only in LE during the estrous cycle and pregnancy with highest levels on day (D) 12 of pregnancy. Transcripts of all subtypes of FXYD subunit were primarily localized to LE and GE during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that all subtypes of Na+/K+-ATPase subunits, except ATP1A2, ATP1A3, and ATP1B2 mRNAs, were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that Na+/K+-ATPase subunits are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase subunits may be involved in the establishment and maintenance of pregnancy by coordinate regulation of absorption and secretion of nutrients such as glucose, amino acids, and ions at the maternal-fetal interface in pigs.

Three species of Nortamea concinua (NC) and Haliotis discus hannai (HDH) from Tongyeong and Sulculus diversicolor supertexta (SDS) are widely distributed on the coast of the Yellow Sea, southern sea and Jeju Island in the Korean Peninsula under the innate ecosystem. There is a need to understand the genetic traits and composition of three mollusk species in order to evaluate exactly the patent genetic effect. PCR analysis was performed on DNA samples extracted from a total of 21 individuals using seven decamer oligonucleotides primers. Amplification products were separated by electrophoresis in 1.4% agarose gels (Bioneer Corp., Daejeon, Korea) with TBE (0.09 M Tris, pH 8.5; 0.09 M borate; 2.5 mM EDTA), using 100 bp DNA ladder (Bioneer Corp., Daejeon, Korea) as DNA molecular weight marker and detected by staining with ethidium bromide. Seven primers, BION-55 (5’-GTCACGGACG-3’), BION-50 (5’-CAAGCGA GGA-3’), BION-75 (5’-GAGGTCCACA-3’), BION-35 (5’-AGCGGCTAGG-3’), BION-61 (5’-GAC CGCTTGT-3’), BION-69 (5’-GCATCCACCA-3’) and BION-66 (5’-TGGTGGACCA-3’) were shown to generate the unique shared loci to each species and shared loci by the three species which could be clearly scored. A hierarchical clustering tree was constructed using similarity matrices to generate a dendrogram, which was facilitated by the Systat version 10 (SPSS Inc., Chicago, IL, USA). 236 specific loci, with an average of 56.3 per primer, were identified in the NC species. 142 specific loci, with an average of 44.7 per primer, were identified in the HDH species. Especially, 126 numbers of shared loci by the three species, with an average of 18 per primer, were observed among the three species. Especially, the decamer primer BION-75 generated 7 unique loci to each species, which were identifying each species, in 700 bp NC species. Interestingly, the primer BION-50 detected 42 shared loci by the three species, major and/or minor fragments of sizes 100 bp and 150 bp, respectively, which were identical in all samples. As regards average bandsharing value (BS) results, individuals from HDH species (0.772) exhibited higher bandsharing values than did individuals from NC species (0.655). In this study, the dendrogram obtained by the seven decamer primers indicates three genetic clusters: cluster 1 (CONCINNA 01~ CONCINNA 07), cluster 2 (HANNAI 08~HANNAI 14), cluster 3 (SUPERTEXTA 15~SUPERTEXTA 21). Comparatively, individuals of HDH species were fairly closely related to that of SDS species, as shown in the hierarchical dendrogram of genetic distances.

Olive flounder (Paralichthys olivaceus) is a most important aquaculture species in Korea. Like most marine fishes, olive flounders are stomachless at first feeding and aquired gastric function during the metamorphosis, so food was mainly digested by pancreatic enzyme from first feeding to premetamorphosis. However, comprehensive analysis of pancreatic and gastric digestive enzyme of olive flounder at early developmental period is still unclear. In the expression study of pancreatic and gastric digestive enzyme by real-time PCR at early developmental stage, pancreatic enzyme such as chymotrypsinogen 2, preproelastase 2 and 4, pancreatic protein somatomedin-B domain (PPSB) mRNA expression were initiated at first feeding and strongly expressed in the pancreas developmental stage, while gastric digestive enzyme signal was not at all detected during same period. Although, trypsinogens were secreted from pancreas and have similar amino acid sequence, trypsinogen 3 expression induction was detected both pancreas and stomach developmental stage, while trypsinogen 2 expression was significantly increased only post-metamorphosis period. Pepsinogen mRNA expression was only detected at metamorphosis according to stomach differentiation. Lipid digestive enzyme, lipase and intestine fatty acid binding protein 1 (I-FABP 1), were already reached a certain level at beginning of hatching and more increased during early developmental stage and then gradually decreased before metamorphosis. These results suggested that feed ingestion of olive flounder was exclusive charged by pancreatic digestive enyme, lipid digestive enzyme and trypsinogen 3 from first feeding and then fully swiched by gastric digestive enzyme and trypsinogen 2 from metamorphosis period.

For the study of population genetic structure with mtDNA, it is essential to measure genetic diversity at each mtDNA regions. Also, to evaluate the variation according to the each region should follow as well as to see if there are differences. In this study, we delved into the variations and dendrogram among samples of seven mtDNA regions (NDⅡ, NDⅤ, NDⅣ, NDⅣL, NDⅥ, NDⅠ, 12SrRNA) from wild Pacific abalone, Haliotis discus hannai collected in Yeosu, Korea. The region with the highest genetic variation was NDⅣ region (Haplotype diversity = 1.0000, Nucleotide diversity = 0.010823) with two to five times higher variation than the others. Furthermore, the study to see if there is a difference between the regions of samples showed that similar aspects of dendrogram in NDⅡ and NDⅠ(divergence of 90% and 87%), which forms a group with hd4, 7, 8 and 10 at bootstrap support, based on 1000 replications. Also, pair-wise FST between clusters within the regions showed high values; 0.4061 (P=0.0000), 0.4805 (P=0.0000) respectively. Therefore we can infer that it is the most efficient and accurate way to analyze the region of NDⅣ with the highest variation in addition to the regions of NDⅡ and NDⅠ, which formed clusters with high bootstrap value, for study of population genetic structure in this species.

After vasectomy (VAX), the flux and composition of the epididymal fluid are modified, leading to sequelae to the epididymis. In an effort to understand molecular pathophysiology of the epididymis following VAX, we investigated the changes of gene expression and sperm functions in the epididymis of vasectomized mice. After VAX, the epithelial cell height was significantly decreased in cauda epididymis, resembling the those of vas deference. This suggests that VAX evokes alteration of segmental characteristics of epididymal epithelium. Of note, these was an increase in luminal diameter in corpus and cauda epididymis, indicating the alteration of fluid homeostasis in epididymal lumen and protein synthesis and secretion. Also, the formation of sperm granuloma and infiltration of inflammatory cells were noted in lumen of epididymis at 8 weeks postvasectomy, indicating the activation of immune response in epididymis. The serum TNF-α levels and epididymal TNF-α and IL-1β mRNA levels were significantly increased in VAX mice. Microarray analysis demonstrated that claudin (CLDN) 10 and cystic fibrosis transmembrane conductance regulator (CFTR) were downregulated after vasectomy. In contrast, angiotensin II receptor type 2 (Agtr2) was up-regulated after vasectomy. Taking into account the functional importance of angiotensin system in epididymal epithelia and muscle tissue, VAX may alter the secretory function of epithelia and sperm transport via alteration in angiotensin system. Importantly, the IgG type of anti-sperm antibody (ASA) were markedly increased in the blood of vasectomized mice. Together this indicates that VAX provokes local inflammation in epididymis as well as systemic inflammation, which in turn change the blood epididymal barrier, an important element for epididymal immunological privilege. In addition, the number of sperm in cauda epididymis was increased but the sperm motility was decreased after vasectomy. The spontaneous acrosome reaction was increased by vasectomy after capacitation. This suggests that VAX affects sperm functions as well as sperm maturation. In conclusion, bilateral vasectomy lead to local immune response of epididymis, causing immunologic infertility in men.

