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...

The androgen-androgen receptor (AR) system plays vital roles in a wide array of biological processes, including prostate cancer development and progression. Several growth factors, such as insulin-like growth factor 1 (IGF1), can induce AR activation, whereas insulin resistance and hyperinsulinemia are correlated with an elevated incidence of prostate cancer. Here we report that

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.

BackgroundAtrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP.MethodsWe compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1’s role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1–dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII.ResultsAtrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this “orphan” receptor.ConclusionThe current findings are consistent with atrazine’s endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans.