Regulation of steroid-transforming enzymes by endogenous steroids

J. steroid Biochem. Vol. 19, No. 4. pp. 1527~-1556, 1983 Printed in Great Britain. All rights reserved

0022-4731/83 $3.00+0.00 Copyright (c~ 1983 Pergamon Press Ltd

REVIEW REGULATION

OF S T E R O I D - T R A N S F O R M I N G BY E N D O G E N O U S S T E R O I D S

ENZYMES"

D. B. GOWER and G. M. COOKE* Department of Biochemistry, Guy's Hospital Medical School, London, SE 1 9RT, U. K, (Received 14 September 1982)

Summary--The biosynthesis ofCz~, C~,~,and C~ steroids in adrenal, ovary, testis and foeto-placental unit is summarized and the properties of the enzymes involved are described, with particular reference to their substrate specificities and to the possible controlling effects of endogenous steroids. It is concluded that these may exert a considerable regulatory role in vivo in steroid hormone biosynthesis.

INTRODUCTION

During the past three decades, details of the biosynthesis of steroid hormones in adrenals, ovaries, testes and foeto-placental unit have been elucidated as a result of a wealth of experimentation both in vivo and in vitro [1-5]. Once the intermediates of a biosynthetic pathway have been worked out, it is usually the purpose of researchers to study the control of the metabolic sequence, and this may involve a study of the properties of the enzymes concerned. Such has proved to be the case for steroid hormone biosynthesis which is controlled in a multi~'Present address: The Royal Victoria Hospital, The Women's Pavilion, Department of Obstetrics and Gynecology, 687 Pine Avenue West, Montreal, PQ, Canada H3A IAI. This review of the literature was concluded in May 1982. Abbreviations and trivial names: Dehydroepiandrosterone (DHA), 313-hydroxy-5-androsten-17-one; androsterone, 3a-hydroxy-5a-androstan- 17-one; 4-androstenedione, 4-androsten-3,17-dione; 5c~-dihydrotestosterone(5c~DHT), 17[3-hydroxy-5c~-androstan-3-one: epitestosterone, 17c~-hydroxy-4-androsten-3-one; 5-androstenediol, 5androstene-313,1713-diol; 21)a(13)-pregnanediol, 5[3prcgnane-3c~,20a(13)-diol: 20c~-dihydroprogesterone, 2(Ic~hydroxy-4-pregnen-3-onc, 5 a - p r e g n a n e d i o n e , 5c~pregnane-3,20-dione: 17,20~-dihydroprogesterone, 17,20~-dihydroxy-4-pregnen-3-one; oestriol, 1,3,5(101oestratrien-3,16a,17[3-triol; oestradiol-17[3, 1,3,5(10)oestratrien-3, 1713-dio1: oestrone,3-hydroxy- 1.3.51 l(I)oestratrien- 17-one. Enzyme names--C-17,20 lyase, 17a-hydroxy-C~_t-steroid C-17,211 lyase: 11[3-OHSDH,I l[3-hydroxysteroid oxidoreductase (E.C.1.1.1.146): 17-hydroxylase, steroid hydrogen donor: oxygen oxidoreductase (17-hydroxylating), E . C . 1 . I 4 . 9 9 . 9 : 5-ene-313-OHSDH/isomerase, 313hydroxysteroid: NAD oxidoreductase (E.C. 1.1.1.51) and 3-oxosteroid-4.5-isomcrase (E.C.5.3.3.1); 17[3-OHSDH, 17[3-hydroxysteroid: NADP oxidoreductase, (E.C.1.1.l.64): 2 0 a - O H S D H , 20~-hydroxysteroid: N A D ( P ) + 2 0 / o x i d o r e d u c t a s e (E.C.1.1.1.149); 3aOHSDH, 3a-hydroxysteroid NAD(P)' oxidoreductase (E.C.1.1.1.50); 4-en-5cx-reductase, 3-oxo-5a-steroid NADP' A4 oxidoreductase: 21-hydroxylase, steroid hydrogen donor: oxygen oxidoreductase (21-hydroxylating), (E.C. 1.14.99.1111.

factorial fashion, such factors including A C T H , the gonadotrophins, prostaglandins and mono- and divalent cations [6-10]. Several other control factors are recognized but, in recent years, the possibility of regulation of steroid-transforming enzymic activity by endogenous steroids has been established and its importance increasingly recognized. It is the purpose of this review to summarize some of the properties of the various steroid-transforming enzymes in steroid-producing tissues and to survey the wealth of literature that currently exists showing the effects of steroids on these enzymes. It is, of course, potentially dangerous to extrapolate from an in vitro to the in vivo situation, and if a particular steroid inhibits the activity of an enzyme in vitro, it is necessary to consider this in relation to the K,,. value for the enzyme and the K~ value for the putative regulator and to its endogenous concentration. In this respect, although earlier references to inhibitory effects in vitro are cited, it is clear that these may not necessarily be of significance in the in vivo situation now that, in many instances, data on endogenous concentrations of steroids are available. Only a few references exist for effects of steroids on cholesterol hydroxylation and side-chain cleavage [111 and this review therefore concentrates on the abundance of papers published concerned with biosynthesis from C2~ and Ci9 precursors. Although endogenous steroids are known to have effects on steroid metabolism in liver, kidney and accessory sex organs, these data are considered to be outside the scope of the present work. EFFECTS OF STEROIDS ON STEROID BIOSYNTHESIS IN THE ADRENAL CORTEX

S u m m a r y o f steroid h o r m o n e biosynthesis in adrenal cortex

The classical pathways of the biosynthesis of corticosteroids, androgens and oestrogens in the adrenal cortex have been reviewed on numerous

1527

1528

D. B, GOWERand G. M. COOKE progesterone 4-androstenedione ]]B-OH-androstenedione testosterone PREGNENOLONE " PROGESTERONE

ll-deoxycortisol progesterone 4-androstenedione DOC

20~- and 20B-pregnenediol 17-OH-pregnenolone pregnenolone sulphate progesterone

17-OH-PREGNENOLONE- 20a- and 20B-pregnenediol progesterone 17,20-di(OH) progesterone 17-OH-progesterone

- CORTICOSTERONE testosterone DHA(S)

-0H-corticoster0ne 18-OH-CORTI COSTERONE

ALDOSTERONE

17-OH-PROGESTERONE ~ ~

D

O

progesterone C

~,,.~stradi01 II-DEOXYCORTISOL

DHA ~°est~T°estradi°l'

- 4-ANDROSTENEDIONE

regnenolone rogesterone 20a-OH-progesterone OC ndrostenedione terone

CORTISOL

5-ANDROSTENEDIOL

TESTOSTERONE"

Fig. 1. Pathways of steroid biosynthesis in the adrenals. The effects of endogenous steroids arc inhibitory.

occasions and, recently, by Whitehouse and Vinson [41. The C_~l steroid pregnenolone, formed intramitochondrially through the cholesterol side-chain cleavage system, serves as one precursor. The second, progesterone, is formed from pregnenolone by the action of the 5-ene-3/3-OHSDH isomerase system (Fig. 1). Both give rise to corticosteroids but the proportion of 17-hydroxylated steroids and 17deoxysteroids from pregnenolone and progesterone can vary depending on the species of adrenal studied [12]. Figure 1 summarizes the principal pathways and it can be seen that a number of hydroxylases are necessary which catalyse hydroxylations at C-17, C-21, C-11/3 and C-18. In addition, for completion of synthesis of aldosterone with its unique 18-aldehydic function, an 18-OHSDH was at one time thought to be necessary. Recent evidence, however, suggests that, more likely, two successive hydroxylations at C-18 occur [13] without the requirement for the 18-oxidoreductase. For biosynthesis of adrenal androgens, side-chain cleavage of 17-hydroxypregnenolone and 17hydroxyprogesterone is necessary, catalysed by the C-17,20 lyase {14]. The control of this and other enzymes involved in ovarian and testicular biosynthesis will be discussed later.

Effects o f steroids on enzymes o f the adrenal cortex The biosynthetic pathways for androgens, oestrogens and corticosteroids by the adrenal cortex are intrinsically linked, and several researchers have found that Cm and C~a steroids may regulate not only their own synthesis but also that of the corticosteroids. Of particular significance are the inhibitory

effects of Cm steroids on the 17-hydroxylase, C-17,20 lyase and 5-en-3/3-OHSDH/isomerase enzymes, as these could ensure that C2[ steroids are converted to corticosteroids. There appears to be little evidence for the regulation of either androgen or corticosterold synthesis by eorticosteroids themselves. 17-Hydroxylase. The presence or absence of the 17e-hydroxyl group in a corticosteroid is crucial to its hormonal properties and it is perhaps surprising that the metabolic control of 17-hydroxylase activity is not better understood. As is the case in the testis, this enzyme is found in the microsomal fraction, requires NADPH and 02 for activity and cytochrome P-450 is one of the components of this enzyme complex [14]. The 17-hydroxylase activity of the human adult adrenal gland was investigated using pregnenolone as substrate and the results [15] suggested that naturally occurring steroids would not influence this enzyme in vivo. 4-Androstenedionc. testosterone, oestrogens and corticosteroids were all without effect and the Km of the reaction with respect to pregnenolone was 1.04 #mol/l, However, substrate and cofactor availability was suggested as a way of regulating glucocorticoid and androgen biosynthesis [15]. However, in a similar study in which human fetal adrenal microsomes were used as an enzyme source, 17-hydroxytase activity was influenced by several C21 steroids [16} e.g. the addition of 20c~- or 20/3-pregnenediol inhibited the reaction competitively (Table 1). The presence of a 20c~-OHSDH has been determined in adrenal cytosol and thus, apart from the direct effect of 5-pregnene-3/3,20c~-diol on 17-hydroxylase, there is also the possibility of control due to the competition of these two enzymes for pregnenolone. Shibusawa and coworkers [161 also

Steroid-transforming enzymes and steroids observed inhibition of 17-hydroxylase by pregnenolone sulphate, the formation of which has recently been studied [17]. The sulphotransferase responsible for this conversion has similar properties to the adult human adrenal enzyme, and is extremely active towards D H A , pregnenolone, 5-androstenediols and oestradiol-17/3. The effects of the sulphate esters of these steroids on adrenal steroid 17-hydroxylase have yet to be examined. The inhibitory effects of 4-en-3oxo C21 steroids were less than the 5-ene-3/3-hydroxy C q steroids (Table 1). The 17-hydroxylation of progesterone was shown to tbe influenced by the presence of ll/3-hydroxylase in vitro. Ammonium sulphate fractions of rat adrenal acetone powder preparations were obtained one of which contained 17-hydroxylase activity, presumably because the component proteins were separated by the salt precipitation [18]. When two of these fractions were recombined, l l/3-hydroxylated derivatives were formed but the previously active 17-hydroxylase, no longer converted progesterone t o 17hydroxyprogesterone [18]. This may account, in part, for the preferential synthesis of cortisol from 17hydroxypregnenolone rather than progesterone [19]. C-17,20 lyase. This enzyme is also found in the microsomal fraction of the adrenal cortex, and requires N A D P H and oxygen for activity and, like its testicular counterpart, cytochrome P-450 plays an essential role in this side-chain cleavage step [14]. 17-Hydroxylation of the C2~ steroids is a pre-requisite for this enzyme to be active. The control of this enzyme has received some attention because it must be active for androgen biosynthesis but inactive for the biosynthesis of corticosteroids (see Fig. 1). The adult human adrenal C-17,20 lyase was inhibited in vitro by D H A and 17-hydroxyprogesterone when the substrate was 17-hydroxypregnenolone, the in hibition being of the non-competitive type ] 15 ]. In this study 5 a - D H T was a competitive inhibitor, but the magnitude of the inhibition constant (Table 1) led to the suggestion that in vivo, 5a-DHT would not control this step [15]. However, the feedback inhibition by D H A and 17-hydroxyprogesterone was considered possible in vivo and these steroids could regulate C~,~ and C2~ steroid biosynthesis by the adrenal cortex. Androgens, oestrogens and corticosteroids had no effect on the side-chain cleavage of 17-hydroxypregnenolone, and the same was found when Yates and Deshpande [2(I] studied this enzyme using 17-hydroxyprogesterone as substrate. Pregnenolone, progesterone and 17-hydroxyprogesterone were competitive inhibitors (Table 1). The authors [20] suggested that conversion of pregnenolone and progesterone to their 17-hydroxy derivatives by the microsomal preparation and the concomitant competition of these for the active site of the C-17,20 lyase could explain the inhibition observed and thus, in vivo, this would not constitute a regulatory mechanism, However, these findings indicate that

1529

the same enzyme complex accepts both 5-ene-3/3hydroxy and 4-en-3-oxo steroids as substrates. Other steroids that inhibited side-chain cleavage were: 5a-DHT, 4-androstene-3/3,17/3 diol, 5-androstene3/3,17/3-diol and androsterone [20]. Shibusawa and coworkers [16] also studied the side-chain cleavage of 17-hydroxypregnenolone in their investigations of the fetal adrenal cortex and found that several steroids influenced D H A biosynthesis. 20/3- and 20c~-Hydroxy-5-pregnenes were competitive inhibitors. Progesterone and its 20/3-reduced derivative were a little less inhibitory while C1,~ and Cls steroids had little or no effect. However, the cumulative effect of all these endogenous steroids would be to reduce fetal adrenal steroidogenesis considerably. The alternative pathway to the androgens, via progesterone in adult human adrenal cortex is considered to be of minor importance as the isomerization of pregnenolone is negligible [12, 19]. If this situation exists in the fetal adrenal, then inhibition of 17-hydroxypregnenolone side-chain cleavage could result in diminished androgen, oestrogen and mineralocortocoid synthesis. This aspect is considered later in the text. 3[3-OHSDH-Isomerase. The conversion of 5-ene3/3-hydroxy steroids to the 4-en-3-oxo configuration is an essential step in the biosynthesis of biologically active hormones. The human adrenal appears not to convert pregnenolone to progesterone to any great extent [19] but this conversion proceeds readily in other animals [2l]. The enzymes responsible for this step are the NAD+-dependent 3/3-OHSDH, and the isomerase, that requires no cofactor. Both activities are of microsomal origin [22] and the former is the rate-limiting step. There has been considerable debate as to whether the 3J3-OHSDH and the isomerase activities are present on the same protein, and whether several substrate specific 3fl-OHSDH-isomerase complexes exist or one multi-substrate complex. In 1964, Ewald and co-workers [23] found that, during the fractionation of bovine adrenal tissue, the relative activities of the isomerization of Ct9 and C2~ steroids were altered. Furthermore, the 3~-OHSDH activity also changed in a completely different way. These authors proposed that separate enzymes exist which have differing substrate specificities and that the 3//-OHSDH was a separate enzyme. In 1956, an isomerase that was specific for cholestenes was purified to near-homogeneity [24]. Activity towards Ct9 steroids co-purified until the ion-exchange chromatography stage. Further evidence which supports the hypothesis that several substrate specific 3//-OHSDH isomerase enzymes exist was obtained by Handler and Bransome [25] who suggested that the guinea-pig adrenal contains at least two 3fl-OHSDH's. The inhibition studies [20], outlined above, indicated that separate isomerases exist for C2t and C~9 steroids. These same workers [26] proposed that a single complex may perform both the

17-Ott-pregnenolonc

acetone powder

313+OHS"Dff-i.somerase Bovine

~5

<: lfl <10 .

_

_

{ 22

4-A 17.21 -diO H-prcgncnohmc 11.17,21 -triOfl-prcgnenoMnc

DHA

72 83 l{10

67 76 100

72 68

65 6O 7O

progesterone 4-A DttA

F

0.36 2.58

(}.25

28.5

91.4 3.36

9.07

100

% Inhibition

ll,17,21-triOtlpregncnolone

progesterone 4-A

progesterone 4-A 1lfd-OH-4-A testosterone

oestrone oestradio[- 17,8 5c~-DHT

DHA 17-OH-progesterone 5a,-DHT

0.63 0.17

pregnenolone progesterone 17-Ott-pregnenokme 5~-DHT

2.82

0.17 0.116 0.34 0.56 O.76

0.081

0.024

0.031 0.015 0.061

5-pregnene-3,8,20o~-diol 5-pregnene-3,B,20/3-diol progesterone 17,2fl3-dihydroprogesteronc 17-OH-progesterone

cow ovary cytosol

17-OH-pregnenolone pregnenolone S progesterone

5-pregnene-3p,20a-diol 5-pregnene- 3fl,20fl -diol

App. K~

(~mol/I)

progesterone 4-A DHA

m

r

0.4

1.91

0.63

0.12

--

0.013

App. K,,, (umol/I) Effector

17.2l-diOHpregnenolone

21 -OH-pregnenolonc

pregnenol{me

Adulthuman micro- / SOlVeS l" DHA

Human microsomes

17-OH-pregnenolonc

Human foetat microsomes

17-Oft-progesterone

Progesterone

Bovine microsomes

17-Hvdro.o,lase

C-I 7,20 lvase

Pregnenolone

Human foetal microsomes

Substrate

Adrenal preparation

Enzyme

Table 1. Effects of steroids on steroid-transformino enzvn3es in the adrenal cortex

13]}

12ol

[151

[2o]

[161

I481

[16]

Reference

7

e..

Ovine mitochondria

Bovine cultures

Bovine mitochondria

Human foetal mitochoudria

Bovine microsomes

micro-

corticostcrone

1 l-deoxycortisol

1 I-DOC

11-DOC

1l-deoxycortisol

pregnenolone

17-O H-progesterone

8.5

2.4

3.2

13.3

4-A, 4-androstenedione; OH, hydroxy: S. sulphate; SDH, stcroid dehydrogenase.

18-Hvdroxvlase

1l/3-Hydroxylase

21-Hydroxyhtse

Hunlan foetal

somes

[46] 40

18-OH-corticosteronc

[421

[43]

38 16 17

[411

136J

84 84 80 75 96 67 92 79 32

18.0

4-A testostcrouc DHA DHAS

77

54 69

[371

11-dcoxycor tisol cortisol DOC corticosterone 4-A 1 I/3-OH-4-A testosterone 11/3-OH-testosterone oestradiol- 17/3

3.2 23.0

1.6 11.4 24 .(I

20.0 27.5 87.5

11-deoxycortisol progesterone

} 20a-dihydroprogesterone prcgnenolone progesterone

testosterone DHA 5-androstencdiol

4-A

progesterone 1 l-dcoxycortisol oestradiol- 17/3

e.-

3

,7:

3 E

g

1532

D.B. GOWERand G. M. COOKZ

o x i d a t i o n a n d i s o m e r i z a t i o n of 5-ene-3/3hydroxysteroids. Measuring the conversion of pregnenolone and D H A to progesterone and 4androstenedione respectively, Yates and Deshpande [26] obtained similar values for K,,, and V...... in microsomal incubations. Furthermore, cyanoketone ( 2 ~ - c y a n o - 4 , 4 , 17c~-trimethyl- 17/3-hydroxy-5androstene-3-one) inhibited both conversions equally. When an equimolar mixture of DHA and pregnenolone was provided as substrate, both the K,,, and Vm,~ for the conversions were reduced. If separate complexes existed for these substrates, then the V,,,,× would have been between the two values obtained when the substrates were measured singly. In 1974, Ford and Engel [27] purified a 3fl-OHSDH-isomerase complex from sheep adrenal microsomes, that possessed activity towards DHA and pregnenolone. The available evidence [28] suggests, therefore, that either the two enzyme activities are present on one protein or two very closely linked proteins, or that separate adrenal isomerase complexes exist which have limited, if not absolute, substrate specificity. The conversion of DHA to 4-androstenedione in microsomes of human adrenals was studied by Yates and Deshpande [20]. When added to incubations, oestrogens and 5e-DHT reduced 4-androstenedione production. Both oestrone and oestradiol-17/3 were non-competitive inhibitors whereas 5c~-DHT was a competitive inhibitor (Table 11. These interactions may be important with regard to the control of oestrogen biosynthesis. It was assumed that the oestrogens inhibited the 3/3-OHSDH step, preventing the formation of 4-androstenedione and thus reducing further oestrogen biosynthesis. 5~-DHT is unlikely to have a similar effect, as the amount produced by the adrenal would be insufficient to interfere with a reaction which has an apparent K,,, only one-sixth that of its K i. However, in contrast to the human situation, administration of oestradiol benzoate to neonatal rats causes an increase in adrenal 5c~-reductase activity [29]. The bovine adrenal 3¢3-OHSDH-isomerase in homogenates was shown [30,31] to be capable of utilizing D H A , 1 7 - h y d r o x y p r e g n e n o l o n e , 21hydroxypregnenolone, pregnenolone and l l,17,21trihydroxypregnenolone or 17,21dihydroxypregnenolone, ttowever, DHA and 4androstenedione were effective in preventing the isomerization of these Ce~ steriods and thus, low amounts of these Ct,, steroids, in vivo, could impair both corticosteroid and progestin biosynthesis. 21-Hydroxylase. This enzyme is found predominantly in the microsomal fraction of all three zones of the adrenal cortex and NADPH and molecular oxygen are essential for full activity. Kahnt and Neher [321 concluded that the bovine adrenal cortex contained two distinct 21-hydroxylases, one that uses 17c~-hydroxypregnenes, as substrates, and another which accepts 17-deoxypregnenes, and furthermore

that only this latter enzyme was cytochrome P-450dependent. The regulation of this enzyme has recently been investigated intensively [32-35]. Components of adrenal cytosol were shown to stimulate the microsomal 21-hydroxylase activity [32] and subsequent research [34,35] revealed that these activators were the oxidized and reduced forms of glutathione. The relationships between ascorbic acid (which inhibits 21-hydroxylase), ACTH and these activating principles have been reviewed [28]. Steroidal inhibitors of 21-hydroxylase have been examined, and the bovine e n z y m e was f o u n d to be s u s c e p t i b l e to 4androstenedione, testosterone and DHA when 5pregnenes were supplied as substrates. When 4pregnenes were used however, these Cv~ compounds did not inhibit the reaction [36]. This control may be important for two reasons: first, if pregnenolone were 21-hydroxylated, then glucocorticoids would not be formed because 17-hydroxylation could not occur thereafter, and secondly, this explains in part why isomerization to the 4-en-3-oxo configuration occurs before 21-hydroxylation in most species studied. The human foetal adrenal 21-hydroxylase was shown to be inhibited by steroids known to be present in the foeto-placental unit. The conversion of 17hydroxyprogesterone to 1l-deoxycortisol was inhibited by progesterone, 1l-deoxycortisol and oestradio1-1713 (Table 1). However, other steroids such as cortisol, DHA and DHA sulphate, which are present in high concentration, were without effect [37]. The significance of this inhibition may lie in the role that corticosteroids play in pregnancy. Foetal corticosteroids are produced at the onset of labour and reach a peak immediately after delivery [38]. If these corticosteroids are important in the processes of birth, then the inhibition of their production by endogenous steroids would be of considerable value to the maintenance of the pregnancy prior to delivery. ll[3-Hydroxylase. This enzyme has received much attention by research workers, and it is known that it consists of three components all of which are associated with the inner membrane of adrenal cortex mitochondria [37,40]. The three components are: a flavoprotein (adrenoxin reductase), adrenodoxin and cytochrome P-450~t~. Together with NADPH and oxygen this enzyme requires Mg s~ ions tbr activity, and 21-hydroxylated pregnenes are the preferred substrates, although C1,~steroids have been shown to be l l/3-hydroxylated. ACTH stimulates this enzyme, and the histological examination of adrenal cortex has shown that the stimulation was greatest in the zone fasciculata-zona reticularis junction [12]. Shibusawa and coworkers [41] observed that foetal adrenal cortex mitochondria could I l f l - h y d r o x y l a t e l l - d e o x y c o r t i s o l , l l deoxycorticosterone and 4-androstenedione with relative activities of 3.3:3:1 respectively, the apparent K,,, values being 3.2, 2.4 and 2.5 p~mol/l, respectively.

Steroid-transforming enzymes and steroids These substrates inhibited 11/3-hydroxylation of each other, suggesting a single enzyme with multiple s u b s t r a t e affinities. The c o n v e r s i o n of l ldeoxycorticosterone was inhibited competitively by 20c~-dihydroprogesterone, pregnenolone and progesterone and, further, 20~-dihydroprogesterone and testosterone were inhibitors of the conversion of 11-deoxycortisol to cortisol. The presence of these inhibitors in the foetal adrenal mitochondrion would control the foetal output of glucocorticoids and mineralocorticoids, the possible significance being as described for 21-hydroxylase inhibition (see above). Using the supernatant from a sonicated bovine adrenal mitochondrial preparation as an enzyme source, Sharma and coworkers [42] found that 1l/3hydroxylation of 1 l-deoxycorticosterone was inhibited by 4-androstenedione, testosterone, DHA and DHA sulphate but that ll/3-hydroxy-4androstenedione was without effect. The levels of DHA and DHA sulphate in the body may be high enough to control 1 l/3-hydroxylase activity in vivo. More recently, the activity of this enzyme was found to decrease when bovine adrenocortical cells were cultured, the half-life of the enzyme being 40h [32]; addition of cortisol to the culture medium reduced this half-life to 9h. Similar effects were exerted by l l - d e o x y c o r t i s o l , l l - d e o x y c o r t i c o s t e r o n e , 4a n d r o s t e n e d i o n e , testosterone and their 11/3hydroxylated derivatives. Of interest also was the inhibition by oestradiol-17/3 which, although lower than that of the Cl~ steroids, may have some importance during those stages of the menstrual cycle when oestrogens are synthesized in much greater quantities. Thus, when the oestradiol level decreases prior to menses, l l/3-hydroxylase activity would be enhanced, cortisol levels would rise and could help combat the premenstrual tension syndrome observed in many human females. It was suggested that administration of androgens could prevent overproduction of corticosterone, thereby decreasing the possibility of hypertension [43]. 18-Hydroxylase. Aldosterone is produced by cells of the zona glomerulosa and 18-hydroxylase is found here. The zona fasciculata also contains 18hydroxylase activity, but aldosterone cannot be produced here as the presumed 18-OHSDH is lacking. Both 18-hydroxylase and the presumed 18-OHSDH (but see refs [41 and [131) are found in the mitochondria of zona glomerulosa cells and, in common with many other hydroxylases, NADPH, oxygen and cytochrome P-450 are all necessary for aldosterone production from corticosterone [14]. It has been tentatively suggested that 18- and 1l/3-hydroxylation are performed by the same enzyme complex in humans [441. The effects of steroids on aldosterone production have been reviewed [45]. In a study of the biosynthesis of 18-hydroxycorticosterone and aldosterone from corticosterone in sheep adrenal mitochondria [46], the presence of added 18hydroxycorticosterone was markedly inhibitory but

1533

aldosterone i t s e l f a n d 1 8 - h y d r o x y - 1 ldeoxycorticosterone were without effect. The formation of aldosterone in rat adrenal mitochondria was also inhibited by cortisol [451. These findings are in keeping with more recent ones [471 in which the ACTH-induced corticosteroidogenesis in rat and bovine adrenal cortex was shown to be inhibited in vitro by exogenous corticosterone and cortisol at physiological concentrations. EFFECTS OF STEROIDS ON STEROID BIOSYNTHESIS IN THE OVARY

Summary of steroid hormone biosynthesis in the ovary

The study of oestrogen biosynthesis in the mammalian ovary is complicated by the constantly changing population of cells, which occurs throughout the menstrual cycle. During the development of the follicle the cells of the theca interna are provided with an extensive capillary network, while the granulosa cells are completely avascular. Following ovulation, the granulosa cells become vascularized and hypertrophy and in some species, e.g. primates, form the bulk of the corpus luteum. Hormonal output from the ovary during the menstrual cycle varies in a precisely controlled manner. Before ovulation, the follicle produces oestradiol-17,8, the plasma level of which decreases markedly shortly before release of the mature ovum. Post-ovulation, both oestradiol17/3 and progesterone levels increase, but tbe latter to a greater extent. These two hormones are believed to act in concert to prepare the female reproductive tract for the reception of a fertilized ovum [491. The initial growth of the follicle is probably controlled by follitropin and, as the component cells develop, sensitivity to lutropin appears and the theca cells produce androgens. During early follicular development, the granulosa cells do not produce large amounts of steroid hormones. However, under the influence of follitropin, the aromatase system is induced and the granulosa cells produce large quantities of oestrogen, probably using the thecal androgen output as precursors. The oestrogens cause rapid follicular development and possibly exert an influence which enables the granulosa ceils to become sensitive to lutropin and therefore to produce progesterone and some 17-hydroxyprogesterone. Following release of the ovum, the cells of the corpus luteum secrete progesterone which will maintain the reproductive tract during early pregnancy, should fertilization take place. If this does not occur, then steroid hormone secretion decreases and follitropin levels increase in order to begin the next cycle [49]. Ovarian theca and granulosa cells are both capable of utilizing acetate to synthesize oestrogens, via the formation of C~,l and CI,~steroids, the pathway being the same as that found in the testis (Fig. 2). The conversion of C.9 steroids to oestrogens is performed by aromatization of ring A of 4-androstenedione or

1534

D.B. GowlzRand G. M. CooK~ oestradiol

testosterone

.

PROGESTERONE

PREGNENOLONE

20a-OH-PROGESTERONE

oestradiol *

l

oestradiol

17-OH-PREGNENOLONE

-

\

testosterone oestradiol

~

4-ANDROSTENEDIONE

DHA

testosteronp oestradiol

5-ANDROSTENEDIOL

17a-HYDROXY-5~-PREGNANED!ONE

17-OH-PROGESTERONE

.

OESTROGENS

~-DHT

5,~-Andeostanealone

L TESTOSTERONE~

Fig. 2. Pathways of steroid biosynthesis in the ovaries. The effects of endogcnous steroids arc inhibitory except for those denoted by *, indicating augmentation. of testosterone. This cytochrome P-450 dependent enzyme complex is located mainly in the microsomal fraction, requires molecular oxygen and NADPH for maximal activity [14]. Current ideas on the mechanism of aromatization favour two sequential hydroxylations at C-19 of 4-androstenedione, followed by 2/?-hydroxylation. Isolatable intermediates would be 19 - h y d r o x y a n d r o s t e n e d i o n e , 19-oxoandrostenedione and 2#3-hydroxy-19-oxoandrostenedione. This collapses non-enzymically to form oestrone. The first two hydroxylations may occur at a single active site, the third being at a site remote from this and therefore being the ratelimiting step [501. The non-enzymic collapse of synthetic 2fl-hydroxy-19-oxo-androstenedione has been demonstrated [51] and the oestrone produced retained the le-hydrogen, as is the case in oestrogen biosynthesis. Oestrone is converted to oestradiol-17/3 by a 17/3-OHSDH, that requires a nicotinamide eofactor for activity [52]. As outlined above, the peptide hormones are instrumental in the control of the menstrual cycle, but steroid hormones could also contribute to the regulation of oestradiol-17/3 and progesterone biosynthesis. Lutropin has been shown to raise ovarian c-AMP levels when administered to immature female rats and this was followed by an increase in progesterone [53]. This work verifies earlier observations [54], in which lutropin was shown to cause an increase in rat ovarian progesterone biosynthesis and secretion. Follicular development, was accompanied by elevations of 5c~-reduced pregnanes suggesting the activation of 5c~-reductase activity. However, the post-ovulatory progesterone increase was not accompanied by these 5c~-reduced metabolites [54]. In other tissues 5c~-pregnanedione has been shown to inhibit competitively the sidechain cleavage of C~ steroids [55] but it is not yet known if the ovarian lyase is similarly affected.

More recently, Hillier and De Zwart [153} have provided evidence suggesting the involvement of androgens in the induction/activation of granulosa cell aromatase by follitropin. When granulosa cells were isolated from ovaries of oestrogen-pretreated immature rats, aromatase activity was negligible and remained so, even after a 48h culture period with the addition of human follitropin. However, if testosterone was included in the culture medium with the gonadotrophin, then aromatase activity was stimulated to levels equivalent to those found in proestrous follicles of cycling adult rats. Furthermore, the degree of stimulation was dependent upon the testosterone concentration. Other androgens which stimulated aromatase were 4-androstenedione, 5c~-DHT and 3a- and 3/3-androstanediols, although these were not as effective as testosterone. These and other findings indicated that the induction and/or activation of granulosa cell aromatase by follitropin is an androgen receptor regulated process in vitro. This leads to the interesting possibility that granulosa cell aromatase induction is controlled by extracellular androgens through a receptor mechanism, and its activity is regulated by intracellular androgens by direct interaction with the enzyme protein. Ef#ects of steroids on enzymes in the ovary. It is of interest, therefore, that naturally-occurring steroids have been shown to influence the activities of some of the steroid-transforming enzymes. Recently [591. oestradiol-17/3 was shown to inhibit androgen production by directly affecting the 17-hydroxylase activity (Table 2) but it did not interfere with the binding of hCG to ovarian cells, nor did it cause the number of androgen-producing cells to change (as measured by 3/~-OHSDH staining). However, a 90"/o decrease in 4-androstenedione and testosterone synthesis was observed. C o n c o m i t a n t l y , 17hydroxypregnenolone levels were diminished but progesterone levels increased 30%. It is possible that

17-OH-progesterone

75-day old rat ovarian microsomes

Rat corpus luteum cytosol

Rat ovarian granulosa cells

progesterone

testosterone

rat ovarian cell suspen- 17-epitestosterone sion

testosterone

prcgnenolone

pregnenolone

75-day old rat ovarian microsomes

Rat corpus luteum microsomes

OH, hydroxy; SDH, steroid dehydrogenase.

2&x-OHSDH

Aromatase

5e~-Reductase t

17~,-OHSDH

denovosynthesis

Substrate

Bovine ovarian micro- progesterone somes

Rat ovarian cell suspension

Ovarian preparation

Human corpus luteum 3~-OHSDH-isomerase minces

17-Hydroxylase

Enzyme

0.(14

0.78

Induction of 20ocOHSDH inhibited

app. K,,, changed to 0.11 5c~-androstanedione oestradiol administration

app. K,,, changed to 0.06

47 49 35

5a-DHT

progesterone 17-OH-progestcrone oestradiol-17[3

61)

neonatal oestradiol propionatc

-

-

6O

neonatal testosterone propionate

-

neonatal oestradiol propionate

--

59

neonatal testosterone propionate

--

/

[64]

[62]

[611

[29]

t[29]

[64]

Enzyme activity maintained

oestradiol- 17[5administration 63

[6o]

40-50

[4Sl

100

cow ovary cytosol testosterone

[59]

Reference

7O

% Inhibition

ocstradiol- 17,8

App. (/xmol/l) Effector

K,,,

Table 2. Effects of steroids on steroid-transforming enzymes in ovarian tissues

v.

N

rg~

p,

1536

D.B. GOWERand G. M. CooKl

oestradiol-17/3 interferes with the cytochrome P-450 of 17-hydroxylase but this has not been confirmed. The isomerase of human corpora lutea may be influenced by steroids. When minces of this tissue were incubated with pregnenolone as substrate, and with additional hCG (10/} U/ml), progesterone production was inhibited 4(}-50% when testosterone (3.5 /xg) was present and 5-ene-3/3-hydroxysteroids accumulated, compared with control incubations in which either hCG, testosterone or both were omitted [601. As mentioned previously, 5c~-reduced steroids are synthesized during follicular development but are s u b j e c t to c o n t r o l by s t e r o i d s . W h e n 17epitestosterone was supplied as substrate, to rat ovarian suspension cultures, the conversion to 5orreduced metabolites was inhibited by progesterone and 17-hydroxyprogesterone by almost 50% [61] but testosterone, oestrogens and 5c~-reduced metabolites were less inhibitory. When homogenates of these cells were examined using the same substrate, only oestradiol-17/3 had no effect whereas the other steroids were still inhibitory. In order to explain this observation, it was proposed that oestradiol- 17/3 may interfere with membrane transport or may be cytotoxic at the inhibitory concentrations. However, it must be remembered that follicular fluid contains appreciable quantities of oestrogen and this may be important in regulating the synthesis of 5~-reduced metabolites. The possibility that C21 and CL~j steroids are reduced by the same 5c,-reductase has some significance, as Hillier and coworkers [621 demonstrated that 5c+-reduced Ct{+ steroids inhibit the aromatization of androgens by rat ovarian granulosa cells. These cells were isolated and incubated with t e s t o s t e r o n e , 4 - a n d r o s t e n e d i o n e a n d 19hydroxyandrostenedione, all of which proved to be suitable substrates for oestrogen bioswlthesis. The apparent K,,, for testosterone was increased to different extents when 5c~-DHT or 5c~-androstanedione ( 1(}0nmol/l) were included in the incubation medium (Table 2). It would appear that steroids may attenuate the activities of ovarian enzymes, and control the a m o u n t s of o e s t r o g e n , p r o g e s t e r o n e , 17hydroxyprogesterone and androgen produced by this tissue. The relationship between the aromatase and 5et-reductase may be particularly significant in that the products of these enzymes can exert inhibitory effects on steroid biosynthesis as explained above. They may be involved in the age-related changes in ovarian steroidogenesis in rodents [154, 1551 where immature animals produce predominantly 5~reduced steroids, but in adult rats 4-en-3-oxo steroids and oestrogens are synthesized in greater quantities. The inhibition of lutropin release by steroids with the 5-erie configuration may also contribute to the control of ovarian steroidogenesis in vivo [631. However, there is need for further investigation

along these lines as it is not yet known precisely which steroids are produced by the theca cells and granulosa cells at the various stages of the menstrual cycle. The interactions between these cells and the hormones they produce may provide important control mechanisms. V;FFEUTS0~"STEROIDSONSTEROIDBIOSYNTHESISIN PLACENTAANDFOETALTISSUES Summa U qf steroid horntone biosynthesis in ./beto placental unit The development of the placenta is essential for the maintenance of mammalian pregnancy. It has many functions, e.g. it supplies oxygen and nutrients from the maternal circulation, it excretes foetal CO: and other waste products, it serves as a barrier against infection, and of particular interest here, it produces peptide and steroid hormones in appreciable quantities. Human chorionic gonadotrophin (hCG) is released by the developing placenta to stimulate the corpus luteum to produce oestradiol17/3 and progesterone, which are needed to ensure that the endometrium will retain the pregnancy. When the placenta is established it produces these steroid hormones itself and the corpus luteum quiesces. The quantities of progesterone and oestrogens synthesized by the placenta are much greater than those evident during the menstrual cycle, and are sufficient to prevent ovulation [49]. The placenta does not synthesize cholesterol from acetate, but uses maternal plasma cholesterol for s t e r o i d o g e n e s i s . The side-chain cleavage of cholesterol occurs readily and the pregnenolone produced is converted to progesterone (Fig. 3}. 17-Hydroxylase and C-17.20 lyase enzyme activity is not evident and therefore the placenta does not synthesize (7~,~steroids. Instead, the progesterone is metabolised by foetal and maternal tissues, while pregnenolone is converted to its sulphate ester and to DHA sulphate by the foetal adrenal gland. Enzymes of the placenta cleave the sulphate group and cause isomerization of these steroids so that the pregnenolone sulphate gives rise to progesterone; DHA sulphate is a precursor of oestrogens. Thus, the placental unit synthesizes large quantities of progesterone and oestrogens. The maternal liver reduces progesterone to 5c~-pregnanediol which is excreted as the glucuronide conjugate, whilst DHA is 16~hydroxylated by the foetal liver and converted to oestriol by the placenta, and this oestrogen is also excreted in maternal urine. Placental and foetal well-being can thus be monitored by the maternal plasma progesterone and oestriol concentrations, and their urinary metabolites [65 l. Throughout gestation, maternal plasma progesterone and oestrogen levels increase steadily, progesterone reaching a maximum shortly before parturition (>170 ng/ml) and oestradiol-17/3 still rising to 10 ng/ml at parturition [66]. Steroid hormones are thought to initiate the onset of labour by virtue of

Steroid-transforming enzymes and steroids

1537

FOETUS

/

/

CHOLESTEROL

PREGNENOLONE~ SULPHATE

PREGNENOLONE ....

