We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.

Androgen receptor (AR) null male mice (AR L؊/Y ) revealed late-onset obesity, which was confirmed by computed tomography-based body composition analysis. AR L؊/Y mice were euphagic compared with the wild-type male (AR X/Y ) controls, but they were also less dynamic and consumed less oxygen. Transcript profiling indicated that AR L؊/Y mice had lower transcripts for the thermogenetic uncoupling protein 1, which was subsequently found to be ligand-dependently activated by AR. We also found enhanced secretion of adiponectin, which is insulin sensitizing, from adipose tissue and a relatively lower expression of peroxisome proliferatoractivated receptor-␥ in white adipose tissue in comparison to AR X/Y mice. Both factors might explain why the overall insulin sensitivity of AR L؊/Y mice remained intact, despite their apparent obesity. The results revealed that AR plays important roles in male metabolism by affecting the energy balance, and it is negative to both adiposity and insulin sensitivity. Diabetes 54:1000 -1008, 2005 T he etiology of obesity is extremely heterogeneous, in that it is the final result of interactions among genetic, environmental, and psychosocial factors. The androgen receptor (AR) gene may be one of these genetic factors. AR gene repeat variation was shown to be strongly associated with central obesity indexes in older adults (1). Testosterone is an important factor for determining body composition in males. Abdominal obesity is inversely correlated with serum testosterone levels in men but not in women (2). Steady increases in body fat mass accompany the age-dependent decrease in serum testosterone levels in men (3,4), leading to greater morbidity (5). Pathologically hypogonadal men also have a significantly higher fat mass (3,6), which is reversed by testosterone administration (7,8), whereas suppression of serum testosterone in healthy young men increased the percent fat mass and decreased lipid oxidation rates and resting energy expenditure (9).We generated an AR null (ARKO) mouse line, using a Cre-loxP system (10 -12), and found that male ARKO mice (AR LϪ/Y ) developed late-onset obesity, whereas neither heterozygous nor homozygous female ARKO mice were affected (10), suggesting a male-specific AR effect on adiposity.Herein we report the underlying mechanism of lateonset obesity in AR LϪ/Y mice. Despite a lack of hyperphagia, AR LϪ/Y mice had lower spontaneous activity and a decreased overall oxygen consumption ratio. We also observed a concomitant decrease in expression of the thermogenic uncoupling protein 1 (UCP-1). In addition, a unique lack of insulin resistance in AR LϪ/Y mice, despite the obese phenotype, suggests it was related to an enhanced secretion of adiponectin from adipose tissue. RESEARCH DESIGN AND METHODSAn ARKO mutant mouse line was established and maintained as described previously (10 -12). Heterozygous females were bred to wild-type males (C57BL/6NCrj; Charles River Japan, Tokyo, Japan) to produce ARKO male mice (AR LϪ/Y ) and heterozygous females. Their diet (CLEA rod...

To establish the novel approach in order to distinguish the transcriptionally active androgen receptor (AR) from the transcriptionally inactive AR, we performed the three-dimensional construction of confocal microscopic images of intranuclear AR. This method clearly distinguished the subnuclear localization of transcriptionally active AR tagged with green fluorescent protein (AR-GFP) from the transcriptionally inactive AR-GFP. Transcriptionally active AR-GFP mainly produced 250 -400 fluorescence foci in the boundary region between euchromatin and heterochromatin. Although the AR-GFP bound to such antiandrogens as hydroxyflutamide or bicalutamide translocated to the nucleus, they homogeneously spread throughout the nucleus without producing any fluorescence foci. Antiandrogenic environmental disrupting chemicals, such as 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene, vinclozolin, or nitrofen, also disrupted the intranuclear fluorescence foci. A point mutation (T877A) resulted in the loss of ligand specificity in AR-GFP. Even in this mutant receptor, agonists, such as dihydrotestosterone, hydroxyflutamide, or progesterone, produced the fluorescence foci in the nucleus, whereas the transcriptionally inactive mutant binding bicalutamide was observed to be spread homogeneously in the nucleus. Taken together, our findings suggest that, after nuclear translocation, AR is possibly located in the specific region in the nucleus while demonstrating clustering tightly depending on the agonist-induced transactivation competence.

