G.J. Van Der Vusse

Assessment of fatty acids in dog left ventricular myocardium

The concentration and composition of fatty acids in four lipid classes in biopsies of dog left ventricular myocardium were determined, using gas-liquid chromatography. When precautions were taken to minimize lipolysis during storage of the tissue and the homogenization process, the following results were obtained: 29 +/- 10 nmol non-esterified fatty acids, 2.98 +/- 2.41 mumol triacylglycerol, 149 +/- 51 nmol cholesteryl esters and 23.76 +/- 3.38 mumol phospholipid (expressed as fatty acid moiety per gram of wet tissue). The concentration of non-esterified fatty acids was 15 to 300 times lower than reported in literature. The main constituents of the non-esterified fatty acids were palmitic, stearic and oleic acid. Triacylglycerol consisted of approximately 40% esterified oleic acid. Linoleic acid accounted for 40% of the fatty acids in the cholesteryl-esters class. More than half of the fatty acid moiety of total phospholipids was linoleic acid and arachidonic acid.

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.

Endogenous production of steroids by subcellular fractions from total rat testis and from isolated interstitial tissue and seminiferous tubules

Abstract The endogenous production of pregnenolone and testosterone in subcellular fractions of rat testis tissue was estimated. A medium containing cofactors was used and the incubations were carried out for 120 min at 33 °C. Only mitochondrial fractions produced the estimated steroids. Seminiferous tubules and interstitial tissue were isolated from rat testis tissue using a wet dissection technique. Mitochondrial fractions were isolated from the homogenized isolated tissue compartments and production of pregnenolone and testosterone after incubation of these fractions was estimated. The interstitial mitochondrial fraction produced 1200–2600 pmoles per mg mitochondrial protein per 2 h which was at least 60 times higher than the production in similar fractions obtained from seminiferous tubules. Production in the mitochondrial fraction of whole testis tissue was 300–600 pmoles per mg protein per 2 h.

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes

The effect of Ca2+ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca2+ 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, Ca2+ 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 fraction, incubated in the presence of initial Ca2+ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.

Distribution of Steroids, Steroid Production and Steroid Metabolizing Enzymes in Rat Testis

In the present paper some results are presented on: 1. the occurrence and age dependence of some enzyme activities in whole rat testis tissue as related to the development of spermatogenesis; 2. enzymes for steroid metabolism in isolated dissected rat testis tissue fractions with special emphasis on the age dependent variations of steroid 5α-reductase activity, and; 3. concentrations and production of steroids in isolated cellular and subcellular fractions of rat testis. These data are presented mainly to illustrate the limitation of results on enzyme activities, steroid concentrations and steroid production in total testis tissue and in isolated tissue fractions for the understanding of the regulation of specific processes in particular cell types, such as the regulation of spermatogenesis.

Dynamics of steroid uptake in rat testis studied by quantitative autoradiography

Localization of radioactive steroids in rat testis was studied by autoradiography of tissue section. For autoradiography small tissue samples were frozen, freeze-dried under vacuum, fixed with osmium vapor and embedded in epon. The transfer of radioactive steroids was studied after in vitro perfusion of radioactive steroids in the testes isolated from hypophysectomized animals, thereby excluding the interference of endogenous steroids. Quantitative autoradiography on the basis of grain densities after perfusion of testes with tritiated pregnenolone or testosterone, revealed an accumulation of the label in the Leydig cell cytoplasm. After longer perfusion periods the amount of label in the seminiferous tubules increased and a preferential localization was observed in the basal cytoplasm of Sertoli cells and in lipid droplets. Perfusion of testes with estradiol-17 beta resulted in a distinctly different pattern of radioactivity in the autoradiographs. A high labeling of the Leydig cell nuclei was observed in combination with a low general labeling of all the other cell structures. The results suggest that different steroids are localized in different specific areas of the rat testes in vivo.

Endogenous steroid production in preparations of rat testis after long-term treatment with HCG

Abstract The effect of prolonged treatment with Human Chorionic Gonadotrophin (HCG) on endogenous steroid production in homogenates and mitochondrial fractions of rat testis tissues was investigated. Following HCG treatment (5 days, 100i.u.s.c. per day) testosterone production in homogenates of whole testis tissue and of isolated interstitital tissue was increased by a factor of 2.5 and 2.4 respectively. In the presence of cyanoketone mitochondrial fractions obtained from whole testis tissue and isolated interstitial tissue of HCG treated rats produced respectively 2.4 times and 3.3 times more pregnenolone than comparable mitochondrial preparations obtained from control testes. The production of steroids from endogenous precursors in homogenates and mitochondrial fractions of isolated seminiferous tubules was very low and contributed very little to steroid production in preparations of whole testis tissue. After HCG treatment the activity of 3β-hydroxysteroid-dehydrogenase was increased in whole testis tissue (from 140 to 320 μU/mg), in isolated interstitial tissue (from 1030 to 2200 μU/mg) as well as in isolated seminiferous tubules (from 8 to 20 μU/mg). These findings indicate that the interstitial compartment is the main site of steroid production in rat testis and that enhanced testicular steroid production after treatment with HCG is caused by an activation of the steroid production in the interstitial tissue.

Biochemical Functions of Isolated Interstitial Tissue and Seminiferous Tubules from Rat Testis

Publisher Summary This chapter discusses the separation and the characterization of interstitial tissue and seminiferous tubules from whole rat testis tissue. The esterase activities in seminiferous tubules and interstitial tissue reflect the sum of activities of different isoenzymes. The concentrations of testosterone have been measured during in vitro incubation of these tissues. Low concentrations of testosterone were found in the seminiferous tubules and no evidence was obtained for the synthesis of testosterone in this tissue in vitro. Purified mitochondria of isolated interstitial tissue produce at least 60 times as much steroids—pmol pregnenolone + testosterone per mg protein/2 h—as mitochondria of seminiferous tubules. In a total rat testis, mitochondria of interstitial tissue can produce at least 10 times as much steroid (pregnenolone + testosterone) as mitochondria of seminiferous tubules. Testis tissue of mature and immature rats contains a specific cytoplasmic receptor for estradiol with a sedimentation constant of 8S on a sucrose gradient and an affinity constant of 1010 (M−1). This receptor can be labeled with estradiol both after in vivo and after in vitro incubation. The receptor is localized in the interstitial cells of the testis.