• Korean Journal of Ichthyology
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• v.18 no.4
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• pp.311-318
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• 2006

Ultrastructure of germ cells, the cyst epithelial cells and interstitial cells during spermatogenesis of the stone flounder, Kareius bicoloratus (Pleuronectidae) sampled on the west coast of Korea were investigated by electron microscopic observations. In the primary spermatocyte, the synaptonemal complexes appear in the zygotene stage of the prophase during maturation division. In the growing testis, especially, the interstitial cells (Leydig cells) appear near the primary, secondary spermatocytes and spermatids. Well-developed interstitial cells (steroid hormone secreting cells) which are located in the interlobular space in growing testis have three morphological characteristics of a vesicular nucleus, mitochondria with tubular cristae and smooth endoplasmic reticulum. During spermatogenesis, the primary and secondary spermatocytes attach to the cyst epithelial cell (Sertoli cell) having an elongated ovoid or triangular nucleus and several mitochondria in the cytoplasm. In the growing testis, lipid droplets, the mitochondrial rosettes and glycogen particles appear in the cytoplasm of the cyst epithelial cells near the secondary spermatocytes and spermatids. Particularly, the mitochondria, endoplasmic reticulum, little lipid droplets and the large amount of glycogen particles are present in the cytoplasm of the cyst epithelial cell in the late growing testis. In the late stage of spermiogenesis, the proximal centriole is joined to the nuclear envelope, the distal centriole forms the basal body of the flagellum and gives rise to the axial filament of the flagellum. No acrosome of the sperm is formed as seen in other teleost fish. The head of the spermatozoon is approximately $3{\mu}m$ in length and its tail is about $30{\mu}m$ in length. The axoneme of the tail flagellum of the spermatozoon consists of nine outer doublet microtubules at the periphery and two centrial singlet microtubules at the center. The spermatozoon of this species has two axonemal lateral fins. Especially, the cyst epithelial cells which located near groups of gametes in the various stages, show three functions: nutrition, phagocytosis and steroidogenesis. Especially, the nuclei of cyst epithelial cells in the recovery stage of the testicular developmental stages appear to be irregular in shape after spermiation. Of three functions of the cyst epithelial cell, several characteristics of phagocytosis are showed in the cytoplasm of the cyst epithelial cells in the recovery stage of the testicular developmental stages. At this stage, therefore, it is assumed that the cyst epithelial cells are involved in degeneration and resorption of undischarged germ cells after spermiation.

• Radiation Oncology Journal
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• v.17 no.4
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• pp.299-306
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• 1999

Purpose : The human genetic disorder ataxia-telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. The recent identification of the gene mutated in AT, ATM, and the demonstration that it encodes a homologous domain of phosphatidylinositol 3-kinase (PI3-K), the catalytic subunit of an enzyme involved in transmitting signals from the cell surface to the nucleus, provide support for a role of this gene in signal transduction. Although ionizing radiation was known to induce c-fos transcription, nothing is known about how ATM or PKCI mediated signal transduction pathway modulates the c-fos gene transcription and gene expression. Here we have studied the effect of PKCI on radiation sensitivity and c-fos transcription in normal and AT cells. Materials and Methods: Normal (LM217) and AT (AT5BIVA) cells were transfected with PKCI expression plasmid and the overexpression and integration of PKCI was evaluated by northern blotting and polymerase chain reaction, respectively. 5 Gy of radiation was exposed to LM and AT cells transfected with PKCI expression plasmid and cells were harvested 48 hours after radiation and investigated apoptosis with TUNEL method. The c-fos transcription activity was studied by performing CAT assay of reporter gene after transfection of c-fos CAT plasmid into AT and LM cells. Results: Our results demonstrate for the first time a role of PKCI on the radiation sensitivity and c-fos expression in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells but reduced apoptosis in AT cells. The basal c-fos transcription activity is 70 times lower in AT cells than that in LM cells. The c-fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos transcription in LM cells but not in AT cells Conclusion: Overexpression of PKCI increased radiation sensitivity and repressed c-fos transcription in LM cells but not in AT cells. The results may be a. reason of increased radiation sensitivity of AT cells. PKCI may be involved in an ionizing radiation induced signal transduction pathway responsible for radiation sensitivity and c-fos transcription. The data also provided evidence for novel transcriptional difference between LM and AT cells.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.6 no.1
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.31 no.2
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• pp.129-142
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• 1986

Fifty-five local collections of buck wheat, Fagopyrum esculentum, were investigated their ratios of long-styled (LS) and short-styled (SS) flowers, fertility, meiosis of megaspore and microspore mother cell, female and male gametogenesis, and egg apparatus in accordance with the sowing seasons (spring, summer), altitudes (20m, 50-100m, 300m), and parent style types (L, S). Also they were embryologically investigated the fertility, fertilizing phenomenon and proembryogenesis by the legitimate and illegitimate pollination. There were no differences in the ratios of long-styled and short-5tyled flowers along with altitudes, but more irregularness was observed in plain area than that in the mountaineous or coastal area. LS versus SS ratios by sowing seasons were significantly separated into 1 : 1 in the summer sowing (P 0.1), but they were irregularly separated in the spring sowing. The segregating ratios by parent style types showed more number of short-styled flower in the spring sowing, and were statistically seperated into 1 : 1 in the summer sowing (P 0.25), regardless to parent style types. In the artificial legitimate union, the seed setting rates of the summer sowing (59-61%) were much higher than those of the spring sowing (about 30%), but in the artificial illegitimate union the seed setting rates were only fructified about 0.8-1.8% in the spring sowing. The seed setting rates in accordance with flowering stages were larger in turn early, middle, late, in the summer sowing. The grain number and grain weight per plant of short-styled flower were more than those of long-styled one regardless to style types. The 1,000 grain weight of long-styled flower was heavier than that of short-styled one in large grain, but it was lighter than that of short-styled flower in small or medium grain. The percentage of normal female and male gametogenesis in the summer sowing were higher than those in the spring sowing. The ovule was atropous and two polar nuclei were a synkarion before flowering. The pollens germinated at 30 minuts after pollination and the pollen tube grew continually and penetrated into micropyle at 1.5-2 hours and the two male nuclei fertilized with egg nucleus at 3 -5 hours after pollination. Flertilizing times in summer were shorter than in autumn. The fertilized egg was divided in a small apical cell toward the interior of the embryo sac and a large basal cell toward the micropyle cell at 15-24 hours after pollination, and division times in summer were shorter than in autumn. The proembryo began the embryogenesis at 7-8 days and formed itself into the perfect embryo at 15 days after pollination.