,- PROGESTERONE

progesterooe

I

I

\

l 7-OH-pregesterone / pregnenolone sulphate/

[

\

\

5-pregnenediol (200 /

l 7-HYDROXYPREGNENOLONE SULPHATE ,

and 20B) 17 OHPR -- - - EGNENOLONE

\

/ / ~

X ~

5e-PREGNANEDIOL

\ \ ~ IT_OH_PROGESTERONEX

/ progesterone DOC / l 7-OH-progesterone I / 20~- and 208-0HIpregnenolone

DHAS,.

X

\

\

\n~ ~ ~. . . . . \ / pregneno]one Jprogesterone \ P - ~ ~....... . \ / ZOs-OH-progesterone |ll-deoxvcortisol ~ - eoxycortisol\ / DHA ~20~-OH-progesterone \ oestradiol \ / 5-androstenediol I X / 16-OH-OHA I " ~ / testosterone ~ ~ ~(oestradiol CORTICOSTERONE ~ - - DHA I ]I-DEOXYCORTISOL ~g~tosterone regnenolone (sulphate) 18-OH-CORTICOSTERONE 20e-OH-progesteron( OHAS stenedioI A!OOSTERONE CORTISOL

i

/

~-OHAS PLACENTA

PROGESTERON[" progesterone [20~-OHIprogesterone IDHA / cortisone oestrone

PREGNENOLONE

~

OHAS

~

16~-OH-DHAS

CORT[SOL i progesterone ll~-OH-progesterone cortisone

,pregnenolone I (sulphate) 4-androstenedione

DHA

gl-OH-pregnenolone

20~-OH-progesterone progesterone !oestradiol 'oestrone sulphate

corticosterone

CORTISONE 16e-OH-DHA

\

16~-0H-4- ANDROSTENEDI ONE DHA prognenolone progesterone 20a-OH-progesterone 4-androstenedione ll9-OH-4-androstenedione testosterone

l

OESTRIOL

/ 5~-PREGNANEDIOL

- OESTETROL

I

4-ANDROSTENEDIONE testosterone ll9-OH-4-androstenedione ]l,4-androstadienedione 15~-DHT Ioestrone

5e-androstandedione

OESTRONEq

oestradiol, oestriol, oestrone

~ OESTRADIOL

Fig. 3. Pathwaysof steroid biosynthesisin the foeto-placentalunit. The effectsof endogenoussteroids are inhibitory. The foetal adrenal isomerase for pregnenolone to progesterone is of very low activity.

1538

D.B. GOWERand G. M. CooKi

their metabolism being altered in the later stages of gestation [156]. In the sheep it is believed that progesterone levels decrease and oestrogen and cortisol increase and the relationship between these alterations releases prostaglandin F2~, causing labour contractions. The situation in the human is unclear. Maternal urine thus contains large quantities of pregnandiol (approx. 40 rag/day) and oestriol (approx. 30 rag/day) at term. Oestrone and oestradiol171/ urine levels are lower ( < 5 rag/day) [67]. The placenta is therefore both a very large and very active endocrine gland. The relationship between the foetal, placental and maternal steroidogenesis is complex and a simplified scheme is presented in Fig. 3. The enzymes catalysing these reactions have been studied and some naturally occurring steroids may regulate their activity in vitro. Effects of steroids on enzymes of placental and fbetal tissltes 5-ene-3/3-OHSDH/lsomerase. An important step in placental steroid metabolism in the placenta is the conversion of pregnenolone to progesterone. The activity is found predominantly in the microsomes and there is some evidence suggesting that the enzyme complex is unstable to freezing and thawing, and that in placentae that are sulphatase-defieient, this instability is more pronounced I681. It has been suggested that sulphatase deficiency is caused by membrane defects [69] and this may account for the relative instability of the 3/3-OHSDH-isomerase in these placentae, especially if these two activities (isomerase and sulphatase) are closely linked in the endoplasmic reticulum. Using the 10,1100 g supernatant of human term placenta as enzyme source and D t t A as substrate, Townsley I701 observed that 3/3-OHSDH activity (eatalysing the rate-limiting step) was inhibited by 4-androstenedione, progesterone and pregnenolone. The two former inhibitors were non-competitive while pregnenolone was a competitive inhibitor with an apparent K~ appreciably higher than the apparent K,,, for DHA (Table 3). Of interest also was the finding that other 3/3hydroxysteroids (e.g. 5-pregnene-3//,20a-diol, 3//hydroxy-5~-androstan-17-one, oestradiol-17fi and oestrone) had very low inhibitory properties, suggesting that the enzyme is relatively specific for D t t A and pregnenolone. At concentrations in excess of those found in the human placenta (5/xmol/l) testosterone, 19-hydroxy-4-androstenedione and 20c~dihydroprogesterone inhibited conversion of DHA by more than 65')~;. Townsley [70] suggested that progesterone could regulate oestrogen biosynthesis, as placental levels would be higher than the apparent K i of 1.5 /\moll1. It was also considered that oestrogen biosynthesis from conjugated precursors would be regulated by the effects of steroids on the sulphatase, and that control of 3/3-OHSDH would be necessary to prevent oestrogen biosynthesis from free steroids.

An enzyme with similar properties to the placental 3/3-OHSDH has been examined in foetal membranes. The chorion was found to be more active than the amnion with both DHA and pregnenohmc as substrates [71]. The enzyme was inhibited by 4-cn-3oxosteroids (including cortisone) and, to a lesser extent, by oestrogens (Table 3). The conversion ol pregnenolone was affected to a greater extcnt than that of D | t A , suggesting that progesterone biosynthesis would be diminished more than that of 4androstenedione. Sehwarz and co-workers 1721 haxc intimated that progesterone has a role in initiating the onset of labour, and foetal membrane steroid metabolism may bring about the necessary change. The inhibition of progesterone biosynthesis by cortisone is a possible method, because the cortisone would be produced by the placental 11/3-O|tSDt[ (see later) from cortisol, and a progesterone -withdrawal'" in the foetal membranes may trigger prostaglandin release from the decidua [73]. Further research is required to elucidate the complex relationships between foetal, maternal and placental steroid biosynthesis and prostaglandin release at the onset of labour before the process is made clear. Sulphatase. The foetal adrenal possesses an active sulphotransferase which is responsible for the high levels of DHA sulphate in the foetal circulation. This ester follows either of two major metabolic routes [49]. It can be converted to DHA by the placental sulphatase and thence to oestrone and ostradiol, ol it can be C-t6 hydroxylated by the foetal liver first, and then converted to oestrioi under the influence of sulphatase, isomerase and aronaatase (Fig. 3). Great importance is attached to these two major routes as the former generates oestrogens with considerable hormonal activity but the latter results in a relatively ineffective hormone which is easily excreted in maternal urine, As steroid sulphates have no endocrine activity per se, these pathways protect the developing foetus from exposure to potent viri/iAng and feminizing influences. It follows that the sulphatase step should be strictly controlled, and the human placental sulphatase has been shown to be influenced by endogenous steroids which will assist in the regulation of steroid sulphate levels. Townsley and co-workers [74] assayed sulphatase, 3fl-OHSDH isomerase and aromatase activities m human placental microsomes and found the relative enz\ me activities to be 700:100:2(} nnlol product/rain mg protein respectively. The sulphatase was hrhibited by many steroids which are present m cord blood and placenta. Progesterone. 20~-dihydroprogesterone, 16-hydroxyDHA and oestriol were all t\mnd to inhibit sulphatase activity at concentrations equivalent to the placental endogenous levels (Table 3). Furthermore, if two or more effectors were added to the in vitro incubations, then cumulative inhibition was observed. The authors [741 suggested that. m vitro regulation of sulphatase activity would be

Steroid-transforming enzymes and steroids exerted by potential substrates, progestagens, corticoids and by the products of sulphatase activity, acting in concert. In a later study, it was proposed [75] that inhibitors of placental sulphatase should have the following characteristics: an oxygen function on ring D or the ring D side-chain, a sulphate binding site (C-3 or C-17) and an oxygen function, and the 5-ene or 5~-configuration, since planar steroids were considered to be generally more inhibitory. Steroids with apparent K i values the same or lower than the apparent K,, for D H A sulphate were D H A , 4-androstenedione, oestradiol-17/3, progesterone, pregnenolone sulphate and oestrone sulphate (see Table 3). Regulation of placental sulphatase activity by endogenous steroids could maintain steroid sulphate levels and control the amounts of oestradiol-17/3 and oestrone produced by the placenta during gestation. However, the rise in oestrogen content prior to parturition could be due to de-inhibition of this enzyme and it is noticeable that oestrogens, oestrogen precursors and cortisol are less inhibitory than C2~ steroids, but some control over sulphatase activity would still be evident. Oestrogen biosynthesis itself would therefore be increased in a controlled manner. As mentioned previously, placental sulphatase deficiency is possibly caused by a defect in membrane structure rather than by the absence of enzyme protein or by the presence of cytosolic inhibitors [69]. When sulphatase activity was detergent-solubilized from "sulphatase-deficient' placentae, the low activity could be enhanced by the addition of phosphatidylcholine but not by several other phospholipids. 'Normal' placental sulphatase activity could also be enhanced in a similar fashion. It was also observed [69] that "sulphatase-deficient' placentae possessed a single sulphatase enzyme whereas in normal placentae a second, albeit less active, sulphatase of lower molecular weight was evident. However, little significance was attached to this finding by the authors, presumably because of the low specific activity (4.2 compared to the major peak 83.3 nmol DHAS hydrolysed/min/mg protein). Aromatase. Following the quiescence of the corpus luteum, the placental aromatase is responsible for the biosynthesis of oestrogens. This microsomal enzyme complex has been studied by several workers, and the most recent mechanism, elucidated using placental tissue, is that already outlined earlier [50, 51]. DHA and 16~-hydroxyDHA, released from their sulphate conjugates by placental sulphatase activity, are thought to provide the major sources of substrate for aromatization, provided they are converted to the 4-en-3-one configuration. D H A leads to oestrone and oestradiol-17/3 but 16a-hydroxyDHA is metabolized to oestriol. The aromatase of human term placental microsomes is inhibited in vitro by several C,~ steroids, especially by those with the 4-en-3-oxo configuration [76]. Oestrogen precursors were less inhibitory and oestrogen and progesterone were poor s n 19/4

K

1539

inhibitors (Table 3). The authors [76] concluded that compounds which inhibit 3/3-OHSDH (e.g. C2~ steroids. see above) were poor inhibitors of aromatase. The observation that 5 a - D H T inhibited aromatase activity in this study has been verified by Siiteri and T h o m p s o n [77] who s h o w e d that this nonaromatizable androgen reduced activity by 82% when 4-androstenedione was supplied as substrate (Table 3). Testosterone similarly inhibited aromatization of 4-androstendione by 88%. These workers [77] also observed that ovarian aromatase and 5ereductase activities vary when hCG and PMSG are administered to rats. The competition between these two enzymes for 4-androstenedione and testosterone, coupled with the inhibition of aromatase by 5a-reduced C~9 steroids could provide control over oestrone and oestradiol-17/3 biosynthesis in the placenta. More recently, the substrate specificity of the human placental aromatase has been studied [781 and it was concluded that the same complex utilized 4-androstendione and testosterone, since the apparent K, for the inhibition of 4-androstenedione aromatization by testosterone was the same as the apparent Km of testosterone aromatization. Testosterone also markedly inhibited (90%) the conversion of D H A to oestrogens in perfused human placenta [791 with oestriol inhibiting to a slightly lesser extent [75%). This conflicts with earlier work [76] when oestriol was found to be a poor inhibitor of microsomal aromatase in vitro using 4-androstenedione as the substrate. Since oestriol was a poor inhibitor of 3/3-OHSDH in placental microsome incubations [70], this steroid may prevent oestrogen synthesis at a different level. The control of the relative activities of sulphatase, 3/3-OHSDH-isomerase and aromatase by placental steroids may be of fundamental significance to foetal and maternal well-being, but further research is necessary before this importance can be fully appreciated. 17~-OHSDH. The placental enzyme that catalyses the conversion of a 17-oxo group to a lT/3-hydroxyl group is quite different from the testicular enzyme. It is quite specific for phenolic steroids and has been isolated from the cytosol rather than the microsomes [81]. In the interconversion of oestrone and oestradio1-17/3 similar K,, values were obtained for both ()estrogens [81, 821 and inhibition by endogenous steroids appears to be limited. 4-Androstenedione and progesterone inhibited oestrone biosynthesis from oestradiol-17/3 and product inhibition was also evident. Oestriol was found to inhibit oestradiol-17~ production slightly so that, at high concentrations, oestriol may not prevent over-production of oestradio1-17/3 in the placenta during gestation. If this were so, since oestriol levels increase markedly as the pregnancy progresses, one would expect that oestradio1-17/3 levels would decrease. In fact, this does not occur, so that this level of control must be treated with caution.

