9
ELSEVIER Molecular and Cellular Endocrinology104 (1994) 103-111 ~ Molecular and Cellular Endocrinology Widespread tissue distribution of steroid sulfatase, 3fl-hydroxysteroid dehydrogenase/A5-A 4 isomerase (3fl-HSD), 17fl-HSD 5a-reductase and aromatase activities in the rhesus monkey C61ine Martel a, Michael H. Melner b,l, Donald Gagn6 a, Jacques Simard a, Fernand Labrie a,* aMRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, Laval, Qugbec, G1V 4G2, Canada bOregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA Received 18 March 1994; accepted 27 May 1994 Abstract Dehydroepiandrosterone-sulfate (DHEA-S), the main secretory product of the human adrenal, requires the presence of steroid sulfatase, 3fl-hydroxysteroid dehydrogenase/AS-A 4 isomerase (3fl-HSD), 17fl-hydroxysteroid dehydrogenase (17fl-HSD), 5a-reductase, and aromatase to form the active androgen dihydrotestosterone (DHT) and the estrogens 17fl-estradiol (E2) and 5-androst-ene-3fl,17fl- diol (AS-diol) in peripheral target tissues. Because humans, along with non-human primates are unique in having adrenals that secrete large amounts of DHEA-S, the present study investigated the tissue distribution of the enzymatic activity of the above-mentioned steroidogenic enzymes required for the formation of active sex steroids in the male and female rhesus monkey. Estrone and DHEA sulfatase activities were measured in all 25 tissues examined, and with the exception of the salivary glands, estrogenic and androgenic 17fl-HSDs were present in all the tissues examined. The adrenal, small and large intestine, kidney, liver, lung, fat, testis, prostate, seminal vesicle, ovary, myometrium, and endometrium all possess the above-mentioned enzymatic activities, thus suggesting that these tissues could possibly form the biologically active steroids E 2 and DHT from the adrenal precursor DHEA-S. On the other hand, the oviduct, cervix, mammary gland, heart, and skeletal muscle possess all the enzymatic activities required to synthesize E 2 from DHEA- S. The present study describes the widespread tissue distribution of steroid sulfatase, 3fl-HSD, 17fl-HSD, 5a-reductase, and aromatase activities in rhesus monkey peripheral tissues. Such findings support the importance of developing therapeutic approaches to the treatment of sex steroid-sensitive diseases which take into account the formation of androgens and estrogens in peripheral target tissues. Keywords: Steroidogenesis; Intracrinology; Dehyroepindrosterone; Sex steroids; Rhesus monkey 1. Introduction Humans, along with other non-human primates, are unique in having adrenals that secrete large amounts of the precursor steroids dehydroepiandrosterone (DHEA) and especially DHEA-sulfate (DHEA-S), which are con- verted into androstenedione (A4-dione) and then into po- tent androgens and estrogens in peripheral target tissues (Adams, 1985; Labrie et al., 1985). Having a high secre- tion rate of adrenal precursor sex steroids is thus com- * Corresponding author. Laboratory of Molecular Endocrinology CHUL Research Center, 2705 Laurier Boulevard Qu6bec, GIV 4G2, Canada. Tel. (418) 654-2704. Fax (418) 654-2735. I Present address: Vanderbilt University, School of Medicine, De- partment of Obstetrics and Gynecology, Division of Reproductive En- docrinology,Nashville, TN 37232-2515, USA. pletely different from animal models currently used in the laboratory, namely, rats, mice, guinea pigs, and others (except monkeys), in which the formation of androgens and estrogens takes place exclusively in the gonads (Labrie et al., 1985; B61anger et al., 1989). DHEA-S, the most abundant adrenal precursor requires the action of sulfatase, 3fl-hydroxysteroid dehydrogenase/A5-A 4 isom- erase (3fl-HSD), 17fl-hydroxysteroid dehydrogenase (17fl-HSD), 5a-reductase, and aromatase to form the ac- tive sex steroids dihydrotestosterone (DHT) and estradiol (E2) (Fig. 1) as well as androst-5-ene-3fl,17fl-diol (A 5- diol), which possess intrinsic estrogenic activity in breast cancer cells (Poulin and Labrie, 1986) and normal tissues (Adams, 1985). The adrenals of the rhesus monkey secrete high levels of sex steroid precursors, at a rate that increases from 0167-8140/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03337-J

Widespread tissue distribution of steroid sulfatase, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4isomerase(3β-HSD), 17β-HSD5α-reductase and aromatase activities in the rhesus monkey

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E L S E V I E R Molecular and Cellular Endocrinology 104 (1994) 103-111

~ Molecular and Cellular Endocrinology

Widespread tissue distribution of steroid sulfatase, 3fl-hydroxysteroid dehydrogenase/A5-A 4 isomerase (3fl-HSD), 17fl-HSD 5a-reductase and

aromatase activities in the rhesus monkey

C61ine M a r t e l a, M i c h a e l H. M e l n e r b,l, D o n a l d G a g n 6 a, J a c q u e s S i m a r d a, F e r n a n d L a b r i e a,*

aMRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, Laval, Qugbec, G1V 4G2, Canada bOregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA

Received 18 March 1994; accepted 27 May 1994

Abstract

Dehydroepiandrosterone-sulfate (DHEA-S), the main secretory product of the human adrenal, requires the presence of steroid sulfatase, 3fl-hydroxysteroid dehydrogenase/AS-A 4 isomerase (3fl-HSD), 17fl-hydroxysteroid dehydrogenase (17fl-HSD), 5a-reductase, and aromatase to form the active androgen dihydrotestosterone (DHT) and the estrogens 17fl-estradiol (E2) and 5-androst-ene-3fl,17fl- diol (AS-diol) in peripheral target tissues. Because humans, along with non-human primates are unique in having adrenals that secrete large amounts of DHEA-S, the present study investigated the tissue distribution of the enzymatic activity of the above-mentioned steroidogenic enzymes required for the formation of active sex steroids in the male and female rhesus monkey. Estrone and DHEA sulfatase activities were measured in all 25 tissues examined, and with the exception of the salivary glands, estrogenic and androgenic 17fl-HSDs were present in all the tissues examined. The adrenal, small and large intestine, kidney, liver, lung, fat, testis, prostate, seminal vesicle, ovary, myometrium, and endometrium all possess the above-mentioned enzymatic activities, thus suggesting that these tissues could possibly form the biologically active steroids E 2 and DHT from the adrenal precursor DHEA-S. On the other hand, the oviduct, cervix, mammary gland, heart, and skeletal muscle possess all the enzymatic activities required to synthesize E 2 from DHEA- S. The present study describes the widespread tissue distribution of steroid sulfatase, 3fl-HSD, 17fl-HSD, 5a-reductase, and aromatase activities in rhesus monkey peripheral tissues. Such findings support the importance of developing therapeutic approaches to the treatment of sex steroid-sensitive diseases which take into account the formation of androgens and estrogens in peripheral target tissues.

Keywords: Steroidogenesis; Intracrinology; Dehyroepindrosterone; Sex steroids; Rhesus monkey

1. I n t r o d u c t i o n

Humans, along with other non-human primates, are unique in having adrenals that secrete large amounts of the precursor steroids dehydroepiandrosterone (DHEA) and especially DHEA-sulfate (DHEA-S), which are con- verted into androstenedione (A4-dione) and then into po- tent androgens and estrogens in peripheral target tissues (Adams, 1985; Labrie et al., 1985). Having a high secre- tion rate of adrenal precursor sex steroids is thus com-

* Corresponding author. Laboratory of Molecular Endocrinology CHUL Research Center, 2705 Laurier Boulevard Qu6bec, GIV 4G2, Canada. Tel. (418) 654-2704. Fax (418) 654-2735.

I Present address: Vanderbilt University, School of Medicine, De- partment of Obstetrics and Gynecology, Division of Reproductive En- docrinology, Nashville, TN 37232-2515, USA.

pletely different from animal models currently used in the laboratory, namely, rats, mice, guinea pigs, and others (except monkeys), in which the formation of androgens and estrogens takes place exclusively in the gonads (Labrie et al., 1985; B61anger et al., 1989). DHEA-S, the most abundant adrenal precursor requires the action of sulfatase, 3fl-hydroxysteroid dehydrogenase/A5-A 4 isom- erase (3fl-HSD), 17fl-hydroxysteroid dehydrogenase (17fl-HSD), 5a-reductase, and aromatase to form the ac- tive sex steroids dihydrotestosterone (DHT) and estradiol (E2) (Fig. 1) as well as androst-5-ene-3fl,17fl-diol (A 5- diol), which possess intrinsic estrogenic activity in breast cancer cells (Poulin and Labrie, 1986) and normal tissues (Adams, 1985).

The adrenals of the rhesus monkey secrete high levels of sex steroid precursors, at a rate that increases from

0167-8140/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03337-J

104 C. Martel et al. / Molecular and Cellular Endocrinology 104 (1994) 103-111

DHEA-S

DHEA A5-DIOL

A4-DIONE ~ '~ TESTO

E1 ~ = ~ 1 "~ E2

I u.atu e I - I - I E1-S

[ 5c~-reductase I ~DHT

Fig. 1. Biosynthetic steps involved in the formation of the estrogens A5-diol (androst-5-ene-3fl,17fl-diol) and estradiol (E2) and of the an- drogens testosterone (testo) and DHT (5a-dihydrotestosterone) from the precursor DHEA-S in gonadic and peripheral tissues.

birth to sexual maturity with no adrenarche (Cutler et al., 1978; Pepe and Albrecht, 1990). The rhesus monkey is one of the closest available models of the human, thus permitting studies of the biosynthesis of active androgens and estrogens in peripheral target tissues. The present manuscript describes the widespread tissue distribution of steroid sulfatase, 3fl-HSD, 17fl-HSD, 5a-reductase, and aromatase activities in gonadal and peripheral tissues of the male and female rhesus monkey. Such data could support the physiological importance of the formation of sex steroids in extragonadal tissues.

2. Materials and methods

2.1. Tissues Tissues from adult male and female rhesus monkeys

(Macaca mulatta) were collected and freed from fat and other adhering tissue immediately after the monkeys were killed. The tissues were quickly frozen in dry ice and kept a t - 8 0 ° C until assayed. Female tissues were obtained from an ll-year-old (no. 305-10577), a 7-year-old (no. 291-12147), and a 14-year-old (no. 305-08381) rhesus monkey, with the exception of the ovaries obtained from two different rhesus monkeys (no. 305-10587 and no. 305-15089). The male tissues were obtained from two 7- year-old rhesus monkeys (no. 305-12575 and no. 305- 14768) and one 10-year-old (no. 305-10553) rhesus mon- key. Animal handling was performed according to the 'Guidelines for Care and Use of Experimental Animals' at the Oregon Regional Primate Research Center.

Tissues were homogenized with a Polytron in phos- phate buffer (20 mM KH2PO4, 0.25 M sucrose, 1 mM EDTA, pH 7.5) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 5/zg/ml each of pep- statin A, antipain, and leupeptin) and centrifuged for 30 min at 1000 × g to remove cell debris. Protein content of tissue homogenates was measured by the method of

Bradford using bovine serum albumin as standard (Bradford, 1976).

