Effect of heavy metals (Cd, Cu, Zn) on the survival and population growth rates (PGR) of marine rotifer, Brachionus plicatilis were examined. B. plicatilis were exposed to Cd, Cu and Zn for 24 h to determine their survival and 72 h to determine their PGR. Survival rates in the control groups were greater than 90%. They were decreased with increasing concentrations of Cd, Cu and Zn. Survival rates were reduced in a concentration-dependent manner. Significant reduction in survival rates after exposure to Cd, Cu and Zn at concentration greater than 40.00, 0.13 and 10.00 mg L-1, respectively. PGR in the control groups were greater than 0.50. They were decreased with increasing concentrations of heavy metals. PGR were reduced in a concentrationdependent manner. Significant reduction in PGR after exposure to Cd, Cu and Zn occurred at concentration greater than 12.5, 0.06 and 1.00 mg L-1, respectively. The order of heavy metal toxicity based on PGR was Cu>Zn>Cd, with EC50 (50% Effective Concentration) values of 0.12, 6.15 and 21.41 mg L-1, respectively. The lowest-observed-effective-concentrations (LOEC) of PGR after exposure to Cd, Cu and Zn were 12.50, 0.06 and 1.00 mg L-1, respectively. The No-observedeffective- concentrations (NOEC) of PGR after exposure to Cd, Cu and Zn were 6.25, 0.03 and 0.01 mg L-1, respectively, in marine ecosystems have toxic effects on PGR of B. plicatilis. These results suggest that the PGR of B. plicatilis are useful tool to assess the effect of heavy metals on primary consumers in marine natural ecosystems.

CD26, also known as Dipeptidyl peptidase IV (DPP-4), is a cell surface glycoprotein that belongs to the serine protease family and has wide spread organ distribution throughout the body. CD26 was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the effect of CD26 in porcine parthenogenetic embryos. We attempted CD26 downregulation of porcine embryos by siRNA, and evaluated CD26 suppression of developmental competencies. Although the porcine embryos injected with CD26 siRNA were able to develop to the early stage, these embryos were decreased to form blastocysts. Our results indicated that CD26 is one of factors for the regulation of development of porcine embryos.

Bisphenol‒A (BPA) is a known endocrine‒disrupting chemical used extensively to manufacture plastic bottles, canned food linings, thermal receipts, and other commonly used items. BPA is capable of inducing chromosomal alterations in germ cell line, thereby produced transgenerational effects on brain function, social recognition, reproductive diseases, sperm quality, gene expression, and obesity. Here, we aimed to investigate the transgenerational effects of BPA on murine male fertility. Six-week-old male mice (F0) were gavaged with corn oil (control), two different doses of BPA (5 mg, and 50 mg·kg bw-1·day-1),andethinylestradiol(EE,0.4mg·kg bw-1·day-1), dailyfor6weeks. Treated male mice were mated with wild‒type female and sibling pairs were bred up to the third generation (F3) in a similar manner with no further BPA exposure. Testes and spermatozoa were collected from 14-week-old males of all generation (F0 to F3) to evaluate testis weight, sperm function, and fertility. We found that high concentration of BPA significantly increased testicular weight in F2. Although the sperm viability, capacitation status, and intracellular ROS levels were not affected by BPA, however, sperm count, motility, hyperactivated motility, and intracellular ATP levels were significantly altered by BPA, dose dependently. In majority of the cases the effects were prominent in F2 followed by F1 and F0, whereas the effects were diminished in F3 generation. Simultaneously, high concentration of BPA significantly decreased cleavage and blastocyst formation rate in both F1 and F2. Similar inhibitory effects on cleavage and blastocyst were also noted in F1 by low dose of BPA. Depending on these findings we conclude that BPA decreases the fertility potential of exposed males and has an adverse impact on sperm function and fertility in subsequent generations.

For the establishment and maintenance of successful pregnancy the maternal immune system must tolerate semi-allogenic fetus during pregnancy. Several mechanisms explaining immune tolerance have been proposed. Among those, it has been suggested that the CD40/CD40L system is involved in immune tolerance in several tissues. However, expression and function of CD40/CD40L in the maternal-fetal interface during pregnancy have not been studied in pigs. Thus, this study determined expression and localization of CD40 and CD40L in the uterine endometrium during pregnancy in pigs. We obtained uterine endometrial tissue samples from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Quantitative real-time PCR analysis showed that levels of CD40L mRNA expression during pregnancy increased on D15 of pregnancy and decreased thereafter whereas levels of CD40 mRNA was highest on D30 of pregnancy. Localization of CD40 and CD40L proteins by immunohistochemistry showed that CD40 was localized to vascular endothelial cells with strongest signal intensity on D15 of pregnancy, and CD40L was localized to luminal epithelial cells on D15 of pregnancy and amniotic membrane during mid- to late pregnancy. To determine the effect of IFNG on CD40 and CD40L expression, we took advantage of endometrial explant culture using tissues from D12 of the estrous cycle, and found that CD40 was up-regulated by IFNG in a dose-dependent manner. These results showed that CD40 and CD40L were expressed in the uterine endometrium in a cell-type and stage-specific fashion during pregnancy, and IFNG induced CD40, indicating that the CD40/CD40L system may be important for establishment and maintenance of pregnancy in pigs. [Supported by the Next Generation BioGreen21 Program (#PJ01110301), Rural Development Administration]

