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HISTOCHEMICAL LOCALIZATION OF a5-3P- HYDROXYSTEROID DEHYDROGENASE AND

“SECONDARY ALCOHOL DEHYDROGENASE” ACTIVITY IN WHOLE BODY SECTIONS FROM MICE

The histochemical demonstration of hydroxysteroid dehydrogenases, to indi- cate the sites of steroid production has been extensively used in the last ten years (for review see Baillie et al., 1966). Wattenberg (1958) was the first to make histological localization of these enzymes possible. H e incubated unfixed tissue sections in a medium containing the hydroxysteroid to be investigated, NAD“, a tetrazolium salt and buffer. Hydrogen ions removed from the hydroxysteroid by the enzymes are transferred to NAD. The reduced NAD is then transformed to NAD by reduced NAD-tetrazolium reductase (diaphorase) while the tetrazolium salt is reduced to coloured formazan deposits. Previous experiments using this technique have been made on isolated organs from different animals. The use of whole body sections for incubation offers an interesting way for direct comparison of the localization of hydroxysteroid dehydrogenases in different organs in the body.

Pathways from cholesterol to Cnl-derivatives involve scission of the side- chain to form pregnenolone and isocaproic aldehyde or acid (Shimizu et al., 1961). 20a-hydroxycholesterol and 20a-22~-dihydroxycholesterol are involved in this pathway from cholesterol to pregnenolone (Shimizu et al., 1962). The enzymes involved in this pathway cannot be shown histochemically and Baillie et al. (1966) claim that the presence of a cholesterol type side-chain totally impedes histochemical investigation of 3/3-hydroxysteroids. However, Hardonk (1 965) has shown the presence of a “secondary alcohol dehydrogen- ase” in human steroid producing cells and suggests that this enzyme might be involved with the oxidative cleavage of the cholesterol side-chain. He showed “secondary alcohol dehydrogenase” in the Leydig cells of the testis, in the theca lutein cells in corpora lutea and in the zona reticularis of the adrenal in human biopsies. Hardonk got the strongest histochemical reaction when using isopro- panol as a substrate to demonstrate this “secondary alcohol dehydrogenase”. In the present investigation incubations have been made with isopropanol using whole body mice sections to possibly indicate sites of cholesterol side- chain scission.

When pregnenolone is formed in or injected into the body it may be con- verted to progesterone by A5-3/3-hydroxysteroid dehydrogenase. This enzyme has been shown histochemically in many steroid producing cell systems in various animals using e.g. pregnenolone as a substrate. In the present investiga- tion histochemical experiments with pregnenolone or cholesterol as a substrate have been made with mouse whole body sections for direct comparison with the distribution of labelled pregnenolone in the autoradiograms. ’ NAD = nicotinamide-adenine dinucleotide

MATERIALS A N D METHODS

Whole body, 10 IJ, thick, freeze sections from NMRl mice were taken on tape 688 (Minnesota Mining and Manufacturing C o . . USA) according to the method described earlier (p. 9). Two female mice. 1 male and 1 pi-egnant mouse (late gestation) were used. T h e sections were allowed to dry in the freeze-room temperature (- 12" C). Otherwise the sections easily came off the tape during the incubation.

For demonstration of "secondary alcohol dehydrogPriasc" activity incubation with isopropanol was made according to Hardonk (1965) for 2 hours at + 3 i - C, but omitting the acetone treatment prior to incubation. The incuba- tion medium consisted of 0.5 ml isopropanol, I .O ml Nitro Blue 'Ietrazoliuin salt'" ( 1 mgiml), 0.5 ml nicotinamide-adenine dinucleotide". (NAD) (5 mg/nil). 2 mi phosphate buffer. pH 7 . 1 (0.2 M) and 2.3 ml distilled water. Adjacent control sections were incubated in the same medium but omitting the isopro- panol or substituting i t for ethanol.

For demonstration of A"-,Yi3-hyd,-oxysteroid d&ydrogrnase incubation with pregnenolone" or cholesterol'" was made with the substance dissolved in di- methyl forniamide (0.23 mg/ml) as suggested by Baillie et al. (1966). T h e same incubation medium as described above was used but replacing the isopi-opanol with 0.3 ml of the pregnenolone o r cholesterol solution. When coiitrol sections were incubated the steroid solution was replaced by 0.5 ml dimethyl forniamide. Some sections were briefly rinsed in acetone before incubation to dissolve possihly occurring endogenic hydroxysteroids.

As the last step in these enzyme reactions is dependent on diaphorase some incubations with reduced NAD". were made to study the distribution of the diaphorase.

After incubating sections for 60 minutes to 2 hours they were briefly rinsed in phosphate buffer and fixed for 10 minutes in formalin ( 1 0 "/o) and embedded in glycerol-gelatine.

RESULTS

"Srcoiidnry alcohol dehydrogenasr"

When isopropanol was used as a substrate no specific enzyme reactions weix seen in adrenals, ovaries, testes and placenta. Formazan deposits were regist- ered in Harder 's gland, bronchi, liver and brown fat (Fig. 48, 49) .

"- S igma (:hemica1 C o m p a n y . Missouri. USA

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Brain Brown fat

Harder’s gland Liver Bronchi Heart Liver

F i g . 48. alcohol activity

. L e f t . Detail of a whole body freeze section from a mouse showing the “secondary dehydrogenase” activity using isopropyl alcohol as a substrate. Note the high enzyme (dark areas) in Harder’s gland.

F i g . 49. R i g h t . Detail of a whole body freeze section from a mouse showing the “second- ary alcohol dehydrogenase” activity (dark areas) in the bronchi.

AS-3/3-hydroxysteroid dehydrogenase

P r e g n e n o l o n e

With pregnenolone as a substrate formazan deposits were seen in the adrenal cortex, ovaries and testicles but also in some other tissues.

Adrenal cortex. The colour of the adrenal cortex was very strong and was evenly distributed through the whole cortex. No clear differences between the sexes were seen (Fig. 50, 51).

Ovaries. The strongest enzyme activity was registered in the corpora lutea from the pregnant mouse, but enzyme activity was also registered in the interstitial tissue and some follicles. In the ovaries from the nonpregnant mice the interstitial tissue had the strongest enzyme activity while corpora lutea and some follicles had less heavy formazon deposits. Most of the precipitates in the follicles were confined to the theca cell layers (Fig. 53, 54).

Testes. Weak A5-3f3-hydroxysteroid dehydrogenase activity was observed in the interstitial parts of the testes (Fig. 55). In the epididymis no enzyme activity was registered.

Placenta. Weak enzyme activity was observed in some cells in the decidua and yolk sac epithelium.

Sebaceous glands. Strong enzyme activity was seen both in the pregnenolone incubated and control sections. In some sections rinsed briefly in acetone prior to incubation, there was somewhat higher activity in the pregnenolone incub- ated sections than in the control sections (Fig. 52).

Brain. The brain showed weak A5-3/3-hydroxysteroid dehydrogenase activity in the white matter (Fig. 31 D).

In the liver and brown f a t weak enzyme activity was also observed.

Livcr

F ig . ,jO. Whole body section from a lemale muuse showing the / \ ~ ~ - 3 / j - h y d r o x v s t e r ~ ) i ( l tie- hydrogenase activity using pregnenolonc as a substrate. h'ote the ahundant enzyme activit>- ((Park areas) in the adrena l cor tex.

Sebaceous qland

Fi,q. i l . 1, c t t . I k t a i l oi' Fig. 5 0 showing the in thc adrena l c o r k s . ( x 2 0 ) .

F i g . ;2 . R i g I i t . Detail o l a whole body- section lrorn a mouse 5howing the . -~- :4 /? -hyt l roxy- itcroid dehydrogenase activity in the sebaceous glands. Prcgnenolonc was used as a substrate arid the section was rinsed briclly in acetone prior to incubation. (x 130).

, " - ' ( ~ - h ~ d r o x y s t e r o i ~ tiehytirogenasr activity

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Stomach Ovary

Fetus Placenta

Fig. 53. Whole body section from a pregnant mouse showing the Aj-3p-hydroxysteroid dehydrogenase activity with pregnenolone as a substrate. Note the high enzyme activity in the ovary.

Corpus luteum Interstitial Follicle tissue

Fig. 54. L e f t. Detail of Fig. 53 showing the A5-3P-hydroxystcroid dehydrogenase activity in the ovary. Note the high enzyme activity in the corpora lutea. The interstitial tissue and some follicles also show activity. (x 18).

Fig. 55. R i g h t. Detail from a whole body section of a mouse showing the a5-3P-hydroxy- steroid dehydrogenase activity in the testis using pregnenolone as a substrate. Note the enzyme activity in the interstitial cells. (x 75).

6-Appetgren

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Lung Adrenal cortex

Hnrdrr's gland I.ivrr Fctuse?,

F i g . , j / j . Whole body scctioii I'rom a pregnant mouse showing- the distribution ot diaphoraac a l t e r incubat ion with reduccd NAII. Note the reaction in almost cverv t i s u e in thc hod!. Rather weak diaphorasc act ivi ty was noted in white mat te r in the bra in .

(: h o 1 e s t e r o I

When cholesterol was used as a substrate no formazan deposits were reg-istered that did not appear in the control sections.