2.2. Measurement of 3fl-HSD, 17fl-HSD, aromatase and 5a-reductase activities

Aliquots of the 1000 × g supernatant were incubated for 90 min at 37°C in a total volume of 0.5 ml phosphate buffer (12.5 mM KH2PO 4, 1 mM EDTA, pH 7.5) contain- ing 0.5/~M of 14C-labeled substrate and 1 mM of the ap- propriate cofactor(s). Three fl-HSD activity was measured with [4-14C]DHEA (spec. act. 51 mCi/mmol) and NAD ÷ while [4-14C] t e s tos t e rone (spec. act. 51.4 mCi/mmol) and NADPH were used for measurement of 5a-reductase. Aromatase activity was measured with [4-14C]Aa-dione (spec. act. 51.4 mCi/mmol) and NADPH. On the other hand, estrogenic 17fl-HSD activity was measured using [4-14C]estrone (El) (spec. act. 51.4 mCi/mmol), and an- drogenic 17fl-HSD was studied with [4-14C]Aa-dione (spec. act. 51.4 mCi/mmol), both substrates being used with the cofactors NADH + NADPH. Labeled radioactiv- ity was purchased from New England Nuclear/Dupont (Markham, Canada) and purified by thin-layer chroma- tography (TLC) before use.

The enzymatic reactions were stopped by chilling the incubation mixtures in an ice-water slurry before adding 3 ml of diethyl ether and mixing. The components were then frozen in a dry-ice/ethanol bath. The liquid organic phase was kept and the aqueous phase re-extracted once with diethyl ether. The two organic phases were then pooled and evaporated to dryness under a nitrogen stream. The products of enzymatic activities were sepa- rated by TLC using 60 F254 silica gel plates (E. Merck, Darmstadt, Germany) and a mixture of toluene/acetone (4:1, v/v) before autoradiography for 2 days. The meta- bolites revealed by autoradiography were identified by comparison with standard steroids characterized by high- performance liquid chromatography (HPLC) as described previously (Th6riault and Labrie, 1991). The different metabolites separated by TLC were identified as DHEA, A4-dione, El, E2, testosterone, DHT, androstane-3,17- dione, androstane-3a,17fl-diol, and androstane-3fl,17fl- diol. The TLC areas corresponding to the substrate and product(s) of the enzymatic reaction were collected and transferred into scintillation vials containing 0.5 ml etha- nol. After adding scintillation fluid (10 ml), radioactivity was calculated in a scintillation spectrometer. Control samples were processed identically except that buffer replaced the homogenate.

All measurements of enzymatic activities were per- formed with individual tissue samples obtained from three male and female macaques as mentioned. The re- covery of 14C radioactivity varied between 80 and 90% of the radioactivity initially added as substrate and was taken into consideration in calculating the rates of product for- mation. Moreover, dilutions of the homogenate were used for some tissues so that, under the conditions of the assay,

C. Martel et al. /Molecular and Celhdar Endocrinology 104 (1994) 103-111 105

no more than 30% of the substrate was metabolized. Hy-

droxylated products and water-soluble metabolites, such as sulfates and glucuronides, were not studied.

2.3. Measurement o f sulfatase activity

Aliquots of the supernatant were incubated in a shak-

ing water bath for 90 min at 37°C, in 0.5 ml Tris-acetate buffer (0.t M Tris-acetate, 5 mM EDTA, pH 7.0, 10% glycerol) containing 1 0 n M of [1,2,6,7-3(N)] DHEA-S (spec. act. 75.8 Ci/mmol) or [6,7-3H(N)]E1-S (spec. act.

49 Ci/mmol). The enzymatic reaction was stopped by chilling the incubation mixture in an ice-water slurry and

adding 320/zM of DHEA-SO4 or El-SO4 to saturate the enzyme and 4 ml of diethyl ether. After centrifugation,

the components were frozen in a dry-ice/ethanol bath. The liquid organic phase containing [3H]DHEA or [3H]E1

was transferred into scintillation vials containing scintil- lation fluid (10 ml), and the radioactivity was measured in a spectrometer.

3. Results

3.1. Androgen formation

As illustrated in Fig. 1, formation of the active andro- gen DHT from the adrenal precursor DHEA-S requires

the action of four enzymes: DHEA-sulfatase, 3fl-HSD,

androgenic 17fl-HSD, and 5a-reductase. In humans and

non-human primates, plasma levels of DHEA-S far ex- ceed the concentration of the free steroid DHEA. Cleav- age of the sulfate group from DHEA-S by sulfatase is thus likely to represent an important step in the formation of active sex steroids from circulating DHEA-S. Table 1 shows that all the male tissues studied, with the exception of the salivary gland, can hydrolyze DHEA-S into DHEA. High levels of DHEA-S sulfatase activity, ranging from 10 to 100 fmol/mg protein per h, were found in the adre- nal, seminal vesicle, kidney, liver, pituitary, and three areas of the brain (cerebral cortex, cerebellum, and cere- brum). Intermediate DHEA sulfatase activity (from 1 to 10 fmol/mg protein per h) was found in the testis, pros-

tate, small and large intestine, lung, spleen, and foreskin and low levels (0.1-1 fmol/mg protein per h) in the heart, mesenteric fat, and skeletal muscle. In the female ma-

caque, DHEA sulfatase was also present in all the tissues examined except the salivary gland. The highest levels

(10-100 fmol/mg protein per h) of enzymatic activity were found in the adrenal, cervix, kidney, spleen, cerebel- lum, and cerebrum (Table 2). As in male tissues, the heart, mesenteric fat, and skeletal muscle showed the lowest level of DHEA sulfatase activity.

