scholarly journals HISTOCHEMICAL DEMONSTRATION OF 3α-HYDROXYSTEROID DEHYDROGENASE ACTIVITY

1966 ◽  
Vol 14 (1) ◽  
pp. 77-83 ◽  
Author(s):  
KÁROLY BALOGH
Keyword(s):  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Histochemical Demonstration ◽  
Female Rats ◽  
Ventral Prostate ◽  
Tetrazolium Salt ◽  
Rat Tissues ◽  
Final Electron ◽  
Nucleotide Specificity ◽  
Final Electron Acceptor

The reversible oxidation of 3α-hydroxysteroids to their corresponding 3-keto forms comprises an important step in the metabolism of C19-steroids. The described techniquue demonstrates the activity of the enzyme catalyzing this reaction, with the use of androsterone as a substrate and a tetrazolium salt as the final electron acceptor. The enzyme is specific for 3α-hydroxysteroids; there was no histochemical reaction with epiandrosterone, the β isomer of androsterone. Since 3α-hydroxysteroid dehydrogenase is soluble in aqueous solutions, it was necessary to increase the osmolarity of the incubation medium by adding polyvinylpyrrolidone in a final concentration of 20%. Although the enzyme has a dual nucleotide specificity, no appreciable differences were seen in its distribution pattern in rat tissues with either NAD or NADP as a coenzyme. In adult female rats, enzyme activity was present in the liver, kidneys and clitoral glands. In mature males, diformazan deposits were observed in the liver, kidneys, preputiai glands, epididymis, ventral prostate and Leydig cells.

HISTOCHEMICAL DEMONSTRATION OF 20β-HYDROXYSTEROID DEHYDROGENASE

1965 ◽  
Vol 32 (3) ◽  
pp. 337-339 ◽  
Author(s):  
A. H. BAILLIE ◽  
K. C. CALMAN ◽  
M. M. FERGUSON ◽  
D. McK. HART
Keyword(s):  
Dehydrogenase Activity ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Histochemical Demonstration ◽  
Mouse Testis ◽  
Zona Fasciculata ◽  
Mouse Ovary ◽  
Theca Interna ◽  
Human And Mouse

SUMMARY NAD-dependent 20β-hydroxysteroid dehydrogenase activity can be demonstrated histochemically using Nitro-BT. 20β-Hydroxysteroid dehydrogenase activity was found in the Leydig cells of human and mouse testis, in the zona fasciculata of the mouse adrenal and in the theca interna of the mouse ovary.

New Methods for the Ultrastructural Demonstration of Oxidoreductase Enzymes

1974 ◽  
Vol 32 ◽  
pp. 218-219
Author(s):  
W. Allen Shannon ◽  
Yoshinobu Hoshino ◽  
Arnold M. Seligman
Keyword(s):  
Ultrastructural Localization ◽  
Hydroxysteroid Dehydrogenase ◽  
Tetrazolium Salt ◽  
Rat Tissues ◽  
Tetrazolium Chloride ◽  
New Methods ◽  
Oxidoreductase Enzymes

The ultrastructural localization of 3-β-hydroxysteroid dehydrogenase (3βHS-DH), β-hydroxybutyrate dehydrogenase (βHB-DH) and α-hydroxybutyrate oxidase (αHB-O) is demonstrated in various rat tissues via nonosmiophilic tetrazolium salt, 2-(2’-benzothiazolyl)-5-styryl-3-(4’-phthalhydrazidyl) tetrazolium chloride (BSPT)(1), reduction to osmiophilic formazan (Fig. 1).

scholarly journals Hexose-6-Phosphate Dehydrogenase and 11β-Hydroxysteroid Dehydrogenase-1 Tissue Distribution in the Rat

Endocrinology ◽  
2007 ◽  
Vol 149 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Elise P. Gomez-Sanchez ◽  
Damian G. Romero ◽  
Angela F. de Rodriguez ◽  
Mary P. Warden ◽  
Zygmunt Krozowski ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Dehydrogenase Activity ◽  
Leydig Cells ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Hydroxysteroid Dehydrogenase ◽  
Interstitial Cells ◽  
Rat Tissues ◽  
Rt Pcr ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
The Brain

Intracellular concentrations of the glucocorticoids cortisol and corticosterone are modulated by the enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD) 1 and 2. 11β-HSD1 is a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal reductase that converts the inactive glucocorticoids cortisone and 11-dehydrocorticosterone to their active forms, cortisol and corticosterone. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme that generates NADPH from oxidized NADP (NADP+) within the endoplasmic reticulum. In the absence of NADPH or H6PDH to regenerate NADPH, 11β-HSD1 acts as a dehydrogenase and inactivates glucocorticoids, as does 11β-HSD2. A monoclonal antibody against H6PDH was produced to study the possibility that 11β-HSD1 in the absence of H6PDH may be responsible for hydroxysteroid dehydrogenase activity in tissues that do not express significant amounts of 11β-HSD2. H6PDH and 11β-HSD1 expression was surveyed in a variety of rat tissues by real-time RT-PCR, Western blot analysis, and immunohistochemistry. H6PDH was found in a wide variety of tissues, with the greatest concentrations in the liver, kidney, and Leydig cells. Although the brain as a whole did not express significant amounts of H6PDH, some neurons were clearly immunoreactive by immunohistochemistry. H6PDH was amply expressed in most tissues examined in which 11β-HSD1 was also expressed, with the notable exception of the renal interstitial cells, in which dehydrogenase activity by 11β-HSD1 probably moderates activation of the glucocorticoid receptor because rat renal interstitial cells do not have significant amounts of mineralocorticoid receptors. This antibody against the H6PDH should prove useful for further studies of enzyme activity requiring NADPH generation within the endoplasmic reticulum.

HISTOCHEMICAL DEMONSTRATION OF 11β-HYDROXYSTEROID DEHYDROGENASE

1965 ◽  
Vol 33 (1) ◽  
pp. 119-125 ◽  
Author(s):  
A. H. BAILLIE ◽  
M. M. FERGUSON ◽  
K. C. CALMAN ◽  
D. McK. HART
Keyword(s):  
Dehydrogenase Activity ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Histochemical Demonstration ◽  
Postnatal Life ◽  
Interstitial Tissue ◽  
Corpora Lutea ◽  
Histochemical Reaction ◽  
Mouse Ovary ◽  
Theca Interna

SUMMARY 11β-Hydroxysteroid dehydrogenase can be demonstrated histochemically by incubating tissues with nitro blue tetrazolium (2,2′-di-p-nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-diphenylene) ditetrazolium chloride), NAD or NADP and an appropriate steroid. Suitable steroid substrates are: (1) 11β-hydroxyandrost-4-ene-3,17-dione (11β-hydroxyandrostenedione), (2) 3,11β-dihydroxyoestra-1,3,5(10)-trien-17-one (11β-hydroxyoestrone), (3) 3α,11β-dihydroxy-5α-androstan-17-one, (4) 3α,11β-dihydroxy-5β-androstan-17-one and (5) 11β-hydroxypregn-4-ene-3,20-dione(11β-hydroxyprogesterone). 11β-Hydroxysteroid dehydrogenase activity was found in the Leydig cells of six human testes from subjects ranging in age from 12 to 57 yr. with all five substrates. The Leydig cells of the mouse testis contain demonstrable 11β-hydroxysteroid dehydrogenase activity and the volume of reactive tissue increases regularly between birth and the end of the 10th week of postnatal life; this growth curve is sigmoid in form. An extremely weak histochemical reaction with human placenta obtained at term was observed, 11β-hydroxyandrostenedione being the only substrate utilized to any extent. A specimen of hydatid mole, however, showed intense 11β-hydroxysteroid dehydrogenase activity with all substrates surveyed. 11β-Hydroxysteroid dehydrogenase was also found in the ova, granulosa, theca interna, interstitial tissue and corpora lutea of the mouse ovary.

