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.

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.

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.

3β-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN THE MOUSE OVARY

1965 ◽  
Vol 32 (3) ◽  
pp. 365-371 ◽  
Author(s):  
M. M. FERGUSON
Keyword(s):  
Dehydrogenase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
The Other ◽  
Interstitial Tissue ◽  
Corpora Lutea ◽  
Mouse Ovary ◽  
Colour Reaction ◽  
Theca Interna

SUMMARY Sections of ovaries from 30 Swiss white mice were incubated with ten steroid substrates to demonstrate 3β-hydroxysteroid dehydrogenase activity histochemically. The substrates were: (1) 3β-hydroxypregn-5-en-20-one (pregnenolone), (2) 3β,17α-dihydroxypregn-5-en-20-one (17α-hydroxypregnenolone), (3) 3β-hydroxyandrost-5-en-17-one (DHA), (4) 3β,17β-dihydroxyandrost-5-ene (androstenediol), (5) 3β-sulphoxypregn-5-en-20-one (pregnenolone sulphate), (6) 3β-sulphoxy-17α-hydroxypregn-5-en-20-one (17α-hydroxypregnenolone sulphate), (7) 3β-sulphoxyandrost-5-en-17-one (DHA sulphate), (8) 3β-acetoxypregn-5-en-20-one (pregnenolone acetate), (9) 3β-acetoxyandrost-5-en-17-one (DHA acetate), and (10) 3β-acetoxy-17β-hydroxyandrost-5-ene (androstenediol acetate). Pregnenolone, 17α-hydroxypregnenolone, DHA and androstenediol gave a colour reaction in the corpora lutea, interstitial tissue, theca interna and stroma of all ovaries examined. The granulosa of many follicles, some thought to be atretic, also contained diformazan granules. 17α-Hydroxypregnenolone did not give as intense a reaction as the other free steroids. No diformazan was deposited with DHA sulphate as substrate. Pregnenolone sulphate and 17α-hydroxypregnenolone sulphate were used by the same tissues as were the free steroids, although they were much less well utilized. Utilization of 3β-acetoxy derivatives was similar to that of the free steroids.

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.

scholarly journals Species differences in the ovarian distribution of 3beta-hydroxysteroid dehydrogenase/delta5-->4 isomerase (3beta-HSD) in two marsupials: the brushtail possum Trichosurus vulpecula and the grey, short-tailed opossum Monodelphis domestica

Reproduction ◽  
2003 ◽  
pp. 65-73 ◽  
Author(s):  
SL Ullmann ◽  
AJ Russell ◽  
JI Mason ◽  
L Selwood
Keyword(s):  
Follicular Development ◽  
Hydroxysteroid Dehydrogenase ◽  
Monodelphis Domestica ◽  
Brushtail Possum ◽  
Interstitial Tissue ◽  
South American ◽  
Trichosurus Vulpecula ◽  
Strong Positive Reaction ◽  
Rabbit Polyclonal Antibody ◽  
Theca Interna

The ovarian distribution of the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase/delta(5-->4) isomerase (3beta-HSD) was investigated by immunocytochemistry in two marsupial species throughout the reproductive cycle, using a rabbit polyclonal antibody raised against human placental 3beta-HSD. In the polyoestrous and polyovular South American opossum Monodelphis domestica, immunostaining was positive for 3beta-HSD in the adrenal cortex, the ovarian interstitial tissue, the corpus luteum and the granulosa cells of antral and atretic follicles. The theca interna was weakly positive for 3beta-HSD, but only in late preantral to early antral stages of follicular development. The adrenal medulla and smaller preantral follicles were completely negative for 3beta-HSD. In contrast, in the polyoestrous and monovular Australian brushtail possum Trichosurus vulpecula, immunostaining showed a strong positive reaction for 3beta-HSD in the theca, whereas the granulosa layer remained predominantly negative for 3beta-HSD except in the largest follicles. The atretic follicles were completely negative for 3beta-HSD. The ovaries of pregnant animals contained grossly enlarged, persistent, antral follicles, which reacted positively for 3beta-HSD. The function of these follicles in T. vulpecula and the 3beta-HSD-positive atretic follicles in M. domestica has not been determined. The differences between the two marsupials represent species variations. The situation in M. domestica does not represent a marsupial-eutherian dichotomy as previously conjectured.

scholarly journals Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads

2001 ◽  
Vol 171 (2) ◽  
pp. 373-383 ◽  
Author(s):  
G Pelletier ◽  
S Li ◽  
V Luu-The ◽  
Y Tremblay ◽  
A Belanger ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Steroid Hormones ◽  
Adrenal Cortex ◽  
Leydig Cells ◽  
Ultrastructural Localization ◽  
Hydroxysteroid Dehydrogenase ◽  
Cytochrome P450 Enzymes ◽  
Steroidogenic Enzymes ◽  
Adrenocortical Cells ◽  
Theca Interna

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

scholarly journals Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

1992 ◽  
Vol 40 (7) ◽  
pp. 903-908 ◽  
Author(s):  
T Suzuki ◽  
H Sasano ◽  
T Sawai ◽  
J I Mason ◽  
H Nagura
Keyword(s):  
In Situ Hybridization ◽  
Leydig Cells ◽  
Cellular Localization ◽  
Cdna Probe ◽  
Simultaneous Detection ◽  
Seminiferous Tubules ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Theca Interna

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.