Following DHEA-S hydrolysis, 3fl-HSD converts DHEA into A4-dione (Fig. 1). The levels of 3fl-HSD ac- tivity measured in crude homogenate from different tis- sues of male and female rhesus monkeys are illustrated in

Figs. 2 and 3, respectively. The highest level of 3fl-HSD

Table 1

Tissue distribution of DHEA and estrone sulfatase activities as well as of androgenic and estrogenic 17fl-HSD activities in a series of classical steroidogenic and peripheral tissues from adult male rhesus monkeys

Male tissues Sulfatase 17fl-HSD

DHEA E 1 Testo E 2 (× 10 -3) (× 10 -3 )

Adrenal 10.6 + 2.6 28.9 + 2.1 124.2 + 10.2 19.8 + 1.8 Testis 6.1 + 1.4 24.0 + 1.8 494.4 + 14.4 97.2 + 54.0 Prostate 2.5 + 0.3 8.2 + 0.3 73.8 +_ 19.2 105.0 + 7.8 Seminal vesicle 43.4 + 9.1 78.3 + 20.7 86.4 + 29.4 534.0 + 25.8 Small intestine 5.0 + 0.2 14.1 + 1.8 34.2 + 0.6 1.2 + 0.1 Large intestine 3.2 + 2.1 22.8 + 3.3 293 + 54 100.2 + 33.6 Kidney 16.4 + 4.5 42.6 + 6.5 15.0 + 8.4 4.2 + 2.4 Liver 16.1 + 5.5 45.1 + 1.0 329 + 70 66.6 + 24.6 Lung 7.4 + 1.8 23.6 _+ 11.3 45 + 27 79.2 + 52.2 Heart 0.3 +0.1 1.9+0.1 3.0+0.6 3.6+ 1.2 Spleen 5.7 + 0.7 20.1 + 7.5 12.0 + 6.0 12.6 + 8.4 Mesenteric fat 0.9 + 0.2 3.5 + 0.2 47.4 + 19.2 75.6 _+ 6.6 Foreskin 2.0 + 0.3 21.5 + 2.7 64.2 + 2.4 10.8 + 5.4 Skeletal muscle 0.7 + 0.1 0.6 + 0.4 8.4 + 4.8 47.4 + 24.6 Salivary gland ND ND 28.8 _+ 8.4 15.6 + 0.6 Pituitary 13.3 + 1.3 39.9 + 5.0 21.0 + 0.3 22.8 + 0.3 Cerebral cortex 30.9 + 5.3 48.5 _+ 5.0 35.4 + 0.3 60.0 + 0.3 Cerebellum 26.4 _+ 16.2 48.2 _+ 6.2 36.6 +_ 6.6 58.8 _+ 38.4 Cerebrum 13.6 + 2.3 51.1 + 5.2 22.2 + 0.3 17.4 + 0.3

Data are expressed as means +_ SEM of pmol of product formed/mg protein per h. DHEA, dehydroepiandrosterone; El, estrone; 17fl-HSD, 17fl- hydroxysteroid dehydrogenase; ND, no enzymatic activity detectable; E 2, estradiol; Testo, testosterone.

106 C. Martel et al. / Molecular and Cellular Endocrinology 104 (1994) 103-111

Table 2

Tissue distribution of DHEA and estrone sulfatase activities as well as of androgenic and estrogenic 17/3-HSD activities in a series of classical steroidogenic and peripheral tissues from adult female rhesus monkeys

Male tissues Sulfatase 17/3-HSD

DHEA E 1 Testo E 2 (x 10 -3) (x 10 -3 )

Adrenal 13.4 + 5.8 265 + 1.0 254.4 +_ 160.8 160 + 107 Ovary 9.2 + 3.2 14.5 + 3.1 79.8 + 37.2 267 + 66 Oviduct 9.3 _+ 0.4 35.4 + 3.4 89.1 + 7.5 61.8 + 22.8 Cervix 13.7 + 2.0 29.6 + 5.0 171.6 + 70.8 342 + 228 Endometrium 10.5 + 2.0 28.1 + 1.7 152.4 + 60.0 330 + 120 Myometrium 4.3 + 0.7 12.6 + 2.7 141.6 + 60.0 162 + 54 Small intestine 6.6 + 1.0 18.2 + 1.6 216.6 + 97.2 199 + 70 Large intestine 6.4 + 0.6 29.3 + 3.1 2740 + 187 2476 + 300 Mammary gland 7.3 + 0.1 5.9 + 1.0 38.4 + 6.0 30.6 + 3.0 Kidney 20.9 + 0.9 35.5 + 2.0 31.2 + 6.6 14.7 _+ 9.0 Liver 7.8 + 1.3 20.6 + 3.1 2296 + 542 1028 + 242 Lung 5.2 + 0.7 20.8 + 2.7 158.4 _+ 78.0 140 + 54 Heart 2.1 +0.9 2.8+_ 1.0 14.1 +3.6 16.8+-0.6 Spleen 29.5 +- 3.2 45.6 +- 3.8 38.4 +- 8.8 27.0 +- 11.4 Mesenteric fat 2.7 _+ 0.4 4.2 + 0.8 69.0 + 17.4 57.6 +- I 1.4 Sex skin 6.8 +- 1.8 17.3 +- 6.7 244 + 75 425 + 47 Skeletal muscle 1.3 + 0.2 0.6 + 0.1 37.8 + 1.2 234 + 16 Salivary gland ND ND 37.4 + 9.6 24.6 +- 6.0 Cerebellum 19.1 +- 1.7 58.0 + 2.1 112 + 42 57.3 + 25.2 Cerebrum 16.5 + 4.1 51.8 + 0.7 24.3 + 13.8 34.8 _+ 16.8

Data are expressed as means + SEM of pmol of product formed/mg protein per h. DHEA, dehydroepiandrosterone; El, estrone; 17fl-HSD, 17/3- hydroxysteroid dehydrogenase; ND, no enzymatic activity dectectable; E 2, estradiol; Testo, testosterone.

