RESUMO
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.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Glucocorticoides/metabolismo , Células Intersticiais do Testículo/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , Animais , Corticosterona/metabolismo , Regulação Enzimológica da Expressão Gênica , Masculino , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Testosterona/biossínteseRESUMO
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 = 0.01) as were serum LH levels (1.31 +/- 0.3 vs. 0.45 +/- 0.08 ng/ml; P = 0.01). Mice that were treated with the pure antiestrogen ICI 182,780 at 100 micro g/kg.d for 7 d, effectively abrogating ER-mediated activity, also had 2-fold elevations in the serum levels of LH (1.15 +/- 0.3 vs. 0.45 +/- 0.2 ng/ml) and T (4.3 +/- 1.1 vs. 2.2 +/- 0.2 ng/ml; P = 0.01). Increased androgen biosynthesis by alphaERKO Leydig cells was associated with higher steroidogenic enzyme activity, especially of cytochrome P450 17alpha-hydroxylase/17-20 lyase (P450(17alpha)) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), as measured by conversion of radiolabeled steroid substrates to T or its precursors. The largest increases in enzymatic activity were observed for P450(17alpha) (423 +/- 45 pmol/min.10(6) cells in alphaERKO Leydig cells vs. 295 +/- 27 pmol/min.10(6) cells in WT cells; P < 0.01). Consistent with steroidogenic enzyme activity, the testis of alphaERKO mice expressed higher steady state mRNA levels for steroidogenic acute regulatory protein and two enzymes involved in androgen biosynthesis, P450(17alpha) and 17beta-HSD type III, as determined by semiquantitative RT-PCR. Compared with the controls, higher steady state mRNA levels for steroidogenic acute regulatory protein and P450(17alpha) were also measured in the testis of ICI 182,780-treated mice. In a second set of experiments estrogen administration reduced serum LH and T levels in WT controls, whereas alphaERKO mice were unaffected. Although exposure of WT and alphaERKO Leydig cells to estrogen in vitro did not affect androgen biosynthesis, incubation with ICI 182,780 reduced T production by WT, but not alphaERKO, Leydig cells. These observations indicate that abrogation of the ERalpha gene by targeted deletion or treatment with an antiestrogen increases Leydig cell steroidogenesis in association with elevations in the serum levels of LH, which presumably is the result of estrogen insensitivity at the level of the hypothalamus and/or pituitary gonadotropes. Furthermore, the decrease in T production by WT Leydig cells and not alphaERKO Leydig cells occasioned by incubation with ICI 182,780 suggests that of the ER subtypes, ERalpha has a regulatory role in Leydig cell steroidogenic function.
Assuntos
Androgênios/biossíntese , Estradiol/análogos & derivados , Células Intersticiais do Testículo/metabolismo , Receptores de Estrogênio/deficiência , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Knockout , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 17-alfa-Hidroxilase/metabolismo , Testosterona/biossíntese , Testosterona/sangueRESUMO
Transgenic expression of the human angiotensinogen (HAGT) gene directed by the mouse kidney androgen-regulated protein (Kap) gene promoter is proximal tubule cell-specific and androgen-regulated in vivo. The same Kap promoter fragment did not support similar regulation of other genes, but a transgene based on the original chimeric KAP-hAGT construct successfully directed NHE3 to kidney, suggesting that sequences within the HAGT gene fragment of the construct contributed to the regulation of its expression in vivo. In the present study, androgen-responsive regulatory sequences in the HAGT gene portions of the transgene were examined in transfected renal cells. A 1.4-kb enhancer between exons 2 and 3 was identified that increased the basal expression of Kap promoter 1.5- to 2-fold, its induction by dihydrotestosterone (DHT) 2- to 3-fold and its induction by dexamethasone (Dex) 4- to 5-fold. Sequence analysis revealed two potential hormone-responsive elements. Mutational assays and electrophoretic mobility shift assay showed one of these elements was androgen-specific. These findings may influence future strategies for the design of inducible, cell-specific transgenes.
Assuntos
Androgênios/farmacologia , Angiotensinogênio/genética , Elementos Facilitadores Genéticos/efeitos dos fármacos , Íntrons/genética , Proteínas/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Di-Hidrotestosterona/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim , Camundongos , Gambás , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/efeitos dos fármacos , TransfecçãoRESUMO
PROBLEM: This study was undertaken to evaluate whether the anti-GnRH antibodies and immune complexes (IC) generated by immunization with GnRH-TT cause cellular damage within the animal. METHOD OF STUDY: Chronic immunization of rats with GnRH-TT injected i.m. was followed by tissue/organ analysis for immune complex deposition by immunofluorescence microscopy. Two groups were studied: (1) those immunized throughout the experiment until their ultimate demise, and (2) those given a chance to recover from the effects of chronic immunization before final analysis. RESULTS: GnRH-TT was effective in stopping spermatogenesis, which resumed after withdrawal of the immunogen. Most tissues from chronically immunized animals were not significantly different than controls, however the kidneys of treated animals exhibited a higher accumulation of IC. Despite increased IC deposition, pathologic effects were not detected at the cellular level. CONCLUSIONS: GnRH-TT is an effective immunocontraceptive although the accumulation of glomerular IC represents a potential deleterious side effect.
Assuntos
Hormônio Liberador de Gonadotropina/administração & dosagem , Doenças do Complexo Imune/patologia , Bloqueadores de Espermatogênese/administração & dosagem , Espermatogênese/efeitos dos fármacos , Animais , Complexo Antígeno-Anticorpo/imunologia , Hormônio Liberador de Gonadotropina/efeitos adversos , Hormônio Liberador de Gonadotropina/imunologia , Doenças do Complexo Imune/induzido quimicamente , Doenças do Complexo Imune/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Espermatogênese/imunologia , Bloqueadores de Espermatogênese/efeitos adversos , Bloqueadores de Espermatogênese/imunologiaRESUMO
The kidney androgen-regulated protein (KAP) is specifically expressed and differentially regulated by androgens and tri-iodothyronine (T(3)) in intact mouse early (PCT) and late (PR) proximal-tubule cells. Until now, detailed characterization of the molecular elements mediating androgen-responsive gene expression in the kidney has been hampered by the lack of appropriate cultured cell systems suitable for DNA transfection studies. In the present study we have analysed the hormone-dependent transactivation of the KAP gene promoter in immortalized differentiated PCT and PR proximal-tubule cells derived from L-PK/Tag1 transgenic mice. Transient transfection studies with different KAP promoter constructs indicated that a 224 bp-truncated fragment was sufficient to mediate cell-specific expression of the KAP promoter. Dihydrotestosterone (DHT) stimulated in an androgen-dependent manner the transactivation of KAP in PCT and PR cells, while mutation of a putative androgen-response element (ARE) sequence located at -39 bp from the transcription initiation site abolished the transactivation induced by DHT. Furthermore, insulin-like growth factor 1 (IGF-1), but not T(3), enhanced the androgen-dependent transactivation of KAP in cultured PCT cells. These results demonstrate that the short 224 bp fragment of the KAP promoter is sufficient to drive the proximal-tubule androgen-specific regulated expression of KAP and reveal synergistic interactions between IGF-1 and androgens for KAP regulation in PCT cells.