The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulates cell growth, differentiation, and apoptosis. While it is clear that loss of Egr1 leads to anovulatory infertility due to LHβ deficiency in female mice, molecular function of Egr1 in male reproduction has not been clearly investigated. Here, we demonstrate that Egr1 acts as an intrinsic transcription factor in Leydig cells to regulate their proliferation and steroidogenesis in the testis as well as an extrinsic factor for male reproduction via LHβ transcription in the pituitary. Egr1 is predominantly expressed in spermatogonia and Leydig cells in immature testes and later detected in some of these cell types in mature testes. The fertility potential of Egr1(-/-) male mice is relatively deteriorated even at 2 month-old age and aggravated with aging. The incidence of abnormalities of seminiferous tubules such as Sertoli cell only was dramatically increased with aging. The number and mean size of Leydig cells were significantly reduced in Egr1(-/-) testes. The impairment of Leydig cells is consistent with significant reduction in levels of testosterone and expression of factors critical for steroidogenesis such as StAR in Egr1(-/-) testes. Exogenous administration of hCG rapidly and transiently induced Egr1 expression in Leydig cells culture in vitro. hCG could reinstate reduced mean size of Leydig cells but not reduced number of Leydig cells and aberrantly low StAR expression, suggesting that Egr1 has critical functions for Leydig cell proliferation and their steroidgenesis. In addition, daily sperm production and in vitro fertilization (IVF) competence were significantly reduced, and apoptosis was facilitated in these mice. Furthermore, hCG administration to compensate for relatively low LH levels in Egr1(-/-) males could not restore the compromised reproductive phenotypes such as IVF competence and apoptosis in these mice. Interestingly, expression of Egr2, a member of Egr family, is significantly elevated in Egr1(-/-) Leydig cells suggesting that genetic compensation of Egr2 may alleviate phenotypic aberration of Egr1(-/-) male testes. Collectively, these results suggest that Egr1 act as an intrinsic transcription factor required for proliferation and steroidogenesis of Leydig cells to govern spermatogenesis in the testis.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfo-conjugation of estrogens at the 3-hydroxyl position. Sulfated estrogens lose their ability to interact with the estrogen receptor (ER). Previous studies have reported that testicular expression of EST is under the regulation of LH and androgen. In an effort to understand the biological significance of estrogens in the testis, we analyzed the EST gene expression in the developing mouse testis and Leydig cells and its regulation by estrogen receptor alpha (ERα). Male mice at postnatal day (PND) 1, 7, 14, 28, and 56 and ERα flox/flox Cyp17iCre male mice which show deletion of ERα specifically in Leydig cells were used for this study. Testes and Leydig cells isolated from these mice were subjected to quantitative RT-PCR analysis and immunohistochemistry. In addition, 17β-estradiol (E2), ERα-selective agonist PPT, ERβ -selective agonist DPN, and ER antagonist ICI 182,780 were treated in primary adult Leydig cell culture. These cultured cells were subjected to quantitative RT-PCR analysis. In testis, EST mRNA level was excessively low by PND 14 and markedly increased from puberty (PND 28) onward. In the interstitium, EST mRNA was not detected by PND 14 but considerably expressed from PND 28 onward. EST immunoreactivity was moderate in the interstitium by PND 14. Strong EST immunoreactivity was found in the interstitium from PND 28 onward. In ERαflox/flox Cyp17iCre mouse testis and Leydig cells, EST mRNA level was significantly lower than wild type (ERαflox/flox). In primary adult Leydig cell culture, the expression of EST mRNA was increased by E2 and PPT, but was not changed by DPN. The expression of EST in the testis is developmentally regulated. In adult Leydig cells, EST could play an important role in the steroidogenesis by modulating the activity of estrogens. Estrogen as well as LH and androgen may play a role in the regulation of EST expression in Leydig cells via ERα signaling.

In oocytes from different species, MPF, a complex of Cdk1 and cyclin B, is the master regulator of cell cycle. The activity of MPF is regulated by the phosphorylations mediated by Wee1B kinase and Cdc25B phosphatase. Although a regulation of MPF activity by these inhibitory phosphorylations are well established, a dogma in the cell cycle is that MPF activity is regulated by the dynamics of cyclin B during the metaphase II (MII) arrest (also known as CSF arrest). However, growing evidences suggest that Wee1B-mediated Cdk1 phosphorylation is also critical to trigger the progression of cell cycle during the onset of anaphase. Therefore, in the present study, we investigated the role of Cdc25B phosphatase during MII arrest. Cdc25B is present in MII arrested oocytes as a hyperphosphorylated form and disruption of its function either by antibody or siRNA injection induces the progression of cell cycle to interphase. Moreover, the hyperphosphorylated form, which has been known as an active form of Cdc25B, is dephosphorylated during the anaphase onset. Interestingly, this dephosphrylation occurred ahead of cyclin B degradation. Conversely, overexpression of Cdc25B prevents metaphase to anaphase transition induced by calcium stimulation. Therefore, our findings provide novel paradigm in cell cycle that MPF activity during metaphase arrest is regulated by the balance between Cdk1 inhibitory kinases, Wee1, and the counteracting phosphatases, Cdc25. When cells exit from metaphase, Cdc25 is inactivated and Wee1 is reactivated and thereby Cdk1 kinase activity is rapidly and transiently decreased. This initial decrease of Cdk1 activity is further promoted by the proteolytic degradation of cyclin B, which ensures irreversible progression of cell cycle to interphase. Thus, the concerted effort of phosphorylation/dephosphorylation of Cdk1 and synthesis/degradation of cyclin B play roles in fine-tuning the activity of Cdk1 during metaphase to anaphase transition.

