We screened human prostate cancer tissues for the presence of somatic mutations in the hormone binding domain of the androgen receptor (AR) gene. Exons E-H were amplified from genomic DNA using the polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments that differ by only a single base. We detected a mutation in exon E of the hormone binding domain in 1 of 26 specimens of untreated organ-confined stage B prostate cancer. The mutation was not detectable in peripheral blood lymphocyte DNA. Lymphocyte DNA (wild-type AR) migrated in DGGE as a single band. The tumor DNA migrated in DGGE as four bands, consistent with the presence of cells with mutant AR plus cells with wild-type AR and indicating that the tumor contained a somatic mutation. To our knowledge, a somatic AR gene mutation has not been reported previously. Sequencing revealed a G --A substitution in codon 730, changing valine to methionine. Codon 730 is in a region highly conserved among all steroid receptors. The abundance of the mutated fragment (about 50% of the DNA in the specimen) indicates its presence in cells with a growth advantage. A somatic mutation could be detected by DGGE if it represented at least 10% of the sample. Failure to detect mutations in other specimens analyzed may be due to this limit of sensitivity, the presence of mutations in other parts of the AR, or a low frequency of mutations in early stage disease.Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in men in the U.S.(1). Recognition that androgen is required for the development of prostate cancer (2) and its growth (3) has been the basis for continuing interest in the role of the androgen receptor (AR) in prostate cancer (1,(4)(5)(6). The AR is a member of the superfamily of genes that code for the steroid and thyroid hormone receptor family of ligand-dependent nuclear transcription factors, all of which have an N-terminal domain that affects transcription efficiency, a central DNA

Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression of HAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice.The renin-angiotensin system (RAS) 1 is a classical endocrine system activated by the release of renin from the kidney and angiotensinogen (AGT) from the liver. In blood, renin proteolytically cleaves AGT to form angiotensin I (Ang-I) which is further processed by angiotensin converting enzyme to form Ang-II, a potent vasoconstrictor and antinatriuretic peptide. The RAS has been implicated in the genetic basis of hypertension and pre-eclampsia (1-4). Our understanding of the RAS in normal and pathophysiological regulation of blood pressure has been complicated by the fact that in addition to its actions as an endocrine system, certain individual tissues, such as the kidney (5-7), heart (8, 9), brain (10), and vasculature (11), contain all the components of the RAS cascade and therefore have the potential for local synthesis and action of Ang-II. In the kidney, for example, renin, AGT and ACE mRNAs, and proteins are synthesized in juxtaglomerular cells, proximal convoluted tubule (PCT) cells, and endothelial and tubular cells, respectively, and Ang-II type-1 (AT-1) and type-2 (AT-2) receptors are localized in glomeruli, collecting ducts, tubules, and vasa recta (12-18). The intrarenal RAS has been postulated to regulate various aspects of renal function including blood flow, natriuresis, and tubular-glomerular feedback, and may therefore participate in the pathogenesis of hypertension (19 -21). Our current understanding of the relative importance of the intrarenal versus systemic RAS comes largely from pharmacological studies (22) which have been limited by the specificity...

Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long-Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2-20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6 -30 cells increased in all treatment groups, with 29 ؎ 2% in control vs. 37 ؎ 3, 35 ؎ 3, and 56 ؎ 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.di-(2-ethylhexyl) phthalate ͉ testosterone ͉ reproduction ͉ endocrine disruptor ͉ steroidogenesis P hthalates, widely used as plasticizers and solvents, are commonly found in a variety of consumer products including cosmetics, toys, medical tubing, and catheters and in the environment as an industrial waste product. Increasing public concern over lack of regulation on their use in the United States, in contrast to the European Union and 14 other countries (1), has arisen in response to reports that exposures to phthalates may be linked to abnormal reproductive development in the human male (2, 3). Epidemiological studies show statistical correlations between serum concentrations of phthalate monoesters, the primary metabolites of phthalates, and the incidence of anomalies such as cryptorchidism and shortened anogenital distance (AGD) (4, 5). Di-(2-ethylhexyl) phthalate (DEHP), the most abundant phthalate in the environment, has been shown to have adverse effects on androgen synthesis in the rodent (6).The Agency for Toxic Substances and Disease Registry reported that, although exposure to DEHP is generally low, the exposures of preterm infants can be as high as 10-20 mg per day (7). Controversy exists over whether DEHP, at the levels found in the environment, is harmful to humans, because most studies have been conducted in rodents administered high doses. In previous studies, we showed that the administration of low-dose (10 mg/kg body weight) DEHP for 28 days during pubertal development caused elevations in testosterone (T) (8, 9)....

Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P

Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.

Phthalates, the most abundantly produced plasticizers, leach out from polyvinyl chloride plastics and disrupt androgen action. Male rats that are exposed to phthalates in utero develop symptoms characteristic of the human condition referred to as testicular dysgenesis syndrome (TDS). Environmental influences have been suspected to contribute to the increasing incidence of TDS in humans (i.e. cryptorchidism and hypospadias in newborn boys and testicular cancer and reduced sperm quality in adult males). In this review, we discuss the recent findings that prenatal exposure to phthalates affects Leydig cell function in the postnatal testis. This review also focuses on the recent progress in our understanding of how Leydig cell factors contribute to phthalate-mediated TDS.

Deletion of the insulin-like growth factor 1 (Igf1) gene was shown in previous studies to result in reduced numbers of Leydig cells in the testes of 35-day-old mice, and in reduced circulating testosterone levels. In the current study, we asked whether deletion of the Igf1 gene affects the number, proliferation, and/or steroidogenic function of some or all of the precursor cell types in the developmental sequence that leads to the establishment of adult Leydig cells (ALCs). Decreased numbers of cells in the Leydig cell lineage (ie, 3β-hydroxysteroid dehydrogenase–positive cells) were seen in testes of postnatal day (PND) 14–90 Igf1−/− mice compared with age-matched Igf1+/+ controls. The development of ALCs proceeds from stem Leydig cells (SLCs) through progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). The bromodeoxyuridine labeling index of putative SLCs was similar in the Igf1−/− and Igf1+/+ mice. In contrast, the labeling index of PLCs was reduced in the Igf1−/− mice on each day of PND 14 through PND 35, and that of more mature Leydig cells (referred to herein as LCs, a combination of ILCs plus ALCs) was reduced from PND 21 through PND 56. In Igf1−/− mice that received recombinant IGF-I, the labeling indices of PLCs and LCs were similar to those of age-matched Igf1+/+ mice, indicating that the reductions in the labeling indices seen in the PLCs and LCs of the Igf1−/− mice were a consequence of reduced IGF-I. On each day of PND 21 through PND 90, testicular testosterone concentrations were significantly reduced in the Igf1−/− mice, as were the expressions of testis-specific mRNAs involved in steroidogenesis, including Star, Cyp11a1, and Cyp17a1. The increased expression of the gene for 5α-reductase (Srd5a1) in adult Igf1−/− testes suggests that the depletion of Igf1 might suppress or delay Leydig cell maturation. These observations, taken together, indicate that the reduced numbers of Leydig cells in the adult testes of Igf1−/− mice result at least in part from altered proliferation and differentiation of ALC precursor cells, but not of the stem cells that give rise to these cells.

The androgen receptor (AR) is a structurally conserved member of the nuclear receptor superfamily. The amino-terminal domain is required for transcriptional activation and contains a region of polyglutamine encoded by CAG trinucleotide repeats. In humans, the number of CAG repeats is polymorphic; the average number is 22 in Caucasian males. Expansion of CAG repeats in the AR has clinical implications for human disease. As androgen influences prostate cancer growth, polymorphisms in CAG repeat length may affect the clinical course of patients with prostate cancer. To test for an association between clinical parameters of human prostate cancer and CAG repeat length, we analyzed normal lymphocyte DNA from 109 patients. The CAG region of the AR was amplified by the PCR. Reaction products were then amplified using end-labeled internal primers, cut at the internal PstI site and assayed on sequencing gels using a sequence ladder as a size standard. Sequence analysis of several samples validated this method for measurement of CAG repeat number. The median age of patients was 63 yr (range, 42-83), with 104 Caucasian, 2 African American, 1 Asian, and 2 other racial origin. The median repeat length was 25 for patients with stage A, 22 for patients with stage B, 22 for patients with stage C, and 23 for patients presenting with stage D disease. A significant correlation between CAG repeat length and age at onset was observed, whereas correlations with stage, level of prostate-specific antigen at diagnosis, and time to prostate-specific antigen relapse were not significant. Shorter CAG repeat lengths may be associated with the development of prostate cancer in men at a younger age. These data suggest that CAG repeat length can affect the risk of developing prostate cancer.