act ivi ty was measured in male (17 1 7 2 + 9 4 0 p m o l / m g

protein per h) and female (26 245 + 1280 pmo l /mg pro-

tein per h) adrenals, fo l lowed by the ovary and the testis

with respec t ive 3 ~ - H S D activi t ies o f 5 6 4 + 191 and

140 + 20 p m o l / m g protein per h. The levels o f 3~ -HSD

act ivi ty in these s te ro idogenic tissues are consistent with

the known capaci ty o f these organs to synthesize high

levels o f steroids to be secreted in the circulation. How-

~ ADRENAL TESTIS

PROSTATE SEMINAL VESICLE SMALL iNTESTiNE I LARGE INTESTINE I

KIDNEY ~ 1 LIVER I LUNG

HEART SPLEEN I

MESENTERIC FAT I FORESKIN I

SKELETAL MUSCLE ' I SALIVARY GLAND ' I

PITUITARY CEREBRAL CORTEX • DHEA ~ A4-DIONE

CEREBELLUM CEREBRUM

0.1 1 1 1000 10000 100000

ANDROSTENEDIONE FORMED (pmol / mg protein / hour)

Fig. 2. Tissue distribution of 3fl-HSD activity in a series of classical steroidogenic and peripheral tissues from adult male rhesus monkeys. 3fl-HSD activity was measured by the formation of A4-dione from DHEA. Incubations were performed at 37°C for 90 min with 0.5/tM labeled substrate and 1 mM NAD +. Data are presented as means + SEM of pmol of A4-dione formed/mg protein per h (log scale).

ever, it is o f special interest to f ind 3f l -HSD activi ty

in many peripheral tissues. Thus, the prostate ( 2 2 +

0 . 6 p m o l / m g protein per h), the male l iver ( 1 4 2 +

3 6 p m o l / m g protein per h), and the pituitary (41 +

6.0 pmo l /mg protein per h) possess fairly high levels o f

3f l -HSD activity (Fig. 2). In addition, a comparab l e level

of activity (4 -8 pmol lmg protein per h) was observed in

the seminal vesicles, small and large intestine, mesenter ic

fat, foreskin, skeletal muscle , and sal ivary gland; low

levels (<1.5 pmo l /mg protein per h) were measured in the

kidney, lung and heart. Unde r the exper imenta l condi t ions

used, no signif icant 3f l -HSD activi ty was detected in the

spleen, cerebel lum, ce rebrum and cerebral cortex. In the

female monkey (Fig. 3), 3 f l -HSD activi ty in the l iver was

also high at 285 + 65 p m o l / m g protein per h; the oviduct ,

small and large intestine, lung, mesenter ic fat, and sex

skin had comparab le but lower 3f l -HSD act ivi ty at about

10 pmo l /mg protein per h. L o w e r levels o f 3f l -HSD ac-

tivity were measured in the cervix, endomet r ium,

myomet r ium, mammary gland, kidney, heart, skeletal

muscle, and salivary gland, and none detec table in the

spleen, cerebel lum, and cerebrum.

The next step in androgen format ion requires that the

steroid Aa-dione be conver ted into tes tosterone by andro-

genic 17fl-HSD (Fig. 1). As shown in Tables 1 and 2,

androgenic 17fl-HSD activi ty was measured in all the

male and female tissues examined. All 25 tissues studied

can t ransform A4-dione into testosterone. Thus, with the

C. Martel et al. / Molecular and Cellular Endocrinology 104 (1994) 103-111 107

ADRENAL OVARY

OVIDUCT CERVIX

ENDOMETRIUM MYOMETRIUM

SMALL INTESTINE LARGE INTESTINE MAMMARY GLAND

KIDNEY LIVER LUNG

HEART SPLEEN

MESENTERIC FAT SEX SKIN

SKELETAL MUSCLE SALIVARY GLAND

CEREBELLUM CEREBRUM

m U J

J D

m

i • DHEA ~ ~4-DIONE

0.1 1 1'0 "1(10 1000 10000 100000 ANDROSTENEDIONE FORMED (pmoI / mg protein / hour)

Fig. 3. Tissue distribution of 3fl-HSD activity in a series of classical steroidogenic and peripheral tissues from adult female rhesus monkeys. 3fl-HSD activity was measured as described in Fig. 2. Data are pre- sented as means _+ SEM of pmol of A4-dione formed/mg protein per h (log scale).

exception of the salivary gland (no DHEA sulfatase ac- tivity) and the cerebellum, cerebrum, cerebral cortex, and spleen (no 3fl-HSD activity), all the other tissues exam- ined possessed the enzymatic activities required to con- vert the precursor DHEA-S into testosterone.

The last step in the biosynthesis of DHT requires the conversion of testosterone into DHT by 5a-reductase (Fig. 1). As shown in Fig. 4, levels of 5a-reductase activ- ity above 85 pmol/mg protein per h were found in the small intestine and liver, whereas the adrenal, testis, prostate, seminal vesicle, large intestine, foreskin and pituitary had 5a-reductase activity ranging between 17 and 60 pmol/mg protein per h. Lower levels of 5a- reductase activity were observed in the cerebrum, cerebel-

O ~ ADRENAL TESTIS

PROSTATE SEMINAL VESICLE SMALL INTESTINE LARGE INTESTINE

KIDNEY LIVEF LUNG

HEART SPLEEN

MESENTERIC FAT FORESKIN

SKELETAL MUSCLE SALIVARY GLAND

PITUITARY CEREBRAL CORTEX

CEREBELLUM CEREBRUM

0.1

I I

I

I I

I I

I • TESTO ~ DHT

I

u 1 110 100 ' 1000

DIHYDROTESTOSTERONE FORMED (prnol / mg protein / hour)