Mesenchymal stem cells (MSCs) are considered to be attractive approaching in gene or drug delivery for cancer therapeutic strategies. In this study, the ability and feasibility of human bone marrow derived MSCs expressing the cytosine deaminase (CD)/5-Fluorocytosin (5-FC) prodrug was evaluated to target human osteosarcoma cell line Cal-72. At first, the fibroblast-like cells were successfully obtained from human bone marrow and demonstrated that they contained full of stem characteristics by the ability of differentiation into adipocyte/osteocyte and expression of typical mesenchymal markers CD90, CD44, while negative for CD34 and CD133 markers. We established the stable CD-expressing MSCs cell line (CD-MSCs) by transfection of pEGFP-C3 containing cytosine deaminase::uracil phos-phoribosyltransferase (CD::UPRT) gene into MSCs, and confirmed that the manipulated MSCs still remained full characteristics of multipotent cells and shown migration toward human osteosarcoma cancer cells Cal-72 as high as origin MSCs. Based on bystander effect, the therapeutic CD-MSCs significantly augmented the cytotoxicity on cancer cell Cal72 in either direct co-culture or conditioned medium in the presence of 5-FC. Moreover, in osteosarcoma cancer- bearing mice, the therapeutic CD/5-FC MSCs showed the inhibition of tumor growth compared with control mice which was s.c injected with only Cal72. Our findings suggest that these therapeutic CD-MSCs may be suitable and viable cellular vehicles for targeting human osteosarcoma cancer.

DGCR8 is a RNA-binding protein working with DROSHA involved in critical processes for microRNA production in the nucleus. To understand function of miRNAs in the uterus, we have produced uterus-specific Dgcr8 conditional knock-out mice using two well-known Cre mouse models, anti-Mullerian hormone receptor 2 (Amhr2)-Cre and progesterone receptor (PR)-Cre. Dgcr8flox/flox;PRcre/+ mice were mainly analyzed and considered as uDgcr8 KO in this study unless otherwise indicated as Dgcr8flox/flox;Amhr2cre/+ mice. Morphological and histological analyses, embryo cultures, genomic DNA PCR, realtime RT-PCR and Western blotting were performed. uDgcr8 KO females bred with fertile males did not produce any offspring, suggesting that these mice are infertile. Vaginal smear analyses showed that these mice do not undergo estrous cycle, whereas Dgcr8flox/flox;Amhr2cre/+ mice exhibited regular estrous cyclicity. In vitro culture of 2-cell stage embryos and histological analyses for CL in uDgcr8 KO demonstrated that they can respond to gonadotrophins to ovulate healthy oocytes with comparable fertilization potentials as compared to those in Dgcr8flox/flox mice (Control). Gross morphology, histology, and weight of uteri of uDgcr8 KO mice were similar to those of control at 3-week-old stage. However, uterus become extremely thinner and shorter from 4-week-old stage onward. Histological examination showed significant reduction in gland numbers and stromal area from 4-week-old stage. Interestingly, this phenotype is reflected by significant increase of PR expression in the uteri of 4-week-old mice. In addition, stromal cell proliferation of uDgcr8 KO is severely impaired. BrdU incorporation experiments showed that while epithelial cells undergo proliferation by E2 treatment, stromal cells do not incorporate BrdU under the uterine conditions provided with E2+P4. Collectively, these results conclude that microRNAs are essential for uterine stromal cell proliferation in mice.