A quantitative cytochemical method for the demonstration of ?5,3?-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary

Abstract

A method for the quantitative measurement of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD+ or NADP+ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP+ dependence is not abolished by inhibiting nonspecific alkaline phomonoesterase. The activity of delta5,3beta-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10(-4) M.

Ultrastructural and histochemical study on the interrenal cells of the male stickleback (Gasterosteus aculeatus, Teleostea), in relation to the reproductive annual cycle

“…To evaluate the steroidogenic capacity of testicular and interrenal tissues, we used histochemical tests for the 3β-and 17β-hydroxysteroid dehydrogenases (3βHSD and 17βHSD) on sections of frozen organs according to the technique of Baillie et al (1966). The incubation medium was prepared as described by Erpino (1971) and Robertson (1979) and contained : 2 ml of phosphate buffer (Millonig) 0n1  pH 7n2, 6 mg of NAD (nicotinamide adenine dinucleotide, Sigma or Fluka), 4 mg of NBT (nitro blue tetrazolium salt, Fluka), 10 mg of EDTA (ethylene diamino tetra acetic acid, Sigma), 2n5 ml 50 % of solution of PVP (polyvinyl pyrrolidone, Merck) in phosphate buffer. For the demonstration of the 3βHSD the substrate was : 2 mg of etiocholan-3β-ol-17-one (Sigma or Aldrich) in 0n2 ml of dimethyl formamide (DMF), Merck).…”

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: Modulation by calcium

“…The percentage of con¬ taminating peritubular myoid cells at this time as assessed by fibronectin synthesis (12), was between 2-5%. In ad¬ dition, there were no Leydig cells present as assessed by the absence of A5, 3ß hydroxysteroid dehydrogenase ac¬ tivity (13) and by the absence of testosterone secretion (<10 nmol/1) after 24 h stimulation of cells by LH (20 IU/1) in the absence of 19-hydroxyandrostenedione.…”

Reaction rate measurements of proteases and glycosidases with chromogenic methods

“…It is suitable, and superior to biochemical and microchemical methods, for the measurement of enzyme activities in subpopulations of skeletal muscle fibres (Nolte & Pette, 1972;Reichmann & Pette, 1982), hepatocytes in different zones of the liver acinus Frederiks et al, 1986), corpus luteum cells (Robertson, 1979) or osteoclasts (Webber et al, 1988; for further references see Altman, 1972;van Noorden & Butcher, 1989) and also in the decidua, which consists of an antimesometrial, mesometrial and mesometrial cell population (Graf & Gossrau, 1985).…”