Fig. 4. Tissue distribution of 5a-reductase activity in a series of classi- cal steroidogenic and peripheral tissues from adult male rhesus mon- keys. 5a-Reductase activity was measured by the formation of DHT from testosterone. Incubations were performed at 37°C for 90 min with 0 .5/ tM labeled substrate and 1 mM NADPH. Data are presented as means + SEM of pmol of DHT formed/rag protein per h (log scale).

lum, kidney, lung and mesenteric fat. In the female rhesus monkey, the liver also showed the highest 5a-reductase activity, at 694 pmol/mg protein per h (Fig. 5). In analogy with male tissues, the large and small intestine show high levels of 5a-reductase activity in the female rhesus mon- key; lower levels were observed in the adrenal, ovary, endometrium, myometrium, kidney, lung, mesenteric fat, sex skin, cerebellum and cerebrum. Under the experimen- tal conditions used, no 5a-reductase was detected in the oviduct, cervix, mammary gland, heart, spleen, cerebral cortex, skeletal muscle and salivary gland, thus suggest- ing that these tissues have a low or no ability to transform testosterone into the active androgen DHT.

3.2. Estrogen formation To synthesize the active estrogen E 2 from the precur-

sor DHEA-S, four enzymes are needed: namely DHEA sulfatase, 3fl-HSD, estrogenic 17fl-HSD and aromatase (Fig. 1). The tissue distribution of DHEA sulfatase and 3fl-HSD has already been described above as these two enzymes are also needed for androgen formation. We next studied the tissue distribution of aromatase, the en- zyme required for the synthesis of estrone (El) from A 4- dione and E 2 from testosterone.

As illustrated in Fig. 6, aromatase activity was meas- ured in all male tissues examined except the foreskin and pituitary, where no significant level of activity could be detected. Aromatase activity was highest in the liver at 59.0 pmol/mg protein per h and comparable levels in the testis and prostate while the values ranged from 0.11 to 49.4 pmol/mg protein h in the other tissues. In the female, aromatase activity was detected in all tissues examined except in the sex skin (Fig. 7). As expected, aromatase ac- tivity was highest in the ovary and the liver, in agreement with the high levels found in the male, and possess the second highest level of aromatase activity in the female.

ADRENAL OVARY

OVIDUCT CERVIX

ENDOMETRIUM MYOMETRIUM

SMALL INTESTINE LARGE INTESTINE MAMMARY GLAND

KIDNEY LIVER LUNG

HEART SPLEEN

MESENTERIC FAT SEX SKIN

SKELETAL MUSCLE SALIVARY GLAND

CEREBELLUM CEREBRUM

0.1

I I

I I

I I

• TESTO----~DHT I

1~ ,~ 1o'oo DIHYDROTESTOSTERONE FORMED

(prnol / mg protein / hour)

Fig. 5. Tissue distribution of 5a-reductase activity in a series of classi- cal steroidogenic and peripheral tissues from adult female rhesus mon- keys. 5a-Reductase activity was measured as described in Fig. 4. Data are presented as means + SEM of pmol of DHT formed/mg protein per h (log scale).

1 0 8 C. Martel et al. / Molecular and Cellular Endocrinology 104 (1994) 103-111

O • ADRENAL TESTIS

PROSTATE SEMINAL VESICLE SMALL INTESTINE LARGE INTESTINE

KIDNEY LIVER LUNG

HEART SPLEEN

MESENTERIC FAT FORESKIN

SKELETAL MUSCLE SALIVARY GLAND

PITUITARY CEREBRAL CORTEX

CEREBELLUM ~ i B B • I I I I I B ~ CEREBRUM I

0.1

I

I I

I

I

• &4-DIONE ~ E1

i ; ,'o ,oo

ESTRONE FORMED (fmol / mg protein / hour)

Fig. 6. Tissue distribution of aromatase activity in a series of classical steroidogenic and peripheral tissues from adult male rhesus monkeys. Aromatase activity was measured by the formation of estrone from A 4- dione. Incubations were performed at 37°C for 90min with 0.5/tM labeled substrate and 1 mM NADPH. Data are presented as means + SEM of pmol of estrone formed/mg protein per h (log scale).

Estrone synthesized by aromatase from A4-dione is next converted into E 2 by the estrogenic 17fl-HSD (Fig. 1). As indicated in Tables 1 and 2, estradiol formation from estrone was demonstrated in all the male and female monkey tissues examined.

Estrone can also be released following hydrolysis of EI-S by steroid sulfatase (Fig. 1). In fact, as observed for the hydrolysis of DHEA-S into DHEA, the enzymatic conversion of E1-S into E 1 was found in all male (Table 1) and female (Table 2) tissues examined with the excep- tion of the salivary gland.

4. Discussion

The present data show a wide tissue distribution of

ADRENAL OVARY

OVIDUCT CERVIX

ENDOMETRIUM MYOMETRIUM

SMALL INTESTINE LARGE INTESTINE MAMMARY GLAND

KIDNEY LIVER LUNG

HEART SPLEEN

MESENTERIC FAT SEX SKIN

SKELETAL MUSCLE SALIVARY GLAND

CEREBELLUM CEREBRUM

m - ~

m l m m 4 i m

I

I /

/

• A4-DIONE ~ E 1

ESTRONE FORMED (fmol / mg protein / hour)

Fig. 7. Tissue distribution of aromatase activity in a series of classical steroidogenic and peripheral tissues from adult female rhesus monkeys. Aromatase activity was measured as described in Fig. 6. Data are pre- sented as means + SEM of pmol of estrone formed/mg protein per h (log scale).

steroidogenic enzymes activities in the rhesus monkey, thus providing the basis for the intracrine formation of androgens and estrogens in peripheral target tissues (Labrie, 1991). In fact, of the 25 tissues examined, 13 possess all the enzymatic activities required to transform DHEA-S into DHT and E2: adrenal, testis, ovary, pros- tate, seminal vesicle, ovary, endometrium, myometrium, small and large intestine, kidney, liver, lung and mesen- teric fat. Enzymatic activities in monkey tissues were measured with different protein concentrations to ensure that more than 70% of the initial substrate used was still present at the end of the incubation period. However, as we did not determine the K m values of enzymes studied in monkey tissues, we cannot certify that the enzymatic as- says were necessarily conducted under saturating/optimal steroid substrate levels in all cases. Data presented in this paper thus principally demonstrate the presence of many steroidogenic enzymes in these tissues (qualitative data) rather than provide complete kinetic data on the levels of enzymatic activities (quantitative data).