II/3-OHSDH

17fl,OHSDH

3(3-OHSDHisornerase

Enzyme

3,2

0.3 0.3

cortisone cortisone

0.3

14.0 12.0

1.8

1.9 0.71 0.71

--

0.33

11-oxoprogesterone tetrahydroeortisone

corticosterone progesterone 11a-OH-progesterone testosterone 5a-DHT

cortisone corticosterone progesterone 11a-OH-progesterone

1,3,5(10), 16-oestratetren-3-ol

1,3,5(10)-oestratrien-3-ol

5,16-androstadien-3/3-ol

oestrone oestradiol-17/3 oestriol

progesterone 20tx-dihydroprogesterone DHA cortisone oestrone

4-A progesterone pregnenolone testosterone 19-OH-4-A 2(/e~dihydroprogesterone

app. K,,; 0*mol/I) Effector

cortisol

cortisol }

Human microsomes

Human choriodecidua microsomes

oestradiol- 1713 oestrone

17/3-oestradiol (3-methyl ether)

Human placenta

Purified enzyme (human)

} oestrone°eStr°ne°eStradi°l-17/3

Pregnenolone

DHA

Substrate

Human cytosol

Human microsomes

Human postmitochondrial supernantant

Placental preparation

Table 3. Effects of steroids on steroid-transforming enzymes in the placenta

m

m

m

m

1.8 6.0 0.04

3.7 3.3 5.0

0.6 1.5 4.5

app. K, (~mol/l)

97 70

100 75 89 50 50

80 100 65 99

m

84 85 80 69 64

70 69 65

% Inhibition

[s41

[84]

I821

[851

[811

(71]

[711]

Reference

© ©

©

e'~

@

©

U~

Steroid-transforming enzymes and steroids

t~tt~

~

~

o

~

IIII ^

.~

II ~

°

~

II

II

o

~

'~,

0

~0

~

~

O

O ~J3

<

<

<

~

~

o

o

E N

© ~

4

<"

-4

1541

ll/3-OHSDH. This enzyme has been found in both the placenta and the decidua and is of microsomal origin [83]. The amnion does not express 11/3OHSDH activity and the chorio-decidual activity is thought to be decidual rather than chorionic. The conversion of cortisol to cortisone was much faster in decidual tissue (3805 + 768 pmol/mg/min) than in placenta (861 _+ 158) but the placental enzyme has a lower Kin. Although the decidual enzyme could operate in both directions, the placental 11/3OHSDH could only convert cortisol to cortisone [84]. In microsomal incubations the enzyme from both tissues was inhibited by progesterone and corticosterone and product inhibition was also evident. The decidual enzyme, however, was much less sensitive to cortisone (20% inhibition) than the placental enzyme (80%) (Table 3). Oestrogens were found to be poor inhibitors of both enzymes but testosterone and 5a-DHT inhibited only the decidual enzyme. The authors [84] concluded, therefore, that the enzymes in these two tissues are quite different. As mentioned previously, corticosteroids are present in relatively high amounts at term, and it has been suggested [73] that cortisol, either directly or indirectly, triggers prostaglandin release. The placental enzyme may thus protect the foetus from exposure to maternal cortiso[ by converting it to the less active cortisone. The decidua, however, may require high cortisol levels for initiation of prostaglandin release and could use placental cortisone, convert it to cortisol and be free of inhibition by its product. Prior to the onset of labour, the decidual enzyme could be inhibited by progesterone and other steroids found in the foeto-placental unit. When the levels of these inhibitors are reduced, perhaps by activation of 17-hydroxylase and C-17,20-1yase by cortisol, this inhibition would be diminished. Thus, cortisol in the placenta would be detoxified and cortisone in the decidua could be converted to cortisol. Prostaglandin Ft,~ release from the decidua could follow and labour commence. High levels of oestrogens would not interfere with this mechanism, and a decrease in progesterone, either by protein binding or by metabolism, would further relieve 11/3-OHSDH from inhibition, thus accelerating this cycle by the generation of even more cortisol. 16a-Hydroxylase of foetal liver. The 16hydroxylation of DHA sulphate by the foetal liver is an important step in foetal steroidogenesis as this directly affects the quantities of oestrone, oestradiol17/3 and oestriol which can be synthesized in the placenta. For this reason it will be discussed here. 16-Hydroxylase is a microsomal enzyme which requires NADPH and O~ for activity [14] and it is likely that cytochrome P-450 is involved in the mechanism. The enzyme in foetal liver at mid-trimester has been examined and DHA and pregnenolone were shown to be suitable substrates [79]. Hydroxylation of both these steroids was inhibited by 5-androstenediol and hydroxylation of pregnenolone was inhibited by

1542

D.B. GOWERand G. M. COOKI

D H A while that of D H A was inhibited by pregnenolone. 16-Hydroxylated steroids and progesterone had apparent Ki values that were much higher than the apparent K,, values for D H A and pregnenolone. Oestrogens were generally poor inhibitors except that oestradiol-17/3 prevented 16-hydroxylation of pregnenolone but not of D H A . This, and other results, suggested the existence of separate binding sites for C21 and C1,~steroids on the enzyme [801. The lack of inhibition by endogenous steroids, found in high concentrations in the foetoplacental circulation, may negate the accumulation of hormonally potent androgens and oestrogens that may be deleterious to the ma:.ntenance of the pregnancy. Foetal adrenal 5-ene-3~-OHSDH-isomeruse. The human foetal adrenal cortex develops within the first few weeks of pregnancy, and becomes differentiated into an inner "foetal" zone and an outer "definitive' zone. The definitive zone remains undifferentiated in utero, but, following birth, forms the three layers of the adrenal cortex. The foetal zone is active during foetal life, and disappears after birth {157,1581. Both zones possess 17-hydroxylase, C-17,20-1yase and sulphokinase but these are thought to be active only in the foetal zone in utero. The inability of the foetal adrenal to convert 5-ene-3/1-hydroxy steroids to the 4-en-3-oxo configuration is thought to be due to suppression of 3/1-OHSDH-isomerase activity or to very low enzyme levels in the tissue 1158]. This enzyme has been detected in human foetal adrenal homogenate incubations. Villee and Driscoll [1591 observed that pregnenolone was converted to 4androstenedione and ll/3-hydroxy-4androstenedione, but that no cortisol or corticosterone were formed. If progesterone was provided as substrate, cortisol and corticosterone were synthesized, and the authors [159] concluded that the 3~-OHSDH for pregnenolone a n d 17hydroxypregnenolone was absent but that the 3/,PREGNENOLONE PREGNENOLONE SULPHATE pregnenolone testosterone 5-pregnenediol testosterone oestradiol

17-OH-PREGNENOLONE SULPHATE

DHAS

pregnenolone

]7-OH-PREGNENOLONE

EFFECTSOF STEROIDSONSTEROIDBIOSYNTHESISINTill'; TESTIS

Summary of steroid hormone biosynthesis and metabolism in the testis It is now recognised that the compartments within the testis which are responsible for steroid hormone biosynthesis are the Leydig cells of the interstitia and the Sertoli cells of the seminiferous tubules. Both compartments possess the necessary enzymes for androgen production, but the interstitial cells posscss much greater quantities of most of these enzwnes [86]. The distribution of testosterone in mature rat testis tissue suggests that the interstitial cells arc thc primary site of testosterone production. Podesta and Rivarola [87] found that the interstitial tissue contained 51 ng per testis of this steroid compared with only 8.5 ng per testis in the seminiferous tubules. However, Sertoli cells are capable of producing some testosterone from progesterone, but the major PROGESTERONE

Oestradiol

]7-OH-PROGESTERONE

/

DHA

DHA testosterone Sa-pregnanedio]s

5-ANDROSTENEDIOL SULPHATE oestradiel

O H S D H for C1,~steroids was evident. This supported the earlier observation that foetal adrenal homogenates converted D H A to 4-androstenedionc with conversions ranging from 3 to 24%. However thc activity did not correlate with foetal age [ 160]. In in vivo studies in which second trimester foetuses were infused with Cat and CI,~ 5-ene-3-hydroxysteroids, no 4-en-3-oxo steroids were produced [158]. This indicates that any 313-OHSDH-isomerase enzyme in the adrenal is inactive. This suppression could be mediated in utero, by circulating levels of progesteronc, oestrogens and eorticosteroids, all of which inhibit the isomerization in other tissues. Itowevcr. as decreased isomerase activity extends into neonatal life, when maternal progesterone and oestrogcns have disappeared from the circulation of the infant, inhibition by these steroids cannot fully account for this lack of enzyme activity.

20m-OH-progesterone 17,20~-diOH-progesterone oestradiel progesterone

4-ANDROSTENEDIONE--

~ OESTRONE

testosterone oestradio~

5-ANDROSIENED]OL

- TESTOSTERONE

OESTRONE oestrone testosterone

--QESTRADIOL~

~ OESTR#O[OL testesSULPHATE terone o~strone sulphate

Fig, 4. Pathways of steroid biosynthesis in rat testes. The effccts of endogenous steroids arc inhibitor v except for those denoted by 4, indicating augmentation.

Steroid-transforming enzymes and steroids metabolites are 20~-dihydroprogesterone (71%), 3e-hydroxy-5~-pregnan-20-one and 50c-pregnane3o~,20o~-diol [88[. Sertoli cell androgenesis may be significant in the maintenance of spermatogenesis, but the importance is not yet fully understood. Androgens are produced from pregnenolone by enzymes of the endoplasmic reticulum, and two possible pathways exist: the 5-ene-3/3-hydroxy pathway and the 4-en-3-oxo pathway [Figs 4-6]. The predominance of either pathway seems to be speciesdependent. The 4-en-3-oxo pathway is predominant in the testes of rat [89] and mouse [90] and the 5-ene-3/3-hydroxy pathway is predominant in the dog [91], the human [92-94] and the rabbit [901. The biochemical stages of testosterone biosynthesis from C2] precursors involve: hydroxylation at C-17, sidechain cleavage to yield the Cl9 17-oxosteroid, isomerization of the 5-ene-3/3-hydroxy configuration and reduction of the C-17 ketone. If the double bond at C-4 of testosterone is reduced to the 5aconfiguration, the product is 5c~dihydrotestosterone. This occurs in target tissues where 5a-dihydrotestosterone is a more potent androgen (for review see refs. [3] and [95]). The order in which these reactions takes place depends on the predominant pathway in the animal, but C-17 hydroxylation must precede side-chain cleavage if an active hormone is to be produced. The control of androgen biosynthesis by the peptide hormones released by the anterior pituitary has been fully documented elsewhere [1,7,9,10] but naturally-occurring steroids have been shown in vivo and in vitro to influence the enzymes of androgen biosynthesis. These effects may be of some significance in the overall regulation of hormone biosynthesis in the testis, by attenuating the activity of existing enzyme protein.

1543

EfCects of steroids on enzymes of the testis 17-Hydroxylase. In the rat testis this microsomal enzyme requires N A D P H and molecular oxygen for full activity [96] and is associated predominantly with s m o o t h - s u r f a c e d microsomes, the distribution approximating that of cytochrome P-450. The involvement of this cytochrome in 17-hydroxylase activity is now widely accepted. The enzyme appears 24h after the plasma follitropin peak in rats (i.e. the 30th day of life) [97] and accordingly, plasma testosterone increases, hCG administration in a single dose depresses activity but repeated administration enhances activity. A protein of the cytosol has also been found which increases testicular 17-hydroxylase activity in microsomal incubations [98[. The apparent K,,, was unchanged and stimulation was specific to testis, as the cytosols of other endocrine tissues did not influence the enzyme. When rats, pretreated with hCG, were treated with testosterone or oestradiol benzoate, the 17hydroxylase activity of the testis was considerably reduced, but these steroids inhibited by differing mechanisms. In hypophysectomised rats, only oestradiol benzoate inhibited, suggesting that plasma t e s t o s t e r o n e regulates 17-hydroxylase activity through the hypothalamo-hypophysial axis whereas oestradiol benzoate has a direct effect on the enzyme complex or its synthesis [97] (Table 4). This effect of oestradiol-17/3 has been examined recently by several researchers. Van Beurden et al. [99] investigated the regulation of androgen biosynthesis in immature rat testis geydig cells. Hypophysectomized rats were injected with lutropin for five days, and the Leydig cells, isolated at the end of this period, were capable of appreciable testosterone biosynthesis. Follitropin was less stimulatory and oestradiol benzoate (5 #g/day) decreased the extent of the stimulation

PREGNENOLONE . ~ PREGNENOLONE 20e-OH-progesteron~ PROGESTERONE SULPHATE 5-pregnenediol 20a-OH-progesterone 17-OH-pregnenolone 5a-androstanediol 5-androstenediol DHA oestrc)ne oestradiol progesterone 17-OH-PREGNENOLONE.

17-OH-PREGNENOLONE

suLpHaTE

1?-OH-PROGESTERONE

/2oo-oH-progo

t

.....

20a-OH-pregnenolone

teZst0ster0ne (sulphate)

DHAS II

,

oestradiol stenediol DHA 4-androstenedione

5-preg . . . . diol iI \ 4.androstenedione 20m-OH-progesterone 5. . . . drostanediol ~ DHAS testosterone, I I 5-andros tenediol. I 1°estradi°l*

5-ANDROSTENEDIOL~ SULPHATE

~ 5-ANDROSTENEDIOL

~

/

/

/ pregnenolone sulphate DHAS S-androstenedi()l (sulphate)

~-ANDROSTENEDIONE ~,~_androstenediol su!phat e ~DHAS testosterone. ~ S ~ - a n d r o s tened i o i , ~ oestradiol

~ OESTRONE

OESTRONE SULPHATE

oestraSiol OESTRADIOL TESTOSTERONE-~oESTRADIqL . 4-androstenedione --SULPHATE DHA(S) testosterer, e 5-androstenediol sulphate

Fig. 5. Pathways of steroid biosynthesis in human testes. The effects of endogenous steroids are inhibitory except for those denoted by *, indicating augmentation.

•Androstadienol synthetase"

C-17,20 lyase

17-Hydroxytase

Enzyme

9.04

136 29 5.0

5et-androst- 16-en-313-ol 5,16-androstadien-3[3-ol oestradiol- 17J3 oestradiol- 17~3 5a-pregnanedione testosterone oestrone 17-Of-l-progesterone

3.3 5.0 1.7 0.6 0.6 10

17-OH-progesterone

Boar microsomes

pregnenolone

3.8 102 184 70

191

5t~-pregnanedione

3.0 3.0 10.0 3.0

3.3

200~-dihydroprogesterone 20~-dihydropregnenolone testosterone 5-androstenediol

17-OH-progesterone

0.59

17.00

17-OH-pregnenolone

17-OH-pregnenolone

17-OH-progesterone porcine testis (immature), purified en17-OH-pregnenolone zyme

Boar microsomes

Human microsomes

17-OH-progesterone

20a-dihydroprogesterone 20a-dihydropregnenolone testosterone 5-androstenedio[ pregnenolone S testosterone S DHAS 5-androstenediol-3-S

2.5 3.2 22.0 3.3

17-OH-progesterone

Rat microsomes

-

--

60i

lli 14i 13i 15i

(i)

4o0

t [1151

[551

I107]

} [ll5]

[55]

[110]

[1081 [29] 1152]

[106]

--

(i)

20a-dihydroprogesterone 17,20a-dihydroprogesterone oestradiol benzoate (neonatal administration) progesterone

1481

cow ovary cytosol

progesterone 1.82

[100l

70i

100i

Oestradiol implants

progesterone

[101 l

60i

Oestradiol implants

} [97]

[98]

[991

85i

43i

65a

50i

--

pregnenolone

App. K i (/xmol/l)

Oestradiol benzoate administration

Oestradiol administration

--

pregnenolone

Rat testis cytosol Testosterone administration

1.4

App. Km 0xmol/I) Effector

progesterone

Substrate

% Change effected (i=inhibition, a= activation) Reference

Rat microsomes

Rat Leydig cells

Rat microsomes

Testis preparation

Table 4. Effects of steroids on steroid-transforming enzymes in the testis

©

e~

©

t~

17f3-OHSDH

313-OHSOHisomerase

synthetase'

• Anclrostacl~enone

progesterone

Human homogenates

oestrogen therapy 4-A DHAS testosterone DHAS testosterone oestradiol- 17[3 testosterone 4-A 5-androstenediol oestradiol- 1713 5ct-DHT DHAS 5-androstenediol-3-S testosterone 5-androstenediol oestradiol- 1713 DHAS

DHA DHA DHA 4-A 4-A 4-A 4-A testosterone 4-A 4-A 4-A 4-A 4-A DHA DHA DHA DHA 8.0 18.0

3,3 3.3 3.3

4-A

Human testis

oestradiol-1713 (implants)

Neonatal testosterone propionate

testosterone

Rat Leydig cells

Neonatal oestradiol benzoate

17-OH-pregnenolone 5-androstenediol DHA 20a-dihydroprogesterone oestrone oestradiol- 17!3 progesterone

oestradiol-1713 oestradiol-1713 4-A Testosterone 5-Androstenediol 4-A DHA testosterone DHAS 5-androstenediol-3-S

testosterone

-t

2.5 2.5 3.0

10.0

0.82 0.82 0.82 2.9 2.9 2.9 2.9 2.9

m

1.16

Rat microsomes

~regnenolone Human testis postmitochondrial supernatant (ammonium sulphate fraction) t7-OH-pregnenolone DHA 5-androstenediol

Human(organculture) DHA 5-androstenediol DHA DHA DHA Human homogenate 5-Androstenediol 5-Androstenediol 5-Androstenediol 5-Androstenediol 5-Androstenediol

Boar mlcrosomes

1.3 2.4 0.74 1.1 0.33 0.87 7.4

i

79i 41i 34a 27i 25a 12a 25a 10a 24a 22a 20a 30i 30i 35a 25a 20a 35i

100i

45i

30i

50i

40i 75i 22i lli 33i 42i 25i 21i 64i 17i

[138]

[1311

I135]

I100l

[29]

[129]

[1311

[130]

[124]

3

o.

Human microsomes

Rat testis

Rat (seminiferous tubules)

Boar cytosol

Testis preparation

1.9

prcgnenolone S

3.85

0.94

5-androstenediol S

20.0

2.04

DIIAS

DHA testosterone

5-pregnene-3[3,20c~-dio[ 20c~-dihydroprogesteronc 5c~-androstane-3[3.17~-diol

63i 48i 40i

47i 33i 4(li r

5-pregnene-313,20u-diol 20c~-dihydroprogeste tone 5e~-androstane-3!3.1713-diol

49i

28i

1.7 3.3 11.8

8.0 22.0 15.(I

28.0

13.0

50i 50i 50i

50i

Vo Change effected (i = inhibition, a = activation)

oestradiol-17[3

oestradiol- 1713 5a-androstane-313,1713-dio[ 5a-androstane-3a, 1713-diol testosterone

testosterone oestrone DHAS

12.(I } testosterone oestrone S

DHAS

oestrone S

oestradiol-3-S

10.0

} 5-pregnenedio! testosterone pregnenolone

3.0

pregnenolone S DHAS

3.0 8.0 8.0

pregnenolonc

3.0

DHA

7.5 7.0

0.3

-r

App. K, (~mol/l)

2.0

20.0

5~-androsb 16-cn-313-yl sulphate 5a-androst- 16-en-3[?,-ol pregnenolone S DHA

App. K,, (~mol/l Effector

DHA

pregnenolone

Substrate

4-A. 4-androstcnedionc: OH. hydroxy: S, sulphate; SDH, steroid dchydrogcnase.

Sulphatase

Sulphotran~ferase

Enzyme

Table 4. (cont.)

[1491

[14(q

I147]

[142]

[1451

Reference

G

©

©

uz

U~

Steroid-transforming enzvmcs and steroids signilicantly. When the cells were provided with i s o t o p i c a l l y l a b e l l e d p r e g n e n o l o n e , the 17hydroxylase activity had been completely inhibited bv the oestradiol benzoate treatment. The authors [99] therefore concluded that oestrogen treatment had a direct effect on 17-hydroxylase. In a more recent investigation (Table 4) it was observed [ 100] that implants of oestradiol- 17/3caused decreases in plasma and testicular testosterone levels in hypophysectomized rats which had been pretreated with hCG and follitropin. The Leydig cell 17-hydroxylasc activity in these rats was found to be considerably reduced compared to controls, but, as the oestradiol-17/3 implants had caused elevated le\els of 17-hydroxyprogesterone, the authors concluded that the C-17,20 lyase was the primary target for oestradiol-17/3. In a similar study, Brinkmann et al. [101] confirmed the earlier findings that corticosterone did not influence testosterone biosynthesis and that testosterone regulates its own production through the hypothalamo-hypophysial axis. Oestradiol-17/3 had a more direct effect, as the Leydig cells e x h i b i t e d d e c r e a s e d 1 7 - h y d r o x y l a s e and C 17,20-1yase activites and cytochrome P-450 levels (however, liver cytochrome P-450 levels were unchanged). These alterations in enzyme activity may have been a result of reduced protein synthesis or inhibition of existing enzyme protein. Oestradiol- 17/~ receptors on the surface of the Leydig cells may be instrumental in the control of protein synthesis but there is some evidence [102] that oestradiol-17/3 interferes with the binding of pregnenolone and progesterone to 17-hydroxylase cytochrome P-450: Moger [103] also concluded that high levels of oestradiol-17/3 may have a direct effect on cytochrome P-450. C-17,20 h'ase. The generation of Cl,~ androgen precursors requires the removal of the two-carbon side-chain from 17-hydroxypregnenolone or 17hydroxyprogesterone. The C-17,20 lyase resides in the endoplasmic reticulum of the interstitial tissue, with the seminiferous tubules possessing only 6-7% of the total testicular activity [86]. Submicrosomal fractionation of rat testis [96] and boar testis [104] has revealed that smooth-surfaced microsomes possess the majority of the testicular lyase activity. It has long been postulated that cytochrome P-450 is inw~lved in this cleavage-step, but only recently has the enzyme been purified and reconstituted. Betz et al. [105] solubilized microsomal protein from rat testis using a mixture of sodium cholate and Emulgen 913. Purification of cytochrome P-450 and the flavoprotein reductase was achieved using ion-exchange and affinity chromatography. Reconstitution of the component proteins yielded lyase activity which could be enhanced by the addition of phospholipid. The authors [105] were unable to demonstrate 17hydroxylase activity but this was attributed to technical difficulties resulting from interference by the detergent with the 17-hydroxylase assay. However, sR 19/4

1547

progesterone could bind to the enzyme causing spectral changes (K,,, (1.6 /xmot/l). Progesterone could also bind to the membrane-bound cytochrome P-450 and the apparent K,,, was 2.8 /xmol/l. which compared favourably with the apparent K,,, for 17-hydroxylase determined previously (1.4 p.mol/l) [98]. Betz and Tsai [1{16] have also found that the cytochrome P-450 of rat testis microsomes was inhibited by 20~-dihydroprogesterone when 17hydroxyprogesterone was supplied as substrate. A highly purified cytochrome P-450 from neonatal pig testis has been shown recently to have 17hydroxylase and C-17,20 lyase activities [107]. Oestradiol-17fl inhibited both activities with 4-en-3-oxo and 5-ene-3fl-hydroxypregnenes as substrates. The inhibition of 5-ene steroid metabolism was noncompetitive but with 4-en-3-oxo steroids, the hvdroxylase activity was inhibited non-competitively while that of lyase activity was inhibited competitively (See Table 4). The lyase activity was considered to be more susceptible to oestradiol-17/3 and the authors [1071 suggested that endogenous oestrogcns would be able to attenuate lyase activity in riro. The influence of pregnane derivatives on lvase activity was investigated when lyophilized rat testis microsomal fraction was incubated with 17hydroxyprogesterone [1081. Several progesterone derivatives inhibited the side-chain cleavage of this steroid. Of particular significance was 17a,2{k~dihydroxyprogesterone which, although less inhibitory than other steroids investigated, was formed in high yield in cytosol incubations, and was not found to be a suitable substrate for the lyase. An apparent K, of 9.04 /*mol/l compared to the apparent K,,, of 1.82 /xmol/l for the lyase suggested that in t'i~,o regulation of androgen production by 17er.20c,dihydroxyprogesterone was possible. This indicated that the 20a-OHSDH would not only compete with the lyase for substrate, but that its product would inhibit the lyase activity. However, later results [109 I disagreed with this hypothesis. The endogenous levels of 17cr,20c~-dihydroprogesterone in rat and rabbit testes were found to be only one-fifth those of 17-hydroxyprogesterone and, further, the 2(Ic~OItSDH resided mainly in the seminiferous tubules. This has been supported by other findings [88], when progesterone was converted to 20c~-dihydroprogesterone (71~) by rat testis Sertoli cells whereas 17-hydroxylase activity was very low. Thus. inhibition of lyase activity would be confined to the Sertoli cells which do not contribute greatly to the total testicular androgen production. However, the local effect may have considerable significance to the regulation of Sertoli cell function. Neonatal administration of oestradiol benzoate to rats was found to cause an increase in the in vitro lyase activity in the testes assayed 75 days later, whereas a similar administration of testosterone propionate was without effect [291. Activation has also been observed when heat-stable cytosolic corn-

1548

D.B. GowEr and G. M. COOKE

ponents were added to in vitro incubations, but the identity of these factors has yet to be elucidated fully

that 17-hydroxyprogesterone a n d 17hydroxypregnenolone are metabolized at distinct loci. The levels of unconjugated androgen precursors in the testis are low due to their rapid metabolism, whereas some of the inhibitors investigated above, are present in much greater quantities (e.g. pregnenolone sulphate 1.74 p,g/g, DHA sulphate 1.34 #g/g and 5-androstenediol-3/3-yl sulphate 0.80 txg/g, compared with 17-hydroxyprogesterone 0.1 /,g/g [ 111]. The cumulative effects of these on C-17,20 lyase activity could regulate androgenesis in the human testis. Furthermore, the sulphation of these 3/3hydroxy-steroids is believed to occur in the Lcydig cells of the interstitia, the same cells in which testosterone biosynthesis occurs. The synthesis of Cl,~ steroids in the boar testis is of considerable interest as pregnenolone can be metabolized to androgens, or, by a different pathway to the 16-androstenes (Fig. 6). These have little androgenic activity, but 5ceandrost-16-en-3-one and 5c~-androst-16-en-3c~-oi are considered to act as pheromones II12, 113]. The findings of Brophy and Gower [55] suggest that 5c~-pregnanedione inhibits both androgen and 16-androstene biosynthesis m vitro. DIIA production by boar testis microsomes was inhibited significantly by several concentrations of 5c~-pregnanedione, and furthermore, this compound was formed in high yield (44%) in incubations of microsomes with progesterone. The 5c~-reductase was not stable to freezing at -20°C and when stored tissue was used, biosynthesis was extensive. 16Androstenes are present in large quantities in boar testis [114] but they do not appear to inhibit C-17,20 lyase. Cooke and Gower [115] investigated the side-chain cleavage of 17-hydroxyprogesterone in microsomal incubations. The apparent K,,, for 17hydroxyprogesterone was 3.3 /zmol/l but the two