11 iuf)lr o rus c

The incubation with reduced NAD gave heavy deposits of forniazan in almost every tissue in the body (Fig. 56). The strongest reaction in the body was seen in thc sebaceous glands. Rather weak diaphorase activity was noted in the white matter of the brain and in the granulosa cells of the ovary.

DISCUSSION

The advantage of using whole body sections in histochemical localization of dehydrogenase activities is that the relative concentration of enzyme activitv in different tissues treated in exactly the same way can be compared in the same section.

The demonstration of dehydrogenase depends on the transfei- o f hydrog-en. removed from the hydroxysteroid, via a pyridine nucleotide to a teti-azoleum salt, which is reduced and deposited at the site of reaction. This last step is dependent on the presence of diaphorase. Therefore control incubations with reduced N A D were made. The negative results in some cell systems were probably not due to lack of diaphorase, as was seen from the incubations with reduced NAD.

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“Secondary alcohol dehydrogennse”

In this investigation “secondary alcohol dehydrogenase” activity could not be established in mice in the same places as has been reported in humans (Hardonk, 1965). Hardonk found very strong enzyme activity in the Leydig cells of the testis, in the theca cells of the ovary and in the zona reticularis of the adrenal in human biopsies. None of these cell systems showed any enzyme activity in the present investigation. The only tissues, where this enzyme activity was demonstrated in mice, were Harder’s gland, bronchi, liver and brown fat. The enzyme activity in these tissues can probably not be correlated to the cholesterol side-chain cleavage. The present autoradiographic investiga- tion of labelled cholesterol showed that cholesterol accumulated in the liver but very little Ci4 was seen in Harder’s gland and brown fat and none in the bronchi. Thus these findings in mouse do not support Hardonk’s theory that the “secondary alcohol dehydrogenase” may be identical with the enzyme that catalyses the cleavage of the cholesterol side-chain.

A5-3P-hydroxysteroid dehydrogenase

The high selective n5-3P-hydroxysteroid dehydrogenase activity in the adrenal cortex and in the ovary is well illustrated by the incubated whole body sec- tions. The enzyme activity in other tissues was difficult to detect without magnification in a microscope.

In the adrenal cortex Wattenberg (1958) found that all cells had enzyme activity, when using pregnenolone as a substrate, but the reaction in zona glomerulosa was weak. In a study of the developing mouse adrenal Allen (1960) found the same but also pointed out that there was enzyme activity in the female X-zone while it was missing from the male X-zone. The present investigation showed that the A5-3P-hydroxysteroid dehydrogenase activity was present in the whole cortex in both male and female mice.

In mouse ouaries A5-3P-hydroxysteroid dehydrogenase has been shown with pregnenolone (Ferguson, 1965) and dehydroepiandrosterone (Deane and Ru- bin, 1965) as substrates. Their findings agree with the present investigation. Enzyme activity was seen in corpora lutea, interstitial tissue and to less extent in theca interna and some granulosa cells. The granulosa cells showing enzyme activity are supposed to be atretic by Deane and Rubin (1965). The enzyme reaction in the granulosa cells may be limited by the low diaphorase concen- tration there, noted in the present investigation and previously observed by Baillie et al. (1966). AS-3P-hydroxysteroid dehydrogenase activity has been reported in the tropho- blastic giant cells in the rodent placenta (Deane et al., 1962; Botte et al., 1966). These authors found the maximum enzyme activity at 15 days after mating in the mouse. The weak enzyme activity seen in this investigation may be due to the late gestation state of the mouse.

II 4

In the Leydig cells from mouse testes, A5-3P-hydroxysteroid dehydrogenase activity, using pregnenolone as a substrate has been found (Hitzeman, 1Sti2: Baillie and Griffiths, 1965). Hitzeman found a much weaker reaction when pregnenolone was used instead of dehydroepiandrosterone. Weak enzyme activity in the interstitial cells was also seen in the present experiments.

i? 5-3,B-hydroxysteroid dehydrogenase activity has been shown using pregn- enolone as a substrate in sehaceoiis gZunds from human biopsies (Baillie et al., 1965) but not from mouse (Baillie, 1966). In the present investigation. how- ever, enzyme activity was demonstrated in mouse sebaceous glands in sections that were rinsed in acetone prior to incubation. T h e strong reaction seen in the control sections not rinsed in acetone may be correlated to the abundant concentration of diaphorase in the sebaceous glands seen in this investigation. Even small amounts of endogenous hydroxysteroids might therefore give strong formazan reaction.

T h e present histochemical results will be further discussed when comparing the autoradiographic distribution of the labelled precursors in General discus- sion.