RESUMEN
Besides gonadotropin release, GnRH stimulates gonadotropin subunit gene transcription and translation in gonadotrophs. In the African catfish, Clarias gariepinus, chicken GnRH-II (cGnRH-II: [His5,Trp7,Tyr8]-GnRH) and catfish GnRH (cfGnRH: [His5,Asn8]-GnRH) are two endogenous forms of GnRH. Studying their effects on LH subunit steady-state mRNA levels, LH de novo synthesis, and LH release in primary pituitary cell cultures of adult males, we found that cGnRH-II hardly influenced the steady-state levels of LH subunit mRNAs or LH de novo synthesis, although it stimulated LH release. Although cfGnRH stimulated LH secretion as well, high concentrations-although apparently still within the physiologic range-reduced LH transcript levels and de novo synthesis in primary pituitary cell cultures. In vivo experiments demonstrated a biphasic response of LH subunit transcript levels after a single GnRH injection: a decrease after 2 h was followed by an increase at 8 h. When the testes were removed before GnRH treatment, however, LH transcript levels remained depressed at 8 h after GnRH injection, indicating that the secondary increase in LH transcript levels depends on the presence of the testes. We conclude that the up-regulation of LH production subsequent to GnRH stimulation in adult male African catfish is mediated by factors originating from the testis. Previous work suggests that aromatizable androgens may play an important role in this context. Under the present experimental conditions, however, GnRHs had no, or an inhibitory, direct effect on LH production in catfish gonadotrophs.
Asunto(s)
Bagres/metabolismo , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/biosíntesis , Animales , Células Cultivadas , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismo , Masculino , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , ARN Mensajero/análisisRESUMEN
In juvenile African catfish (Clarias gariepinus), the pituitary LH content strongly increased after the beginning of spermatogonial proliferation. We hypothesized that a signal of testicular origin is involved in stimulating the gonadotrophs. We investigated the effects of castration and sex steroid treatment on gonadotrophs in juvenile males by quantifying LH production and release and LH subunit transcript levels and by examining gonadotroph morphology and proliferation. Castration reduced but did not abolish the maturation-associated elevation in pituitary LH content. Treatment with testosterone but not with 11-ketotestosterone, an otherwise potent androgen in fish, reversed the castration-induced decrease of pituitary LH levels. An increased pituitary LH content was accompanied by an increased number of cytologically mature gonadotrophs. However, no evidence was found for gonadotroph proliferation, so that quiescent gonadotrophs may have become activated. Although 11-ketotestosterone treatments had no effect in castrated males, this androgen attenuated gonadotroph activation in intact males. Because androgen production in juvenile catfish is downregulated by treatment with 11-ketotestosterone, its inhibitory effects on gonadotrophs in gonad-intact males may be due to suppression of Leydig cell testosterone production, which appears to be a limiting factor for the activation of catfish gonadotrophs. Aromatizable androgens may have opposite effects on fish (stimulatory) and mammalian (inhibitory) gonadotrophs.
Asunto(s)
Bagres/fisiología , Hormonas Esteroides Gonadales/farmacología , Hormona Luteinizante/biosíntesis , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Andrógenos/sangre , Andrógenos/farmacología , Animales , Estradiol/farmacología , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismo , Masculino , Orquiectomía , ARN Mensajero/análisis , Testosterona/análogos & derivados , Testosterona/farmacologíaRESUMEN
Primary pituitary cell cultures from sexually mature adult male African catfish, Clarias gariepinus, were used to study the regulation of LH biosynthesis by sex steroids. The cell cultures were exposed to testosterone (T), estradiol (E(2)), or 5alpha-dihydrotestosterone (DHT), a nonaromatizable analogue of T, and to the likewise nonaromatizable 11-ketotestosterone (KT) and 11beta-hydroxyandrostenedione (OHA), physiologically relevant androgens in fish. Both T and E(2) elevated glycoprotein alpha (GPalpha) and LHbeta steady-state mRNA levels (quantified by RNase protection assay), de novo synthesis (metabolic incorporation of radioactive amino acids and subsequent immune precipitation of LH), and release of preferentially newly synthesized LH, while DHT had no effect. Inhibiting the aromatase activity abolished the stimulatory effects of T. The effects of E(2) on LH mRNA levels and de novo synthesis were dose dependent. Incubation with 10 ng/ml KT elevated GPalpha and LHbeta mRNA levels, while other concentrations of KT or all concentrations of OHA tested had no effect. The amount of newly synthesized LH, on the other hand, was decreased dose-dependently by OHA but not by KT. Since this OHA-induced decrease did not change the specific activity (dpm immune precipitable [(3)H]-LH/ng immune-reactive LH) of LH, we hypothesize that OHA exerted its effect by activating a crinophagic breakdown of secretory granules in catfish gonadotrophs. Electron microscopic examination of gonadotrophs after in vitro exposure to 50 ng OHA/ml revealed that breakdown organelles had increased in size significantly. We conclude that the balanced production of aromatizable (mainly stimulatory) and 11-oxygenated androgens (mainly inhibitory) may be an important factor in regulating the amounts of LH available for secretion in male African catfish.
Asunto(s)
Hormonas Esteroides Gonadales/biosíntesis , Hormona Luteinizante/biosíntesis , Hipófisis/metabolismo , ARN Mensajero/biosíntesis , Androstenodiona/análogos & derivados , Androstenodiona/biosíntesis , Animales , Bagres , Células Cultivadas , Glicoproteínas/biosíntesis , Masculino , Microscopía Electrónica , Hipófisis/citología , Hipófisis/ultraestructura , Biosíntesis de Proteínas , Testosterona/análogos & derivados , Testosterona/biosíntesis , Transcripción Genética/genéticaRESUMEN
The effect of hypolipidaemic compounds on peroxisomal fatty acid beta-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial beta-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial beta-oxidation activities in intact cells. Overall beta-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate beta-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal beta-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate beta-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Clofibrato/análogos & derivados , Ácido Clofíbrico/farmacología , Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Palmitatos/metabolismo , Animales , Catalasa/metabolismo , Células Cultivadas , Clofibrato/farmacología , Activación Enzimática/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Hígado/citología , Hígado/metabolismo , Macaca fascicularis , Masculino , Microcuerpos/efectos de los fármacos , Microcuerpos/enzimología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
In order to study the interactive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated dibenzofurans (PCDFs) and retinoic acid on terminal differentiation of primary cultured keratinocytes derived from human foreskins, the amount of [(35)S]methionine-labelled proteins incorporated into cross-linked envelopes (CLEs) was determined. After isolation of the CLEs with sodium dodecyl sulfate, the amount of radioactivity collected on a filter was considered as a quantitative parameter for keratinocyte differentiation. Treatment of keratinocytes with different concentrations of TCDD resulted in a dose-dependent increase in differentiation, while only a minor decrease in differentiation occurred after treatment with retinoic acid. However, simultaneous application of 10(-8)m TCDD and different concentrations of retinoic acid led to a dose-dependent decrease in CLE formation. PCDFs exerted an effect on CLE formation similar to that of TCDD but with different potencies. We conclude that (1) TCDD induces a dose-dependent induction of human keratinocyte differentiation, (2) retinoic acid is a potent antagonist of TCDD-induced keratinocyte differentiation, and (3) primary cultures of human keratinocytes can serve as a useful model system to study the interactive effects of xenobiotics and hormone-like substances on the regulation of differentiation.