[961. Using the microsomal fraction of human testis tissue as a source of lyase activity, Hosaka et al. [ 110] demonstrated that many naturally occurring steroids influenced the enzyme in vitro. The two substrates competitively inhibited the metabolism of each other with apparent K~ values very close to their apparent Km values (1%hydroxyprogesterone, Km= 17 # tool/1 ; 17-hydroxypregnenolone, K,, = 0.59/lmol/l). The enzyme had a higher affinity and lower capacity for the 5-ene-substrate, consistent with the 5-ene-3/3hydroxy pathway being predominant in human testis [92-94]. When inhibition studies were performed with other steroids it appeared that the metabolism of 17-hydroxyprogesterone would be inhibited to a greater extent than that of 17-hydroxypregnenolone, and that the types of inhibition varied with respect to the two substrates. 20~-Dihydroprogesterone and 20~-dihydropregnenolone inhibited noncompetitively with both substrates [110]. Testosterone and 5-androstenediol i n h i b i t e d 17hydroxyprogesterone metabolism competitively but with 17-hydroxypregnenolone as substrate, testosterone i n h i b i t i o n was u n c o m p e t i t i v e while 5androstenediol exhibited mixed-type inhibition (Table 4). Oestradiol-17/3 was a poor inhibitor of lyase activity under these conditions. The monosulphates of pregnenolone, testosterone, DIIA and 5-androstenediol all inhibited 17hydroxyprogesterone metabolism but had little effect on 17-hydroxypregnenolone metabolism. The disulphate of 5-androstenediol inhibited the metabolism of both substrates equally (see Table 4). The authors [110] proposed that the lyasc may possess two active sites which require similar optimal conditions for catalysis, and the inhibition studies suggest

5a-ANOROST-16-EN-J~-YL SULPHATE 5a-ANDROST-16-EN-3~-UL 5,16-ANDROSTADIEN-3B- ~

E,16-ANDROSTADIEN-36-OL--

I YL-SULPHATE 15a_pregnanedi eon O H A p ; r e g n e n o o l n e sulphate;

PREGNENOLONEPREGNENOLONE SULPHATE 5~-androst-16-enl 3~-ol (sulphate) 17-OH-PREGNENOLONE SULPHATE

/

- 4,16-ANDROSTAOIEN-3-ONE~

/

1

\

SULPHATE

~ 5a-PREGNANEDIONE

t

17-OH-PREGNENOLONE . P 17-OH-PROGESTERONE L oestradiol

DHA

4-ANDROSTENEDIONE~ 4-androstenedione

5-ANDRGSTENEDIOL

,

5a-ANDROST-16-EN-36-OL 5a-ANDROST-16-EN-3B-YL SULPHATE

PROGESTERONE

/5~-preg . . . . dione

DHAS

-pregname~ione

5a-ANORQST-16-EN-3-ONE

5-ANDROSTENEDIOL--

testosterone

OESTROGENS

TESTOSTERONE

Fig. 6. Pathways of steroid biosynthesis in porcine testis. The effects of endogenous steroids arc inhibitory,

Steroid-transforming enzymes and steroids 16-androstenes, 5a-androst-16-en-3/3-ol and 5,16androstadien-3/3-ol had apparent K, values of 191 and 136 ~mol/l respectively, suggesting that these would not regulate androgen biosynthesis in vivo and also that the "lyase'" responsible for 16-androstene formation is not the same as that involved in androgen formation. From a number of studies [116, 117] it appears that 17-hydroxylation of C_,l precursors does not occur prior to side-chain cleavage and that 17-hydroxylated C:t steroids do not serve as substrates for 16-androstene biosynthesis. Further, other workers [118, 119] have provided convincing evidence that the 17c~-hydrogen of progesterone and pregnenolone is retained in the 16-androstenes produced in microsomal incubations of porcine testis. However, as molecular oxygen and NADPH are required for 16-androstene biosynthesis [120], it was suggested that 20fl-dihydropregnenolone may be intermediate [121]. The requirement of cytochrome P-450 in this side-chain cleavage step is open to debate as Brophy and Gower [122] concluded that it was not necessary, but later research by Mason et al. [123], using neonatal testis tissue, suggests that this cytochrome may be involved. The enzyme which catalyses the conversion of prenenolone to 5.16androstadien-3/3-ol (Fig, 7)--"androstadienol synthetase"--has an optimum pH of 6.6 [115] and is inhibited by 5c~-pregnanedione at low concentrations [55] (See Table 4). Several naturally occurring steroids at very high concentration (1 retool/l) inhibited "androstadienol synthetase'" activity in vitro but Lineweaver-Burk analyses revealed that they would be unlikely to regulate i6-androstene biosynthesis in vivo as the apparent K i values were so much greater than the apparent K,,, values for the enzyme (Table 4). These results agreed with those obtained earlier [I241. The only steroid that has so far been found to inhibit "androstadienol svnthetase" at low concentration is 5a-pregnanedione [55] and this steroid also inhibits the production of 4,16-androstadien-3-one from progesterone [124]. However, Cooke and Gower [115] found no inhibition of "'androstadienol synthetase" by 4,16-androstadien-3-one at concentrations less than 100/,tmol/I and that, at this concentration, uncompetitive inhibition was evident. This provides further evidence for "androstadienol syntherase" and the C-17,20 lyase being distinct enzymes. For many years it has been known that boar testis contains large quantities of oestrogens ]125] and these have been detected in the spermatic venous plasma in quantities greater than are evident in oestrous sows [126]. Administration of hCG to boars causes elevation in the spermatic venous plasma values, further suggesting a testicular origin for oestrogens [127]. However, the data using oestrone [115] (Table 4) indicate that "'androstadienol synthetase" would not be the target enzyme for regulation. The lack of attenuation of "androstadienol synthetase" activity by naturally occurring steroids may

1549

account for the large quantities of 16-androstene steroids found endogenously in boar testis [114], but these high levels would not be deleterious to C-17,20 [yase activity, therefore allowing androgen and oestrogen biosynthesis to proceed. 3/3-OHSDH-isomerase. The conversion of 5-ene3/3-hydroxysteroids to the 4-en-3-oxo configuration is essential for androgenesis (Figs 4-6) and is considered to be accomplished by two closely linked microsomal enzyme activities [14]. Testicular isomerization has not been studied to the same degree as the other endocrine organs, but some aspects of the enzymes in human and boar testes have been i n v e s t i g a t e d . The c o n v e r s i o n of 5,16androstadien-3/3-ol to 4,16-androstadien-3-one in boar testis (Fig. 6) was found to be reversible but this was not considered to be of great physiological significance [1281. The comparable stage in androgen biosynthesis in humans is also thought to be reversible [129] and the activity in vitro is influenced by naturally occurring steroids. Yanaihara and Troen [130] examined the effect of oestradiol- 17/3on testosterone biosynthesis in 24 h organ cultures of human testis. Increasing amounts of oestrogen caused a decrease in testosterone biosynthesis and an increase in 5 - a n d r o s t e n e d i o l . W h e n t h i s a n d 4androstenedione were provided as substrates, oestradio1-17/3 inhibited the isomerization of the 3/3hydroxysteroid but had no effect on the conversion of 4-androstenedione to testosterone (see Table 4). In similar experiments, testosterone inhibited its own production from DHA, whilst 5-androstenediol levels were increased, and when the diol was added to incubations with DHA as substrate, the levels of testosterone, 4-androstenedione a n d 5androstenediol all decreased. The authors concluded [1301 that the isomerase would be regulated by its substrates and products. In a later study [131], these effects were examined using cell free homogenates of human testis. Micromolar concentrations of 4-androstenedione, DHA, testosterone, 5-androstenediol, DHA sulphate and 5-androstenediol sulphate inhibited the isomerization of the two substrates. As these sulphate esters are present in human testis in much greater quantities than the free steroids [111], it is possible that these could regulate testosterone biosynthesis in vivo. The 3/3-OHSDH and isomerase of the human testis were both precipitated from the post-mitochondrial supernatant in the 0-40% ammonium sulphate fraction [129] and this preparation catalysed the conversion of pregnenolone, 17-hydroxypregnenolone, DHA and 5-androstenediol to the corresponding 4-en-3-oxosteroids but the apparent K,,, values differed (Table 4). The isomerization of pregnenolone was c o m p e t i t i v e l y i n h i b i t e d by 5-ene-3/3hydroxysteroids, 20~-dihydroprogesterone and oestrogens, with apparent Ki values much lower than the apparent Kin. 17-Hydroxyprogesterone, testosterone

1550

11. B. Gowt~r and G. M. Coo~:t

and 4-androstenedione exhibited mixed type inhibition kinetics whereas progesterone was a noncompetitive inhibitor. Taken together, all these in vitro investigations suggest that the isomerization step (Fig. 5) may be controlled in the human testis in vivo by C1~ sulphates, oestrogens and androgen precursors, all of which are synthesized in the interstitial cells of testis tissue IS6, 111 ]. 17/3-OHSDH. The conversion of 4androstenedione to testosterone, of DHA to 5androstenediol and oestrone to oestradiol-17/3 is catalysed by 17-OHSDHs (Figs 4-6). The reactions are reversible in vitro, the direction depending on the oxidative state of the nicotinamide cofactor. An early observation [1311 demonstrated that, in the testes of newly-weaned rats, 4-androstenedione was produced from pregnenolone via 17-hydroxypregnenolone and DHA, whereas testosterone was produced via progesterone and 17-hydroxyprogesterone, indicating that the conversion of 4-androstenedione to testosterone was not a major metabolic transformation. Inano et al. [1331 found that 17/3-OHSDH activity increases slowly until the onset of maturation, at which time testosterone becomes the predominant androgen. When microsomes were subfractionated in discontinuous sucrose density gradients, the 17/3OHSDH activitv was found predominantly in the smooth-surfaced microsomes, and the distribution approximated that of the 17-hydroxylase and C- 17,2() lyase, and it has been suggested that the androgensynthesizing enzymes exist as multi-enzyme complexes in the microsomal membrane [96]. Neonatal administration of either oestradiol benzoate or testosterone to rats caused the testicular 17/3-OHSDH activity to be depressed when assayed 75 days later [29]. This observation has been supported by other workers I100], who found a 45% decrease in testicular 17/3-OHSDH activity in hypophysectomized rats which had been pretreated with gonadal hormones and provided with implants of oestradiol-17/3 (Table 4). The biosynthesis of oestradiol-17~ in rat Sertoli cells was s t i m u l a t e d when p r o g e s t e r o n e , 4androstenedione, testosterone or oestrone were added to the culture incubations [1341, Oestrone was converted to a much greater extent than the other steroids, suggesting that the 17/3-OHSDH of Sertoli cells is very active towards phenolic substrates, and also that aromatization is the rate-limiting step in oestrogen biosynthesis [134]. Early observations [135] indicated that oestrogen therapy caused the human testicular 17/3-OHSDH to be considerably diminished although the C-17,20 lyase was unaffected. This latter finding was endorsed when it was shown that oestradiol-17/3 was a poor inhibitor of lyase activity [110] (see above). The inhibitory effect of oestradiol-17/3, however, was not observed in human testis organ culture [130] and, when cell-free homogenates were used [13l] slight

activation of 1713-OHSDH activity was evident (Table 4). Activation was also effected by testosterone. Two apparent K,,, values were determined for the enzyme; at substrate concentrations above 5.92 ixmol/l, the apparent K,,, value was 10 p~mol/l whereas, at substrate concentrations below 5.92 p~mol/I, a smaller apparent K,,, value of 3.3 p~mol/l was determined. Thus, 17/3-OHSDH has a higher affinity for substrate at low substrate concentrations and. at high substrate concentrations, a eonformational change in protein structure causes decreased affinity. DtIA sulphate inhibited the enzyme when either DHA ~r 4-androstenedione was supplied as substratc, and this may be due to the ability of some steroidtransforming enzymes to convert one steroid sulphate to another, in this case, DttA sulphate to 5-androstenediol sulphate. Product activation of human testicular 17/3OHSDH was observed by Oshima et al. 11361. In incubations using cell-free homogenates and ~4Clabelled 4-androstenedione, the addition of testosterone (unlabelled) caused a greater amount of radioactivity to be incorporated into the testosterone which was isolated at the end of the incubation period. Unlabelled 4-androstenedione had a similar effect when the reverse reaction was studied. In microsomal incubations, 4-androstenedione not only enhanccd 17/3-OIISDH activity, but the apparent optimal ptl was altered from 8.6 to 8.0: similarly, testosterone altered the optimal pH from 5.8 to 7.4 J137 t. It thus appeared that the presence of the reaction product caused a conformational change in the enzyme structure, thereby enhancing its activity for substrate. In a subsequent investigation [1381, addition of the reaction product altered the kinetics of the reaction, increasing the apparent Vm,,~and decreasing the apparent K,,,. For example, the apparent K,,, for 4-androstenedione was decreased from 8 gmol/1 to 3.8 ixmol/l when testosterone was included in the incubations, and the Vm,,~increased from {).7 to 1.4 mmol/mg protein/20 min. The authors [1381 suggested that 17/3-OHSDH possesses two active sites, one specific for 4-androstenedione and another for testosterone, and that the binding of one steroid to its locus prepares the other for activation. Thus. at low 'product" concentrations, product activation would be evident and, as the product concentration increases, it would compete with the substrate for both binding sites, until it inhibited enzyme activity duc to a mass effect. This hypothesis was supported by the finding that 17/3-OHSDH reductase activity could be enhanced by the addition of other 17/3-hydroxylated steroids, oestradiol and 5-androstenediol. However, the 3 / 3 - y l - s u l p h a t e esters of D H A and 5androstenediol inhibited 17/3-OHSDH with DttA. 4-androstenedione or testosterone as substrate (Table 4). The enzyme of the porcine testis has been purilied from the microsomal fraction, using ultrasound to release membrane-bound protein [139]. The K,,, for