Obesity is associated with insulin resistance and some reproductive abnormalities. Circulating FFAs are often elevated in obese subjects and are also closely linked to insulin resistance. In this study, we demonstrated that saturated FFAs, such as palmitic acid and stearic acid, markedly suppressed the granulosa cell survival in a time- and dose-dependent manner. Polyunsaturated FFA, arachidonic acid, had no effect on the cell survival, even at supraphysiological concentrations. The suppressive effect of saturated FFAs on cell survival was caused by apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effects of palmitic acid and stearic acid were unrelated to the increase of ceramide generation or nitric oxide production and were also completely blocked by Triacsin C, an inhibitor of acylcoenzyme A synthetase. In addition, acylcoenzyme A, pamitoylcoenzyme A, and stearylcoenzyme A markedly suppressed granulosa cell survival, whereas arachidonoylcoenzyme A had no such effect, and this finding was consistent with the effect of the respective FFA form. Surprisingly, arachidonic acid instead showed a protective effect on palmitic acid- and stearic acid-induced cell apoptosis. A Western blot analysis showed the apoptosis of the granulosa cells induced by palmitic acid to be accompanied by the down-regulation of an apoptosis inhibitor, Bcl-2, and the up-regulation of an apoptosis effector, Bax. These results indicate that saturated FFAs induce apoptosis in human granulosa cells caused by the metabolism of the respective acylcoenzyme A form, and the actual composition of circulating FFAs may thus play a critical role in the apoptotic events of human granulosa cells. These effects of FFAs on granulosa cell survival may be a possible mechanism for reproductive abnormalities, such as amenorrhea, which is frequently observed in obese women.

This report describes a design, preparation and evaluation data of a novel polymer electrolyte membrane, which have been successfully developed for the use in fuel cell vehicles (FCV). This membrane is prepared from the aromatic block copolymer, consisting of alternating stiff sulfonic acid-bearing segments and hydrophobic flexible polymeric sub-units. A bicontinuous microphase-separated morphology of the membrane has been attested, contributing to its excellent water resistance with keeping high proton conductivity. The JSR membrane exhibits actually the same chemical stability as a conventional poly(perfluorosufonic acid) one, while outperforming the latter in power output of the fuel cell, life time and temperature range. In particular, a cold start of FCV have been first demonstrated at À20 C, using this material. A manufacturing of the JSR membrane in the semi-industrial scale is established. This technology has been officially approved for the extension through the public road examination. This SPSJ award is given for developing novel aromatic polymer electrolyte membrane with high performance for the practical use. Fuel cells are widely regarded as promising energy sources due to their high energy efficiency in terms of limiting fossil fuel resources. Among them, polymer electrolyte fuel cells (PEFC) can generate electricity with high power density at moderate temperatures, even at room temperature, to offer a significant advantage to other fuel cell systems, e.g., phosphoric acid fuel cells (PAFC), molten carbonate fuel cells (MCFC) and solid oxide fuel cells (SOFC). Many efforts have been devoted into the research and development of PEFC for transportation and stationary applications, as well as in the design of closely related DMFC (direct methanol fuel cells) for the use in portable devices.PEFC consists of two electrodes, which are separated by the polymer electrolyte membrane. On the anode side, hydrogen dissociates into protons and electrons by the electrochemical reaction with the catalyst on the electrode surface. Protons diffuse through the membrane to the cathode, while electrons are transmitted to the cathode through the electron conductor, creating thereby an electrical current. Polymer electrolyte membrane is a key material governing the power generation of the fuel cell, and it should meet strict requirements as possessing a high proton conductivity closely linked to high power generation ability, high stability and durability in the fuel cell environment, excellent mechanical toughness, high heat endurance, as well as impermeability to fuel gas or liquid.Poly(perfluorosulfonic acid) membranes (Figure 1) have been widely used as benchmark materials in PEFC. However, the operation temperature of this type of membranes is limited within the range from 0 C to 80 C, because of the drop in conductivity below the freezing point of water and the poor thermo-mechanical properties above 80 C. Moreover, the durability of membranes is not sufficient to achieve the practical applications in PEFC. There...

