We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N‐omega‐nitro‐L‐arginine methyl ester, L‐NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10−7 M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10−3 and 10−5 M L‐NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four‐cell stage was observed by the addition of high concentration of SNP (10−3 M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10−5 and 10−7 M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose‐dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L‐NAME reversed the inhibitory effect by each SNP or L‐NAME treatment. Furthermore, low concentration of SNP (10−7 M) but not high concentration of SNP (10−3 M) significantly stimulated trophoblast outgrowth, whereas the addition of L‐NAME suppressed the spreading of blastocysts in a dose‐dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner. Mol. Reprod. Dev. 58:262–268, 2001. © 2001 Wiley‐Liss, Inc.

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2 ¶-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner. [Cancer Res 2007;67(15):7095-105]

a1,6-Fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of α1,6 core fucose to the innermost N-acetylglucosamine residue of the N-glycan, has been implicated in the development, immune system, and tumorigenesis. We found that α1,6-fucosyltransferase and E-cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E-cadherin appeared as well as normal sized E-cadherin in cancer samples. To investigate the correlation between α1,6-fucosyltransferase and E-cadherin expression, we introduced α1,6-fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E-cadherin was significantly increased in α1,6-fucosyltransferase-transfected WiDr cells in dense culture, which resulted in an enhancement in cell-cell adhesion. The transfection of mutated a1,6-fucosyltransferase with no enzymatic activity had no effect on E-cadherin expression, indicating that core fucosylation is involved in the phenomena. In α1,6-fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E-cadherin and E-cadherin dependent cellcell adhesion was decreased. The introduction of α1,6-fucosyltransferase into kidney epithelial cells from α1,6-fucosyltransferase -/-mice restored the expression of E-cadherin and E-cadherin-dependent cell-cell adhesion. Based on the results of lectin blotting, peptide Nglycosidase F treatment, and pulse-chase studies, it was demonstrated that the low molecular weight population of E-cadherin contains peptide N-glycosidase F insensitive sugar chains, and the turnover rate of E-cadherin was reduced in α1,6-Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E-cadherin. These results suggest a possible role of core fucosylation in the regulation of cell-cell adhesion in cancer. (Cancer Sci 2009; 100: 888-895) I t is generally accepted that glycosylation affects many properties of glycoproteins, including their conformation, flexibility, and hydrophilicity. As a result, it regulates protein sorting, stability, and protein-protein interactions.(1-5) N-Glycans have a common core structure, and their branching patterns are determined by glycosyltransferases.(6,7) Fut8 is an enzyme that catalyzes the introduction of α1,6 core fucose on the asparagine-branched N-acetylglucosamine residue of the chitobiose unit of complextype N-glycans. (8,9) Fut8 has been investigated especially in terms of oncogenesis, since the α1,6-fucosylation of α-fetoprotein is a well-known marker of hepatocellular carcinoma. (10) In previous studies, our group reported that Fut8 expression is markedly enhanced in several types of cancer cell lines (11) rat hepatoma tissues (12) and in ovarian serous adenocarcinoma cells.E-cadherin is a 120 kDa type I membrane protein, which belongs to the class of calcium-dependent cell adhesion molecules. (14) It mediates cell-cell adhesi...

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We have identified a region with characteristics of a paternal-specific methylation imprint at the human H19 locus. This region, extending from -2.0 kb upstream to the start of transcription, is heavily methylated in sperm and on the paternal allele in somatic cells. This methylation was preserved during pre-implantation. Structural analysis revealed the presence of CpG islands and a large direct repeat with a 400 bp sequence reiterated several times, but no significant sequence homology to the corresponding region of the mouse H19 gene. These findings could suggest a role for secondary DNA structure in genomic imprinting across the species, and they also present a puzzling aspect of the evolution of the H19 regulatory region in human and mouse.