FURTHER OBSERVATIONS ON 3β-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN THE MOUSE LEYDIG CELL

1965 ◽  
Vol 31 (3) ◽  
pp. 207-NP ◽  
Author(s):  
A. H. BAILLIE ◽  
K. GRIFFITHS
Keyword(s):  
Dehydrogenase Activity ◽  
Growth Curve ◽  
Leydig Cells ◽  
Age Groups ◽  
Growth Curves ◽  
Hydroxysteroid Dehydrogenase ◽  
Histochemical Demonstration ◽  
Seminiferous Tubules ◽  
Postnatal Life ◽  
Histochemical Reaction

SUMMARY One hundred and ten male Swiss white mice were killed in batches of ten weekly between birth and the end of the 10th week of postnatal life. To demonstrate 3β-hydroxysteroid dehydrogenase activity histochemically, sections of testis from every animal were incubated with the following steroid substrates: (1) sodium 3β-sulphoxypregn-5-en-20-one (pregnenolone sulphate), (2) sodium 3β-sulphoxy-17α-hydroxypregn-5-en-20-one (17α-hydroxypregnenolone sulphate), (3) sodium 3β-sulphoxyandrost-5-en-17-one (DHA sulphate), (4) 3β,16α-dihydroxypregn-5-en-20-one (16α-hydroxypregnenolone), (5) pregn-5-ene-3β,20β-diol (pregnenediol), (6) androst-5-ene-3β, 17β-diol (androstenediol). Pregnenolone sulphate was rapidly used by the entire interstitium at all ages. 17α-Hydroxypregnenolone sulphate was metabolized by some Leydig cells of all age groups. DHA sulphate was not utilized histochemically by the Leydig cells of the various age groups, but formazan deposition occurred in the mature seminiferous epithelium. This is the only steroid so far investigated to give an histochemical reaction with the germinal epithelium, and 3β-hydroxysteroid dehydrogenase activity has not previously been described in the seminiferous tubules. The utilization of steroid sulphates differently from the free steroids in the histochemical demonstration of 3β-hydroxysteroid dehydrogenase activity suggests that the presence of a sulphate group may affect enzyme-substrate binding. With 16α-hydroxypregnenolone and pregnenediol as substrates, 3β-hydroxysteroid dehydrogenase activity was demonstrable at birth, increased progressively until the 6th week of postnatal life, and subsequently decreased during the ensuing 4 weeks. This growth curve closely resembles the growth curve obtained with pregnenolone. Androstenediol gave a histochemical reaction with the Leydig cells of all age groups studied, and the sigmoid growth curve resembles that obtained with 3β-hydroxyandrost-5-en-17-one (DHA). These differing growth curves are regarded as further evidence of substrate-specific 3β-hydroxysteroid dehydrogenases.

scholarly journals The Cytochemical Localization of Oxidative Enzymes

1958 ◽  
Vol 4 (6) ◽  
pp. 753-760 ◽  
Author(s):  
R. Hess ◽  
D. G. Scarpelli ◽  
A. G. E. Pearse
Keyword(s):  
Oxidative Enzymes ◽  
Tetrazolium Salt ◽  
Differential Centrifugation ◽  
Mitochondrial Localization ◽  
Cytochemical Localization ◽  
Tissue Sections ◽  
Final Electron ◽  
High Concentrations ◽  
Distinct Distribution ◽  
Final Electron Acceptor

Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.