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NDROGEN-insensitivity syndromes in 46,XY fetuses result in various degrees of impairment in genital virilization. 1 These syndromes are caused by mutations in the androgen receptor gene that result in decreased binding of androgen to the receptor. [2][3][4][5][6][7][8][9] As a consequence, the transcriptional activity of the androgen-androgen-receptor complex is reduced, and therefore, genital virilization is reduced. The androgen receptor, like other steroid hormone receptors, has two major transactivation domains 10 -activation function 1 (AF-1) in the N-terminal region 11-13 and activation function 2 (AF-2) in the C-terminal ligand-binding domain 14 -that interact with the target genes directly as well as indirectly by means of intermediary coactivators. 15 We describe a patient in whom the complete androgen-insensitivity syndrome was diagnosed on the basis of phenotypic and endocrinologic findings, but who had no mutations in the androgen receptor gene. Detailed studies revealed that transmission of the activation signal from the AF-1 region of the androgen receptor was disrupted, suggesting that a coactivator interacting with the AF-1 region of the androgen receptor was lacking in this patient. CASE REPORTA 19-year-old woman reported primary amenorrhea. The patient had normal breast development and normal female external genitalia, but she had no pubic or axillary hair, and the vagina was short (6 cm in length) and ended in a blind pouch. Abdominal exploration revealed no uterus, but testes were present, which were resected. Histologic examination of the testes revealed small numbers of immature Sertoli cells and germ cells and a moderate num-A ber of Leydig cells. Preoperatively, the patient's serum testosterone concentration was 614 ng per deciliter (21.3 nmol per liter) and her serum 5 a -dihydrotestosterone concentration was 49 ng per deciliter (1.7 nmol per liter); both values were within the normal range for men. The karyotype was 46,XY. The patient was given a diagnosis of complete androgen-insensitivity syndrome. Her two older sisters were not affected. METHODS Analysis of the Androgen ReceptorThe study was approved by the local institutional review committee, and written or oral informed consent for a genital-skin biopsy was obtained from the patient, another patient with complete androgen-insensitivity syndrome, and five normal men. Primary culture of genital-skin fibroblasts, androgen-binding assays, and sequence analysis of the androgen receptor gene were performed as previously described. 4-9 Tissue concentrations of androgen receptor messenger RNA (mRNA) were determined by a quantitative reverse-transcriptase-polymerase-chain-reaction assay (RT-PCR) as described previously. 16,17 Plasmid Construction and Reporter AssayWe constructed a firefly-luciferase-reporter vector (pGL3-MMTV), which was under the control of the mouse-mammarytumor virus (MMTV) promoter, by inserting the mouse-mammary-tumor virus long terminal repeat promoter 18 into a pGL3 basic vector (Promega). The expression vectors for ...

Our previous studies demonstrated that a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, troglitazone (TGZ),and/or a retinoid X receptor (RXR) ligand, LG100268 (LG), decreased the aromatase activity in both cultured human ovarian granulosa cells and human granulosa-like tumor KGN cells. In the present study, we further found that a combined treatment of TGZ+LG decreased aromatase promoter II (ArPII) activity in both ovarian KGN cells and fibroblast NIH-3T3 cells in a PPARgamma-dependent manner. Furthermore, the inhibition of both aromatase activity and the transcription of ArPII by TGZ+LG was completely eliminated when nuclear factor-kappaB (NF-kappaB) signaling was blocked by specific inhibitors, suggesting NF-kappaB, which is endogenously expressed in both fibroblast and granulosa cells, might be a mediator of this inhibition. Interestingly, activation of NF-kappaB by either forced expression of the p65 subunit or NF-kappaB-inducing kinase up-regulated ArPII activity. Positive regulation of aromatase by endogenous NF-kappaB was also suggested by the fact that NF-kappaB-specific inhibitors suppress basal activity of the aromatase gene. A concomitant formation of high-order complex between NF-kappaB p65 and ArPII was also observed by chromatin immunoprecipitation assay. Although activation of PPARgamma and RXR affected endogenous expression levels of neither inhibitory kappaBalpha nor p65, it impaired the interaction between NF-kappaB and ArPII and the p65 based transcription as well. Altogether, these results indicate that activation of a nuclear receptor system, constituted by PPARgamma and RXR, down-regulates aromatase expression through the suppression of NF-kappaB-dependent aromatase activation and thus provide a new insight in the mechanism of regulation of the aromatase gene.