Effect of Ca 2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes

Abstract

The effect of Ca 2+ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca 2+ in the range of 0.1–0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca 2+ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fractions, incubated in the presence of initial Ca 2+ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria. Pregnenolone production from endogenous substrates in mitochondrial fractions isolated in 0.25 M sucrose from control and luteinizing hormone treated testes incubated in the absence of added succinate and Ca 2+, was maintained during 10–20 min. After longer incubation times no further steroid synthesis took place. Addition of 0.5 mM Ca 2+ to the incubation medium at time zero slightly stimulated initial pregnenolone production rates in control mitochondrial fractions, but had no effect during prolonged incubations. Addition of 0.5 mM Ca 2+ to mitochondrial fractions isolated from luteinizing hormone treated glands showed no effect either on initial production rate or during prolonged incubations. Pregnenolone production rates were maintained during 90 min in the presence of 20 mM succinate in the incubation medium. Under such conditions production rates during the first 20 min in mitochondrial fractions obtained from luteinizing hormone treated glands were approx. 3 times higher than in relevant control samples. Addition of 0.5 mM Ca 2+ to the incubation medium containing 20 mM succinate markedly stimulated initial pregnenolone production rates in control mitochondrial fractions, but gave only a small stimulation of succinate-supported production rates in luteinizing hormone treated testicular mitochondrial fractions. These results indicate that Ca 2+ in vitro can mimic the trophic effect of luteinizing hormone in vivo on mitochondrial pregnenolone production. Ageing of mitochondrial protein for 60 min at 33°C resulted in a marked increase in pregnenolone production rates in mitochondrial fractions obtained from control testes. The same treatement hardly influenced production rates in mitochondrial fractions isolated from luteinizing hormone treated testes. Ageing may have an effect on the ultrastructure of freshly prepared mitochondria, causing a change in the amount of cholesterol readily available for the enzyme complex. The gluco- and mucoprotein specific agent Ruthenium red (50–2000 ng/ml) did not inhibit pregnenolone production in either control or hormone treated testicular mitochondrial fractions, incubated in the absence of added Ca 2+. the presence of 200–2000 ng Ruthenium red per ml incubation mixture. The present results have been discussed in relation to the possible involvement of Ca 2+ in the molecular mechanism of short-term action of luteinizing hormone on testicular androgen production.