HISTOCHEMICAL UTILIZATION OF 3α-, 6β-, 11α-, 12α-, 16α-, 16β-, 17α-, 21- AND 24-HYDROXYSTEROIDS

1966 ◽  
Vol 34 (1) ◽  
pp. 1-NP ◽  
Author(s):  
A. H. BAILLIE ◽  
K. C. CALMAN ◽  
M. M. FERGUSON ◽  
D. McK. HART
Keyword(s):  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Pyridine Nucleotide ◽  
Tetrazolium Salt ◽  
Interstitial Tissue ◽  
Corpora Lutea ◽  
Zona Fasciculata ◽  
Mouse Ovary ◽  
Hydroxysteroid Dehydrogenases ◽  
Theca Interna

SUMMARY The histochemical utilization of 3α-, 6β-, 11α-, 12α-, 16α-, 16β-, 17α-, 21- and 24-hydroxysteroids in human and mouse testis, human placenta, mouse ovary and rat adrenal has been investigated using a coupling method and the tetrazolium salt, Nitro-BT. 3α-Hydroxysteroid dehydrogenase was present in the human Leydig cells and placental syntrophoblast, but there was little in rat adrenal zona fasciculata and in mouse ovary; the enzyme is NAD or NADP dependent. 6β-Hydroxysteroid dehydrogenase was present in human Leydig cells, mouse Leydig cells, placental syntrophoblast, ova, granulosa, theca interna, corpora lutea and interstitial tissue; it is NAD dependent. 11α-Hydroxysteroid dehydrogenase activity was very poorly developed, being NAD dependent and demonstrable only in human Leydig cells. 12α-Hydroxysteroid dehydrogenase could be demonstrated in some human Leydig cells; it was both NAD and NADP dependent. 16α-Hydroxysteroids were very poorly used by all the tissues surveyed. 16β-Hydroxysteroids gave an intense histochemical reaction with NAD in human Leydig and Sertoli cells, in placental trophoblast, in adrenal zonae glomerulosa, fasciculata and reticularis and in all ovarian tissues. 17α-, 21- and 24-hydroxysteroids were poorly utilized by human Leydig cells, but not by the other tissues. The first two were NAD dependent; 24-hydroxysteroid utilization was both NAD and NADP dependent. The techniques used are believed to demonstrate specific hydroxysteroid dehydrogenases because of variations in pyridine nucleotide requirement and in the location in the tissues of different hydroxysteroid dehydrogenases. Moreover, stereoisomers of the same hydroxysteroid behave differently in this system. The role of steroid 5α- and 5β-dehydrogenases is discussed in connexion with the histochemical results.

Aromatase and 11β-hydroxysteroid dehydrogenase 2 localisation in the testes of pigs from birth to puberty linked to changes of hormone pattern and testicular morphology

2008 ◽  
Vol 20 (4) ◽  
pp. 505 ◽  
Author(s):  
A. Wagner ◽  
R. Claus
Keyword(s):  
Blood Plasma ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Blood Collection ◽  
Testicular Development ◽  
Post Mortem ◽  
Cell Numbers ◽  
Testicular Morphology

Oestrogens and glucocorticoids are important for spermatogenesis and are regulated via aromatase for oestradiol synthesis and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD 2) as an inactivator of cortisol. In the present study postnatal changes of these two enzymes were monitored together with testicular development and hormone concentrations. Pigs were assigned to three periods: Weeks 0–5, Weeks 5–11 or Weeks 11–17. In Period 1, groups of four piglets were killed after each week. Blood plasma and testes were sampled immediately post mortem. For Periods 2 and 3, groups of six pigs were fitted with vein catheters for daily blood collection. Testes from all pigs were obtained after killing. Levels of testosterone, oestradiol, LH, FSH and cortisol were determined radioimmunologically. The 11β-HSD 2- and aromatase-expressing cells were stained immunocytochemically. All hormones were maximal 2 weeks after birth. A rise of LH, testosterone and oestradiol occurred again at Week 17. FSH and cortisol remained basal. Parallel to the first postnatal rise, the presence of aromatase and 11β-HSD 2 in Leydig cells increased, together with germ and Sertoli cell numbers. Expression was low from 3 to 5 weeks, was resumed after Week 5 and was maximal at Week 17. The amount of 11β-HSD 2 in germ cells was greatest at birth, decreased thereafter and was absent after Week 3.

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