HYDROXYSTEROID DEHYDROGENASES IN THE HUMAN ADRENAL CORTEX

1966 ◽  
Vol 34 (4) ◽  
pp. 439-NP ◽  
Author(s):  
K. C. CALMAN ◽  
A. H. BAILLIE ◽  
M. M. FERGUSON ◽  
D. McK. HART
Keyword(s):  
Dehydrogenase Activity ◽  
Cushing’S Syndrome ◽  
Adrenal Glands ◽  
Adrenal Adenoma ◽  
Hydroxysteroid Dehydrogenase ◽  
Cushing's Syndrome ◽  
Normal Adult ◽  
Zona Fasciculata ◽  
Adult Human ◽  
Hydroxysteroid Dehydrogenases

SUMMARY The histochemical utilization of 3α-, 3β-, 6β-, 11α-, 11β-, 12α-, 16α-, 16β-, 17α-, 17β-, 20α-, 21- and 24-hydroxysteroids by three normal adult human adrenal glands, two human foetal adrenal glands, three adrenals from patients with Cushing's syndrome and one adrenal adenoma are described. The normal adult human adrenal showed high 16β-hydroxysteroid dehydrogenase activity in the zona glomerulosa. Activity restricted to the outer part of the zona fasciculata was recorded with 3α-, 3β-, 6β-, 11β-, 16α-, 16β-, and 17β-hydroxysteroids. The zona reticularis utilized 3α-, 3β-, 11β-, 16β- and 17β-hydroxysteroids less well than the zona fasciculata. The adrenals of Cushing's syndrome showed activity only for 3β- and 16β-hydroxysteroid dehydrogenases; this activity was noted in all three zones. The activity pattern of the adrenal adenoma resembled that of the normal adult human adrenal except that greater activity for 16α-hydroxysteroid dehydrogenase was noted. The foetal part of the human foetal cortex was extremely active, showing 3α-, 3β-, 6β-, 11β-, 12α-, 16α-, 16β-, 17β-, 20β- and 21-hydroxysteroid dehydrogenase activity. The definitive cortex behaved similarly to the adult gland and possessed 3α-, 3β-, 11β-, 16β- and 17β-hydroxysteroid dehydrogenases; some evidence of zoning of the definitive cortex was seen with the 16β-hydroxysteroid. The relevance of these findings in the light of current knowledge of adrenal zonation is discussed.

Ovarian steroid metabolism and oestrogens in the corpus luteum of the tammar wallaby

1984 ◽  
Vol 101 (2) ◽  
pp. 231-NP ◽  
Author(s):  
M. B. Renfree ◽  
A. P. F. Flint ◽  
S. W. Green ◽  
R. B. Heap
Keyword(s):  
Corpus Luteum ◽  
Follicular Development ◽  
Hydroxysteroid Dehydrogenase ◽  
Tammar Wallaby ◽  
Steroid Metabolism ◽  
Interstitial Tissue ◽  
Corpora Lutea ◽  
Embryonic Diapause ◽  
Probable Source

ABSTRACT Ovaries were obtained from tammar wallabies at various stages of the reproductive cycle to examine the occurrence of oestrogens in corpora lutea, and the synthesis and metabolism of steroids in the corpus luteum and ovarian cortical and interstitial tissues. Corpora lutea contained oestradiol-17β and oestrone during embryonic diapause and at all stages of pregnancy studied after blastocyst activation. Aryl sulphatase, 3β-hydroxysteroid dehydrogenase and 17β-oxidoreductase were shown to be present in luteal and other ovarian tissues by incubation in vitro with labelled substrates. Aromatase was undetectable in corpora lutea or in interstitial tissue, but was present in the ovarian tissues (including follicles) which remained after removal of corpora lutea. The probable source of the oestrogens detected in the corpus luteum is discussed in relation to their role in the inhibition of follicular development during embryonic diapause. J. Endocr. (1984) 101, 231–240

BIOTRANSFORMATION OF PREGNENOLONE AND PROGESTERONE BY RABBIT OVARIAN FOLLICLES AND CORPORA LUTEA

1973 ◽  
Vol 74 (4) ◽  
pp. 775-782 ◽  
Author(s):  
Edward V. YoungLai
Keyword(s):  
Granulosa Cells ◽  
Salt Solution ◽  
Hydroxysteroid Dehydrogenase ◽  
Ovarian Follicles ◽  
Rabbit Serum ◽  
Corpora Lutea ◽  
Normal Rabbit Serum ◽  
Theca Cells ◽  
Theca Interna

ABSTRACT Rabbit ovarian follicles obtained prior to and after mating and corpora lutea (24 h and 48 h post-coitus) were incubated with radioactive pregnenolone and progesterone to determine whether these substrates could serve as precursors of androgens and oestrogens. Incubations were carried out for 3 h in Hanks balanced salt solution: medium 199: normal rabbit serum: 55:30:15. Granulosa cells and corpora lutea converted pregnenolone to progesterone but no labelled oestrogens could be detected. Trace amounts of androgens were synthesized by the granulosa cells. Whole sliced follicles and theca formed progesterone from pregnenolone and androgens from both substrates. Oestradiol-17β was only synthesized by the whole sliced follicles and in one experiment by theca cells. Mating caused an increase in the 3β-hydroxysteroid dehydrogenase for pregnenolone and formation of testosterone but no oestrogens by the theca cells. These results indicate that both theca interna and granulosa cells are needed for oestrogen biosynthesis by rabbit follicles.

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