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The effect of lutropin on specific protein synthesis in tumour Leydig cells and in Leydig cells from immature rats.

ABSTRACT: The amount of (35)S incorporated into the various proteins after separation by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels was used as an estimate of their synthesis in the Leydig cells. Increased synthesis of proteins with apparent mol.wts. 27000 and 29000 was observed 3h after addition of lutropin to tumour Leydig cells. Incubation of Leydig cells from immature rats with lutropin (100ng/ml) for 2h or longer resulted in increased synthesis of proteins with apparent mol.wts. 11000, 21000, 27000 and 29000. At higher concentrations (>/=100ng/ml) of lutropin there was a decrease in the synthesis of a protein with apparent mol.wt. 13000. The amount of lutropin required for the stimulation of protein synthesis in both types of Leydig cells was similar to that needed for stimulation of steroidogenesis. Lutropin-stimulated specific protein synthesis was not due to increased concentrations of testosterone, however, because (1) addition of testosterone to the cells had no effect on the synthesis of the proteins, and (2) inhibition of steroidogenesis with elipten phosphate (an inhibitor of the cholesterol side-chain-cleavage enzyme complex) did not abolish the effect of lutropin. The stimulation of specific protein synthesis was also not due to contaminating follitropin in the lutropin preparation. Addition of actinomycin D to the cells at the start of the incubation prevented the effect of lutropin on specific protein synthesis, indicating that mRNA synthesis may be needed for this effect of lutropin. Incubation of the cells with cycloheximide for 30min after labelling of the proteins did not result in a detectable decrease in the amounts of the lutropin-induced proteins, indicating that their half-life is longer than 30min.

SUBMITTER: Janszen FH

PROVIDER: S-EPMC1185673 | biostudies-other | 1978 Apr

REPOSITORIES: biostudies-other

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Project description:Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED(50) (dose that produces a response that is 50% of the maximum response) 60+/-5.7ng/ml and 8+/-1.8ng/ml (mean+/-s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/10(6) cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific (125)I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4 degrees C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5'-[beta,gamma-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50-60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

Project description:Background: Propofol is a widely used anesthetic. Whether propofol inhibits androgen production by rat Leydig cells and the underlying mechanism remains unclear. The objective of the current study was to examine the effects of propofol exposure to rat primary immature Leydig cells and to define propofol-induced inhibition of steroidogenic enzymes in both rat and human testes in vitro. Methods: Immature Leydig cells were purified from 35-day-old male Sprague-Dawley rats and were exposed to propofol for 3 h. The androgen production by Leydig cells under basal, luteinizing hormone, 8bromo-cAMP, and steroid-substrate stimulated conditions and gene expression of Leydig cells after exposure to propofol were measured. Immature Leydig cells were treated with propofol for 3 h and switched to propofol-free medium for additional 3 and 9 h to test whether propofol-induced inhibition is reversible. 3H-Steroids were used to evaluate the direct action of propofol on cytochrome P450 cholesterol side chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (HSD3B), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), and 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) activities in rat and human testes in vitro. Results: Propofol significantly lowered luteinizing hormone and 8bromo-cAMP stimulated androgen production by Leydig cells after 3-h exposure. Further investigation showed that propofol down-regulated the expression of Cyp11a1 and Cyp17a1 and their proteins at 5 and 50 µM, although it up-regulated Lhcgr expression at 50 µM. Propofol significantly suppressed phosphorylation of ERK1/2 and induced ROS production in immature Leydig cells at 5 and 50 µM. Propofol significantly induced apoptosis of immature Leydig cells at 50 µM. Propofol specifically inhibited rat and human testis HSD3B activities in vitro. The half maximal inhibitory concentrations of propofol for rat and human HSD3B enzymes were 1.011 ± 0.065 and 3.498 ± 0.067 µM, respectively. The mode of action of propofol of inhibiting HSD3B was competitive when pregnenolone was added. At 50 µM, propofol did not directly inhibit rat and human testis CYP11A1, CYP17A1, and HSD17B3 activities in vitro. Conclusion: Propofol inhibits androgen production via both directly inhibiting HSD3B activity and down-regulating Cyp11a1 and Cyp17a1 expression in Leydig cells. Suppression of steroidogenic enzymes is presumably associated with the lower production of androgen by Leydig cells after propofol treatment. However, propofol-induced inhibition on androgen production is reversible.

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The effect of lutropin on specific protein synthesis in tumour Leydig cells and in Leydig cells from immature rats.

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