Hydrolysis of the sulfate group from DHEA-S is the first step required for the transformation of DHEA-S into active sex steroids. As shown in Tables 1 and 2, of the 25 tissues examined, 24 possess steroid sulfatase activity, thus releasing DHEA, which can be further transformed into Aa-dione by 3fl-HSD or into AS-diol by a 17fl-HSD. No definitive information is available on the specificity and nature of sulfatase in various species. Some data sug- gest variable properties of arylsulfatase C(ASC) and steroid sulfatase (SS) among species, ranging from one enzyme having one active site exhibiting ASC, El-S, and DHEA-S sulfatase activities to individual enzymes for each activity. Thus, human placental (Vaccaro et al., 1987) and rat (Kawano et al., 1989) ASC-, EFS-, and DHEA-S-sulfatase activities appear to reside within a single enzyme, whereas three different enzymes in the sheep brain have been suggested as responsible for sul- fatase activities (Balasubramanian, 1976). Data obtained in primates indicate that in the baboon liver, a single en- zyme catalyzes the hydrolysis of the three substrates (Ruoff and Daniel, 1991). The monkey brain (Macaca radiata) has been reported to possess an arylsulfatase with ASC- and El-S-sulfatase activities, and an alkylsul- fatase possesses DHEA-S sulfatase activity (Lakshmi and Balasubramanian, 1981).

The present study does not provide data that indicate whether hydrolysis of E1-S and DHEA-S results from the action of a single or two different enzymes. The present data do, however, show that the tissue distribution of EFS and DHEA-S sulfatase activities is very similar, thus not excluding the presence of one enzyme responsible for both activities. It should, however, be mentioned that the enzyme affinity for E1-S was higher than for DHEA-S. This higher reaction rate for the substrate E]-S as com- pared with DHEA-S has been observed previously in hu- man tissues, such as benign prostatic hyperplasia (Klein et

c. Martel et al. / Molecular and Cellular Endocrinology 104 (1994) 103-111 109

al., 1989), endometrium (Prost and Adessi, 1983), liver (Prost et al., 1984) and lung (Milewich et al., 1983).

The activity of EI-S sulfatase has been described in the rat and human liver (Prost et al., 1984; Eriksson et al., 1989), brain (Connolly and Resko, 1989), pituitary (Connolly and Resko, 1989), and breast carcinoma (Maclndoe, 1988; Pasqualini et al., 1992) and DHEA-S sulfatase activity has been reported in the human ovary, skin, and uterus (Haning et al., 1990) as well as in benign prostatic hyperplasia (Klein et al., 1989) and MCF-7 hu- man breast cancer cells (Maclndoe, 1988), but no such data were available for primates. The present study de- scribes the ubiquitous distribution of the enzyme(s) E1-S and DHEA-S sulfatase in rhesus monkey tissues, while the significance of the absence of detectable sulfatase activity in the salivary gland is unknown.

The enzyme 3fl-HSD catalyzes the conversion of 3/3- hydroxy-AS-steroids into the corresponding 3-ceto-A 4- steroids, a step that is essential to the formation of all ac- tive steroids. Recently, the structure of the cDNAs and genes encoding two types of human 3fl-HSD has been elucidated (Luu-The et al., 1989a; Lachance et al., 1990, 1991; Rh6aume et al., 1991; Labrie et al., 1992a). More- over, the structure of one 3fl-HSD cDNA isolated from macaque ovary has also been reported (Simard et al., 1991). This membrane-bound enzymatic system has been found in classical steroidogenic tissues, such as the pla- centa (Luu-The et al., 1989a; Rh6aume et al., 1991), ad- renal cortex (Lachance et al., 1991; Rh6aume et al., 1991), testis (Simard et al., 1991), and ovary (Labrie et al., 1992a) as well as in several peripheral tissues, includ- ing the prostate (Lacoste et al., 1990), breast (Labrie et al., 1992a), skin (Labrie et al., 1992a; Dumont et al., 1992), and many human fetal tissues (Milewich et al., 1991). In several mice (Bain et al., 1991) and rat (Zhao et al., 1991; Labrie et al., 1992a) tissues, 3fl-HSD activity has also been described. In the rhesus monkey, 3fl-HSD mRNA transcripts have been detected in the ovary, testis, and adrenal as well as in some peripheral tissues, includ- ing the male epididymis, liver, and kidney (Simard et al., 1991); 3fl-HSD activity has been reported in fetal gonads (Sholl, 1983).

The present results thus demonstrate for the first time the widespread tissue distribution of 3fl-HSD activity in all the macaque tissues examined, except the spleen and the brain. The absence of detectable 3fl-HSD activity in the cerebral cortex, cerebrum, and cerebellum is in agreement with a previous study, which reported that no significant conversion of pregnenolone to progesterone could be detected in rat cerebral cortical tissue (Weidenfeld et al., 1980). However, transformation of pregnenolone to progesterone was reported in the rat limbic brain, hippocampus, and hypothalamus (Weiden- feld et al., 1980) as well as in rat glial cells (Jung-Testas et al., 1989). There is thus a wide range of peripheral tis- sues that can transform DHEA and AS-diol into A4-dione

and testosterone, respectively. Although 5a-reductase is required to transform testosterone into the more potent androgen dihydrotestosterone (DHT), testosterone can bind and activate the androgen receptor.