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm–egg fusion, and was therefore selected as candidate gene for boar semen motility and kinematic characteristics. This study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 96 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.358A>T) in intron 6 was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine CD9 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts, and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm–egg fusion, and was therefore selected as a candidate gene to investigate Duroc boar semen motility and kinematic characteristics. This study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 96 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT, 82.27±5.58), Curvilinear velocity(VCL, 68.37±14.58), Straight-line velocity(VSL, 29.06±6.58), the ratio between VSL and VCL(LIN, 47.36±8.42), Amplitude of Lateral Head displacement(ALH, 2.88±0.70)] were used in present study. A single nucleotide polymorphism (g.358A>T) in intron 6 was associated with MOT, VCL, VAP and ALH in Duroc population (p<0.05). Therefore, we suggest that the porcine CD9 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts, and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of IFN-γ. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen post-thawed in boar. Recently, polymorphism (g.358A>T) of cluster-of-differentiation antigen 9 (CD9) gene reported to be significant association with MOT. Also, CD9 gene was expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as candidate gene for boar semen. This study was conducted to evaluate the pig SNP (g.358A>T) of CD9 gene as a positional controlling for semen parameters of post-thawed boar semen. To results, the g.358A>T SNP of the CD9 gene was significantly associated with the traits such as MOT, curve linear velocity, straight line velocity, average path velocity and amplitude of lateral head displacement. Particularly, the g.358A>T SNP significantly has the highest association with MOT and animals with AA genotype (p<0.001). Therefore, we suggest that the g.358A>T in the intron 6 region of the porcine CD9 may be used as a molecular marker for Duroc boar Post-thawed semen quality, although its functional effect was not defined yet.

Genetic polymorphisms within immunity-related candidate genes in pigs have been identified to control variations in immune functions and/or disease resistance. It has become necessary to evaluate the effects of other genetic markers of economically important traits prior to introducing them into marker-assisted selection programs. In this study, polymorphisms of porcine genes coding Interferon-induced Gunylate binding protein 1 (GBP1), GBP2, CD163, and CD169 were investigated for their association with growth and meat quality traits in a Korean native pig breed -Yorkshire inter-crossed F2 pig population (KY-F2). KY-F2 animals (n=346) have been successfully used for linkage mapping to identify quantitative loci that control meat quality, growth, and immunity traits. In our results, polymorphisms in genes GBP1 and GBP2 showed association with pig growth rate as well as meat quality traits such as crude fat, drip loss, and meat color (yellowness) in the KY-F2 population. The polymorphism in gene CD163 only showed association with crude fat, as a meat quality trait. CD169 gene was associated with pork tenderness. In conclusion, four immune-related genetic markers were validated for their association with growth and meat quality traits to gauge their potential use in a swine selection program. The results warrant further studies in other commercial pig populations.

해산규조류 Skeletonema cosatatum의 개체군성장률 (r) 을 사용하여 중금속 3종(Cd, Cu, Zn)의 독성영향 평가를 수행하였다. S. costatum을 Cd (0, 0.63, 1.25, 2.50, 5.00, 10.00 ppm), Cu (0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50 ppm) 그리고 Zn (0, 0.31, 0.63, 1.25, 2.00, 2.50, 5.00 ppm)에 노 출하고 96 h 이후에 r 값을 도출하였다. 대조구(Cd, Cu 그 리고 Zn을 포함하지 않은)에서 r은 0.05보다 높았으며 중금속 농도가 증가할수록 r은 감소하였다. 중금속에 의 해 농도의존적으로 r이 감소되었고 Cd 농도 1.25, Cu 농 도 1.25 그리고 Zn 농도 2.50 ppm 이상의 농도에서 명확 한 감소를 나타냈다. 중금속의 독성영향은 Cu¤Zn¤Cd 순서이며, 이때 EC50 값은 각각 1.11, 2.13 그리고 6.84 ppm으로 나타났다. 중금속 3종(Cd, Cu 그리고 Zn)에 노 출된 r의 최소영향농도는 각각 1.25, 1.00, 2.00 ppm으로 나타났다. 이 결과를 바탕으로, 해양환경에서 S. costatum 의 r은 Cd 농도가 1.25 ppm 이상, Cu 농도가 1.00 ppm 이 상, Zn 농도가 2.00 ppm 이상인 경우에 독성영향을 받았 다. 그러므로 S. costatum의 r은 해양환경에서 중금속 독 성영향 평가를 위한 매우 유용한 방법으로 판단된다.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.

CD63, a member of tetraspanin membrane protein family, plays pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic ‘Cys-Cys-Gly’ motif and ‘Cys188’ residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcript was upregulated the maximum 4.5 fold in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also made significant increase in the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

CD와 DVD에서 발생하는 빛의 회절을 정량적으로 분석하기 위해 본 연구에서는 회절격자와 CD, DVD에의한 회절무늬를 비교하였다. SEM 측정을 통해 격자간격을 비교하였고, 입사각과 회절각을 측정하여 이론적인 격자간격을 계산하였다. 또한, 다양한 파장을 갖는 Ar-ion 레이저에 의해 발생된 1차 회절광의회절각과 세기를 측정하여 그 결과를 정량적으로 비교하였다. 비교결과, DVD는 회절격자와 CD보다 더좁은 격자간격을 가졌으며, 그 회절광은 다른 회절광에 비해 강했다. 또한, 회절격자와 CD에 비해 더 좁은 격자간격과 더 강한 회절광을 가진 DVD에서 보다 우수한 회절성능이 관찰되었다.