Steroid-transforming enzymes and steroids 4-androstenedione was 40 p,mol/l and product inhibition was evident. The enzyme was also capable of accepting D H A and oestrone as substrates, the reduction proceeding at similar rates. Furthermore, 20~-OHSDH activity was evident to the extent of one-seventh of 17/3-OHSDH activity [139, 140]. Cooke and Gower [104] showed that the 17/3OHSDH of porcine testis was evenly distributed between rough and smooth-surfaced microsomes, whereas in the same samples, C-17,20 lyase activity was associated predominantly with the smooth microsomes. The same workers [115] later determined the apparent K,,, for testosterone to be 26 /xmol/l and found that the activity was not influenced bv 16-androstene steroids, which occur in large quantities in boar testis [1141 . Steroid sulphate metabolism in the testis. The enzymes responsible for the synthesis and hydrolysis of steroid sulphate esters are the sulphotransferases and the sulphatases. The physiological role of sulphate conjugated steroids remains unclear, but they have been found in considerable quantities in the testes of humans and boars. They may be a pool of androgen and oestrogen precursors or a means of preventing free steroids from being metabolized to active hormones. As outlined above, they are capable of modifying the activities of some steroidtransforming enzymes in vitro and their high levels in certain endocrine tissues suggests that a regulatory role in vivo may be one of their functions. The sulphotransferases are found predominantly in the cytosol of most tissues investigated, but microsomal activity in pig liver has also been observed [141]. Ruokonen et al. [94] were unable to detect any steroid sulphates in rat testis but there are other reports [1421 of sulphotransferase activity in the seminiferous tubules of rats, the interstitial tissue lacking this activity completely. The apparent Km of the seminiferous tubule enzyme, with respect to D H A , was 3 /xmol/l, and pregnenolone inhibited competitively with an apparent K i of approximately one-tenth the apparent K,,, for D H A , suggesting that pregnenolone was the preferred substrate. The formation of steroid sulphates takes place to a much greater extent in human and boar testis tissue. In human testis, pregnenolone sulphate is quantitatively the major steroid but the sulphates of D H A and 5-androstenediol were also found in large amounts [94]. These sulphates were precursors of unconjugated testosterone and it was shown that one steroid sulphate could be directly converted to another without prior hydrolysis of the sulphate group [ 1431. A similar situation was observed in boar testis, supporting earlier observations [144] showing that pregnenolone sulphate was directly converted to 5,16-androstadien-3/3-yl sulphate in microsomal preparations (Fig. 6). However, in both human and boar testes, the rate of testosterone biosynthesis from sulphated precursors was lower than from unconjugated precursors [ 143], suggesting that the high levels

1551

of steroid sulphates in these organs is due to accumulation and also to sulphatase activity being controlled. Recent research on the sulphotransferase of boar testis cytosol [145] suggests that 5~-androst-16-en3/3-yl sulphate may inhibit the synthesis of pregnenolone sulphate. The former conjugate was present in the testis tissue at a concentration of 49 p,mol/kg wet weight, and when added to pregnenolone sulphotransferase incubations, a 50% reduction in pregnenolone sulphate was observed. Lineweaver-Burk analysis revealed the inhibition to be of the noncompetitive type. Other steroids which inhibited this reaction were pregnenolone sulphate, 5c~-androst16-en-3/3-ol and D H A (Table 4) but D H A sulphate was not inhibitory. When the sulphoconjugation of D H A by boar testis cytosol was investigated, the apparent K,,, was approximately one-tenth that found for pregnenolone (Table 4) and furthermore, 5c~androst-16-en-3/3-yl sulphate had no effect on the reaction. This indicated that pregnenolone and D H A are conjugated by distinct sulphotransferases. The authors [1451 suggested that 5a-androst-16-en-3/3-yl sulphate may regulate the biosynthesis of 16androstenes from pregnenolone sulphate, a reaction shown to occur in vitro [143,1441 , although the yields are lower than when unconjugated precursors are used. D I t A is a poor substrate for 16-androstene biosynthesis [117] and this may be the reason for the lack of inhibition of D H A sulphotransferase by 5~-androst- 16-en-3/3-yl sulphate. The generation of hormonally active androgens and oestrogens from conjugated precursors requires the hydrolysis of the sulphate group. Study of the sulphatases has been hindered by the inability to solubilize active protein from the microsomal membranes, although this has been achieved [691 using Triton X-100 (0.5%. v/v) and the microsomal fractions from normal and 'sulphatase deficient' placentae. In rat testis preparations, it has been shown that the same sulphatase hydrolyses both D H A and 5-androstenediol sulphates and activity towards these was inhibited by 5c¢-androstane-3/~,17/~-diol, 50¢-androstane-3~,17/~-diol and testosterone (Table 4). Kinetic data revealed that hyperbolic competitive inhibition was evident, where the inhibitor binds to the enzyme at a different site to the substrate, altering its affinity for the substrate [146]. Pregnenolone sulphate has the greatest affinity for rat testis sulphatase I1471 and activity was shown to be inhibited by several steroids, including 5-pregnene3/3,20c~-diol and testosterone but not by D H A sulphate. The metabolism of the alternative substrates ( D H A sulphate, oestrogen sulphates and cholesterol sulphate) were all inhibited by testosterone with apparent K i values near or lower than the apparent Km values [147]; the hydrolysis of oestrone sulphate was inhibited by DHA sulphate (See Table 4).

1552

D . B . Gowl~R and G. M. Cooer

T h e c o m p a r t m e n t a t i o n of sulphatase in rat testis was studied by P a y n e and Kelch [148] and activity was evident in b o t h the interstitia and seminiferous tubules, w h e r e a s s u l p h o t r a n s f e r a s e was found only in the latter c o m p a r t m e n t . T h e p r e s e n c e of b o t h the conjugating a n d d e c o n j u g a t i n g enzymes in the same c o m p a r t m e n t suggests t h a t a balance of active horm o n e would be m a i n t a i n e d , a n d that the activities of b o t h e n z y m e s would be a t t e n u a t e d by their products. T h u s , the ideal level of a n d r o g e n necessary for the m a i n t e n a n c e of s p e r m a t o g e n e s i s would be determined. It is of interest that o t h e r e n z y m e s f o u n d in t h e s e m i n i f e r o u s t u b u l e s ( 2 0 o ~ - O H S D H , 5c~reductase, 3c~-OHSDH) give rise to steroids which inhibit sulphatase activity (e.g. 5-pregnene-3/3,20~diol, 5c~-androstane-3c~,l 7/,3-diol). A sulphatase of the h u m a n testis accepts the sulphates of p r e g n e n o l o n e , 4 - a n d r o s t e n e d i o l and D H A as substrates, but 5-prenene-3/3,2(/o~-diol is again inhibitory [149] (Table 4). Inhibition by 5o~r e d u c e d C ~ steroids was also f o u n d and the type of inhibition was hyperbolic competitive, the same as for the rat testis. A l t h o u g h the c o m p a r t m e n t a t i o n of the s u l p h o t r a n s f e r a s e in h u m a n testis r e m a i n s u n c e r t a i n , it has b e e n postulated [ 142] that s e m i n i f e r o u s tubule a n d r o g e n levels could be m a i n t a i n e d by the same m e c h a n i s m as outlined for rat testis, If the Leydig cells possess the h u m a n s u l p h o t r a n s f e r a s e activity, then t r a n s p o r t of sulphated a n d r o g e n precursors to the s e m i n i f e r o u s tubules, (where the majority of sulphatase activity resides [15(1]), would present this c o m p a r t m e n t with a supply of a n d r o g e n precursors as well as active a n d r o g e n . T h u s s p e r m a t o g e n e s i s would be m a i n t a i n e d at a c o n s t a n t level and would not have to rely on the biphasic p a t t e r n effected by peptide h o r m o n e s . In this instance, the control of e n z y m e activity by e n d o g e n o u s steroids would be of param o u n t i m p o r t a n c e . A p a r t from the effects of steroid s u l p h a t e s on enzymes, they have b e e n implicated in the control of lutropin release. Eckstein et al. [631 have d e t e r m i n e d that a n d r o s t a n e d i o l sulphates are capable of inhibiting post-castrational tutropin elewttion in female rats and that the plasma levels in intact rats would be sufficient to control lutropin release in intact i m m a t u r e rats. F u r t h e r research is required to d e t e r m i n e w h e t h e r this situation occurs in the male. T h e above i n f o r m a t i o n suggests that the steroids p r o d u c e d in Leydig and Sertoli cells control b o t h t h e i r own biosynthesis and those p r o d u c e d in the o t h e r testicular c o m p a r t m e n t by influencing the e n z y m e s c o n c e r n e d . It is not possible to give a c o m p o s i t e picture due to species differences in steroid m e t a b o l i s m , but Figs 4 - 6 outline the p r e s e n t situation in the rat, m a n and boar. T h e proximity of the cell types within the testis would allow a close relationship, and a l t h o u g h some of the steroids seem to be regulatory at levels which are greater than those f o u n d e n d o g e n o u s l y , it must be r e m e m b e r e d that microsomes m a y c o n c e n t r a t e such steroids (as has been shown for progesterone [151]) thus presenting

e n z y m e s with a local e n v i r o n m e n t which is quite different from the situation in vitro. Steroid binding p r o t e i n s may also be i n s t r u m e n t a l in this phen o m e n o n , but f u r t h e r research is r e q u i r e d before the situation is m a d e clear. Acknowledgements--Studies performed in the authors" laboratory were supported financially by The Agricultural Research Council (AG 35/29 and 35/35). We also thank Miss R.T.K. Seville and Mrs D.M. Gower for typing the manuscript.

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70. T o w n s l e y J. D.: I n h i b i t i o n of p l a c e n t a l 313hydroxysteroid dehydrogenase by naturally occnrring steroids. Acta endocr.. Copenh. 79 (1975) 7411-748. 71. Gibb W . , Lavoie J. C. and R o u x .1, F.: 3[3Hydroxysteroid dehydrogenase activity in human lctal membranes. Steroids 32 (1978) 365-372, 72. Schwarz B. E., Milewich L , (;ant N. F., Porter J. ('., Johnston J. M. and McDonald P. ( . : Progesterone binding and metabolism in h u m a n fetal membrancs. Ann. N.Y. Acad. Sci. 286 119771 3/14-312. 73. Begley D. J.. Firth J. A. and H o u h J. R.: ltuman Reproduction and Developmental Biology. Macmi[ lan, London and Basingstokc 11981,)) 175-177. 74. f o w n s l e y J. D.. Scheel D, A. and Rubin E. J.: Inhibition of steroid-3-sulphatase by endogenous ~,tcroids. A possible mechanism controlling placental oestrogen biosynthesis from conjugated precursors .1. clin. Endocr. Metab. 31 (1971,1)6711-678. 75. Townsley J. D.: Further studies on the regulation ol h u m a n placental steroid-3-sulphatase activity. Emh)crinology 93 (1973) 172-181. 76. Scharzel W. C., Kruggel W. G, and Brodie It. J.: Studies on the mechanism of oestrogcn biosymhcsb, VIII. The development of inhibitors of the enzyme system in h u m a n placenta. Endocrinolo£,y 92 (19731 861-,-881,). 77. Siiteri P. K. and T h o m p s o n E. A.: Studies of h u m a n placental aromatase. J. ~teroid Bioehem. 6 11975) 317 322. 78. Gibb W. and Lawfie J. ('.: Substrate speciiicit} ol the placental microsomal aromatase. Steroidv 36 (19811t ,>07 DZ(I. 79. Wolf A. S., Musch K. and Lauritzen,: Interference of steroids with the metabolism of D H A in the perfused h u m a n placenta. Acta endocr., Copenh. Suppl. 240, 96 (1981) 105-106. 8t). Sano Y., Shibusawa tt. Yoshida N., Sekiba K., Okinaga S. and Arai K.: Steroid 16~-hvdrox~lasc from h u m a n fetal liver: inhibition by steroids, t ~ t a Obstel. (;vneeol. stand. 59 (1980) 245-24~). 81. Jaraback J. and Sack G. H . . l r . : A sohlblc 1713hydroxysteroid dehydrogenase from h u m a n placenta. The binding of pyridine nucleotides and steroids. Biochemistry 8 (1969) 2203-2212. 82. Karavolas H. J. and EngeI L. L.: t t u m a n placental 176-estradiol dehydrogenase. VI. Substratc spcciliciD of the diphosphopyridine nucleotide linked cnzxmc. Endocrinology 88 ( 1971 ) 1165-1169. 83. l~opez-Bernal A.. Anderson A. B. M.. Flint A. I ' t-. and Turnbull A, C.: l l[3-Hydroxysteroid dchvd rogenase in the h u m a n placenta, fetal m e m b r a n e s and decidua. J. Endoer. 85 (19801 24P. 84. Lopcz-Bcrnal A., Flint A. P. F,. Anderson A. B. M. and Turnbull A. C.: 11[3-Hydroxysteroid dchvdrogenasc (EC 1,1.1.146) in h u m a n placenta and decidua. J. steroid Biochem. 13 119801 1081-11187. 85. Blomquist C. H., Kotts C, E. and tlakanson t',. Y.: Inhibition of placental 17[3-hydroxysteroid dchvdrogcnase by steroids and non-steroidal alcohols: aspects of inhibitor structure and binding speciticity. Archs bioehem. Biophys. Ig6 (19781 35--41. 86. R o m m c r t s F. F. G. and Brinkman A. O.: Moduhilion of stcroidogcnic activitics in testis I,cydig cells. Molec. cell. Endocr. 21 119811 15-28. 87. Podesta E. J. and Rivarola M. A.: ('onccntration oi androgens in whole testis, seminiferous tubules and interstitial tissue of rats at different stages of development. Endocrinology 95 (19741 455 4(~1. 88, Weibe ]. P., Tilbc K. S. and Buckingham K. D.: An analysis of the metabolites of progesterone produced by isolated Sertoli cells at onset ot gametogenesis. Sferoid~ 35 (19811) 561-577. 89. Shikita M., Kakizaki M. and Tamaoki B.: l h c palh-

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