The enzyme 17fl-HSD is responsible for the last step in the formation of all the active androgens and estrogens, which are essential to the development, growth, and function of all structures responsible for reproduction and fertility. The importance of this enzyme in the mainte- nance of physiologic levels of E 2 or T is supported by its ubiquitous distribution in the human (Martel et al., 1992), rat (Martel et al., 1992) and mice (Milewich et al., 1985). The present data show that 17fl-HSD, as measured by the conversion of A4-dione into testosterone (androgenic 17fl- HSD) as well as E 1 into E 2 (estrogenic 17fl-HSD), is pres- ent in all 25 tissues examined. Thus, all tissues except the salivary glands, which lack steroid sulfatase, have the enzymatic activities required to convert DHEA-S into the estrogen E 2. Moreover, all tissues except the spleen and brain, which lack 3fl-HSD, possess the enzymatic activi- ties involved in the formation of the androgen testoster- one from DHEA-S.

Most studies have dealt with estrogenic 17fl-HSD, the enzyme responsible for the interconversion of El and E 2. The structure of the cDNA encoding human estrogenic 17fl-HSD and its corresponding gene has been described by Luu-The and collaborators (1989b, 1990), and recently another cDNA encoding the human 17fl-HSD type II was characterized (Wu et al., 1993). These data confirm the existence of multiple forms of 17fl-HSD as previously suggested (Pittaway et al., 1983; MacIndoe, 1990). In the male adrenal, testis, intestine, kidney, liver and skin, we observed higher androgenic 17fl-HSD, whereas in the seminal vesicle and muscle, estrogenic 17fl-HSD was predominant. On the other hand, estrogenic 17fl-HSD in the ovary and female muscle is higher than the andro- genic counterpart, whereas the liver shows higher 17fl- HSD activity when an androgenic substrate is used. The present data support the hypothesis of the presence, at least in some rhesus monkey tissues, of more than one enzyme having 17fl-HSD activity.

The best known activity of steroid 5a-reductase is the transformation of testosterone into DHT, the most potent androgen, which is responsible for differentiation of the male external genitalia, prostate and other accessory sex organs. Recently, the structure of two cDNAs and the corresponding genes encoding 5a-reductase isoenzymes was characterized in the human (Andersson and Russell, 1990; Jenkins et al., 1991; Labrie et al., 1992b), and in the rat, two cDNAs encoding 5a-reductase isoenzymes have been described (Andersson and Russell, 1990; Normington and Russell, 1992). No data are yet available about the structure of primate 5a-reductase, but the activ- ity of this enzyme has been reported in neural tissues of the fetal rhesus macaque (Sholl et al., 1989) as well as in the fetal heart, muscle and lung (Sholl et al., 1989). The

110 C. Martel et al. I Molecular and Cellular Endocrinology 104 (1994) 103-111

present study shows a relatively large distribution of 5a- reductase, although no activity could be detected in the cerebral cortex, salivary gland, muscle, spleen, heart, mammary gland, oviduct and cervix. The presence of 5a- reductase activity in the prostate, fat, liver, skin and adre- nal has been reported in the human and/or in rat (Liang et al., 1985; Herkner et al., 1986; Zyirek et al., 1987; Lephart et al., 1991).

As shown in the present study, aromatase activity is widely distributed in the monkey peripheral tissues, the enzymatic activity being non-detectable only in the skin and pituitary gland. The significance of the absence of aromatase activity in the pituitary and skin is unknown. In fact, aromatase activity has been reported in fibroblasts of human skin (Emoto et al., 1991). On the other hand, our results confirm the presence of aromatase activity in the rhesus monkey brain (Roselli and Resko, 1989) and prostate (West et al., 1988) and extend previous studies, which have described aromatase activity in the human ovary (Kamiyama et al., 1992), adipose tissue (Frost et al., 1980), liver (Frost et al., 1980), cervix (Taga et al., 1990), endometrium (Taga et al., 1990) and breast (Miller, 1991) as well as in human fetal intestine, adrenal, spleen, muscle, kidney, heart, lung and testis (Doody and Carr, 1989; Tapanainen et al., 1989).

Taking into account the large size of many peripheral tissues, it is likely that these tissues play a major role in the formation of androgens and estrogens from a rela- tively constant supply of precursor steroids provided by the adrenals. These periphera ! target tissues thus possess the ability to adjust the rate of sex steroid formation ac- cording to their individual needs. Such data clearly indi- cate that additional research should focus on intracrinol- ogy (Labrie, 1991) to understand more fully the physio- logical mechanisms controlling local steroid formation and action. The word intracrinology was coined in 1988 when the formation of the androgens testosterone and DHT was demonstrated in the orchiectomized rat from the precursors DHEA and Aa-dione (Labrie et al., 1988). In addition to increasing our knowledge of endocrine physiology, such studies should provide useful informa- tion for the development of novel therapeutic approaches to the treatment of hormonal-sensitive diseases, which should take into account the high proportion of locally made steroids, which are responsible for the growth and function of most of the normal and neoplastic human cells and tissues.

Acknowledgments

This work was supported in part by NIH DK-41035, NIH RR-00163, NIH HD-18185 and Medical Research Foundation of Oregon as well as by the Medical Research Council (MRC) of Canada, Endorecherche and La Soci6t6 d'Investissement R&D Andros Inc. C.M. is holder of a studentship from le Fonds de la recherche en sant6 du Qu6bec (FRSQ).

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