Byung-Nam Cho

Impaired Spermatogenesis and Fertility in Mice Carrying a Mutation in the Spink2 Gene Expressed Predominantly in Testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.

Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line

The F9 cell line, which was derived from a mouse testicular teratoma that originated from pluripotent germ cells, has been used as a model for differentiation. However, it is largely unknown whether F9 cells possess the characteristics of male germ cells. In the present study, we investigated spermatogenic stage- and cell type-specific gene expression in F9 cells. Analysis of previous microarray data showed that a large number of stage-regulated germ cell genes are expressed in F9 cells. Specifically, genes that are prominently expressed in spermatogonia and have transcriptional regulatory functions appear to be enriched in F9 cells. Our in silico and in vitro analyses identified several germ cell-specific or -predominant genes that are expressed in F9 cells. Among them, strong promoter activities were observed in the regions upstream of the spermatogonial genes, Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Tex13 (testis expressed gene 13), in F9 cells. A detailed analysis of the Tex13 promoter allowed us to identify an enhancer and a region that is implicated in germ cell-specificity. We also found that Tex13 expression is regulated by DNA methylation. Finally, analysis of GFP (green fluorescent protein) TEX13 localization revealed that the protein distributes heterogeneously in the cytoplasm and nucleus, suggesting that TEX13 shuttles between these two compartments. Taken together, our results demonstrate that F9 cells express numerous spermatogonial genes and could be used for transcriptional studies focusing on such genes. As an example of this, we use F9 cells to provide comprehensive expressional information about Tex13, and report that this gene appears to encode a germ cell-specific protein that functions in the nucleus during early spermatogenesis.

15-deoxy-Δ12,14-prostaglandin J2 Down-Regulates Activin-Induced Activin Receptor, Smad, and Cytokines Expression via Suppression of NF-κB and MAPK Signaling in HepG2 Cells

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and activin are implicated in the control of apoptosis, cell proliferation, and inflammation in cells. We examined both the mechanism by which 15d-PGJ2 regulates the transcription of activin-induced activin receptors (ActR) and Smads in HepG2 cells and the involvement of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in this regulation. Activin A (25 ng/mL) inhibited HepG2 cell proliferation, whereas 15d-PGJ2 (2 μM and 5 μM) had no effect. Activin A and 15d-PGJ2 showed different regulatory effects on ActR and Smad expression, NF-κB p65 activity and MEK/ERK phosphorylation, whereas they both decreased IL-6 production and increased IL-8 production. When co-stimulated with 15d-PGJ2 and activin, 15d-PGJ2 inhibited the activin-induced increases in ActR and Smad expression, and decreased activin-induced IL-6 production. However, it increased activin-induced IL-8 production. In addition, 15d-PGJ2 inhibited activin-induced NF-κB p65 activity and activin-induced MEK/ERK phosphorylation. These results suggest that 15d-PGJ2 suppresses activin-induced ActR and Smad expression, down-regulates IL-6 production, and up-regulates IL-8 production via suppression of NF-κB and MAPK signaling pathway in HepG2 cells. Regulation of ActR and Smad transcript expression and cytokine production involves NF-κB and the MAPK pathway via interaction with 15d-PGJ2/activin/Smad signaling.

Plasmid DNA of high quality purified by activated charcoal

Demand for plasmid DNA of high purity and safety has increased with rapid advances in gene therapy and DNA vaccines in addition to basic DNA study. Using activated charcoal (AC), we have developed protocols for pure plasmid DNA. Plasmid DNA extracted by the alkaline lysis method was inevitably contaminated with nucleotide fragments. Treatment with AC during purification instead of RNase completely removed nucleotide fragments in the final plasmid DNA and the removing capability of AC was dose dependent on AC quantity. Of note is that nucleotide fragments less than 0.4 kbp were effectively removed by AC and purification up to 500 ml was easily achieved. Taken together, inexpensive AC effectively removed the troublesome nucleotide fragments and practically substituted for expensive RNase. The resultant plasmid DNA has enough quality needed for basic DNA study and application.

Identification and characterization of reproductive KRAB-ZF genes in mice

The mammalian genome contains numerous genes encoding transcription factors that contain Krüppel-associated box (KRAB) and C2H2-type zinc finger (ZF) motifs (KRAB-ZF). In the present study, we identified KRAB-ZF genes expressed in the mouse testis or ovary, and selected three genes that exhibit gonad-specific or gonad-predominant expression. In vitro analyses showed that these gonadal KRAB-ZF proteins are localized in cell nuclei and are able to repress transcriptional activity. We further analyzed one of the gonad-specific reproductive genes, Zfp819, and found that it is expressed exclusively in spermatogenic cells. Overexpression of Zfp819 suppressed cell proliferation and induced apoptosis. Microarray analysis of Zfp819-overexpressing cells allowed us to identify numerous, potential target genes. A number of the down-regulated genes were found to show gene expression levels inversely correlated with Zfp819 during spermatogenesis. Some of the down-regulated genes were previously reported to play significant roles in spermatogenesis and apoptosis. Collectively, our study provides the first comprehensive information regarding the expression of reproductive KRAB-ZF genes in mice and reveals potential functions of Zfp819.

Identification and characterization of promoter and regulatory regions for mouse Adam2 gene expression

ADAM2, a member of the ‘a disintegrin and metalloprotease’ (ADAM) family, is a key protein in mammalian fertilization that is specifically expressed in testicular germ cells. Here, we investigated the transcriptional regulation of the mouse Adam2 gene. An in silico analysis identified two conserved non-coding sequences located upstream of the mouse and human ADAM2 genes. The upstream region of the mouse Adam2 gene was found to lack typical TATA and CAAT boxes, and to have a high GC content. Our in vitro transient transfection-reporter analysis identified a promoter in this region of the mouse Adam2 gene, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Site-directed mutagenesis revealed that the caudal-type homeobox 1 and CCTC-binding factor motifs are responsible for the inhibitory activities of the repressor regions. Finally, electrophoretic mobility shift assays showed putative transcription factor–promoter DNA complexes, and DNA-affinity chromatography and proteomic analyses identified myelin gene regulatory factor as a binding partner of the Adam2 promoter. This provides the first identification and characterization of promoter and repressor regions that regulate the transcription of the mouse Adam2 gene, and offers insights into the regulation of this germ-cell-specific gene.

Longitudinal erythronychia with subungual keratosis: a daughter subungual nail.

radiation-induced BP include cervical, vulval, oesophageal and lung cancers, non-Hodgkin lymphoma, and a lymphnode metastasis from a squamous cell carcinoma. The pathogenesis of radiation-induced BP is unknown, and it has been suggested that radiation damage may alter the BMZ structure or increase autoantibody accumulation because of increased capillary permeability. To our knowledge, this is the first report of radiotherapyinduced BP recurring after rechallenge with radiotherapy. This is a rarely reported side-effect of radiotherapy, which needs appropriate dermatological management.

Parallel regulation of prolactin and c-fos gene expression by 17β-estradiol and stress in the mouse pituitary.

The aim of this study was to investigate expression patterns of the prolactin (PRL) and c‐fos genes by 17β‐estradiol (17β‐E) and stress in the mouse pituitary. In the pituitary, the levels of PRL mRNA were found high with some fluctuation at 30, 60, and 90 min whereas the levels of PRL mRNA were low at 120 min when ovariectomized female mice were injected with 17β‐E or vehicle. PRL mRNA levels began to increase again at 4 h and remained high up to 24 h only in the 17β‐E‐treated mice. The overall changes in c‐fos mRNA by 17β‐E were very similar to those in PRL mRNA in the pituitary. Subsequent study revealed that these high initial levels of PRL and c‐fos mRNAs were caused by stress during injection, not by 17β‐E, since vehicle injection alone into the ovariectomized mice could increase the levels of PRL and c‐fos mRNAs. The stress‐induced elevations of PRL and c‐fos mRNAs were inhibited by bromocriptin, a dopamine agonist, suggesting that the dopaminergic system is involved in the action route of injection stress. In addition, the induced levels of c‐fos mRNA by 17β‐E and stress in the pituitary were very low compared with those in the uterus. The time course changes in c‐fos mRNA level were different between the pituitary and uterus. Taken together, these data indicate that PRL and c‐fos gene expression in the pituitary are regulated by 17β‐E and stress in a parallel manner, supporting the notion that c‐Fos plays a role in regulation of PRL gene expression.