GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
26, 541-549 (1975)
In Vitro Steroidogenesis by the Nonzoned Adrenocortical Tissue of the Skink, Tiliqua rugosa G.P.
VINSON, M. BRAYSHER,
AND B.J.
WHITEHOUSE
Department
of Zoology, St Bartholomew’s Medical College, Charterhouse Square, London, EC1 M 6BQ, Department of Zoology, University of Adelaide, South Australia, and Department of Physiology, Queen Elizabeth College, Campden Hill Road, London, W8 7AH
5000
Accepted March 6, 1975 Steroid formation by adrenocortical tissue from the skink, Tiliqua rugosa, has been studied using established in vitro techniques. Both in conventional incubations, with timed sampling, and in incubations with dialysis, aldosterone, and corticosterone were major products. From endogenous precursors, and from [Ylacetate, yields of the two products were of the same order, whereas from [3H]pregnenolone maximal yields of corticosterone were at least tenfold greater than aldosterone. Maximal rates of steroid formation from the radioactive precursors occurred within the first few minutes of incubation, but maximal rates of steroid formation from endogenous precursors occurred significantly later, between l-2 hr. In incubations with dialysis [‘%]aldosterone was significantly less dialysable than [“Hlaldosterone under all conditions, whereas [“Cl and [3H]corticosterone were homogeneous. In contrast, neither aldosterone nor corticosterone formed from endogenous precursors were bound under control conditions, although binding was increased following dexamethasone pretreatment, and decreased following stimulation with Tiliqua pituitary extract (but not Synacthen), with concomitant changes in yields and specific activities. Inter alia the results suggest that products formed from [%]acetate and from [:‘H]pregnenolone may be maintained in separate pools within the tissue, and this accounts for their different metabolic fates. The bound pool, penetrated only by [3H]acetate, yields more aldosterone than the free, and may be termed a “biosynthetic pool.” In addition there exists a “secretory reserve pool.” This is suggested by the difference between rates of steroid secretion from endogenous and added precursors, and also from the changes in dialysibility seen in steroids formed from endogenous precursors under different conditions of stimulation. In both the compartmental arrangement of steroids, and the production of large yields of aldosterone the adrenocortical tissue of Tiliqua shows similarities to the zona glomerulosa, but not the inner zones of the rat adrenal cortex.
Like other nonmammalian vertebrates, reptiles show no differentiation of their adrenal cortical tissue into zones on the mammalian pattern (Chester Jones, 1957; Deane, 1962; Symington, 1969). Despite this, investigations of adrenocortical functions among the reptiles have shown that the hormonal products are identical with those found among mammalian secretions and include both corticosterone and aldosterone. These are formed in vitro from both added radioactive precursors such as 541 Copyright All rights
@ 1975 by Academic Press. Inc. of reproduction in any form reserved.
pregnenolone or progesterone and also from endogenous precursors (Phillips, Chester Jones, and Bellamy, 1962; Macchi, 1963: 1964; Macchi and Phillips, 1966; Sandor Lamoureux and Lanthier, 1964; Gist and deRoos, 1966; LeloupHatey, 1966; Huang, Vinson, and Phillips, 1969; Tam, Phillips, and Lofts, 1972; Bourne and Seamark, 1973; Mehdi and Sandor, 1974). 17a-Hydroxycorticosteroids have not been reported in reptiles. In rive, corticosterone and aldosterone
542
VINSON,
BRAYSHER
may be present in circulating plasma (Chester Jones, Phillips, and Holmes, 1959; Bradshaw and Fontaine-Bertrand, 1970; Licht and Bradshaw, 1969; Daugherty and Callard, 1972; Vinson, Braysher, and V. H. T. James, unpublished observations) and although early work failed to show stimulatory effects of ACTH added to incubation media in vitro (Gist and deRoos, 1966) more recently good stimulation of corticosteroid formation and secretion by mammalian ACTH has been shown in reptilian preparations both in vitro and in vivo (Huang et al., 1969; Licht and Bradshaw, 1969; Daugherty and Callard, 1972; Callard, Chan, and Callard, 1973). In addition, it is probable that changes in electrolyte balance may also elicit stimulation of adrenal steroidogenesis and secretion (Bradshaw, 1972; IeBrie, 1972). Thus, the adrenocortical tissue of the reptile, undifferentiated into zones, shows many of the functions of the structurally much more complex mammalian gland. In recent work with rat adrenal glands, another functional characteristic of the mammalian gland has emerged. Using a technique of incubation with dialysis it can be shown that in the zona glomerulosa (but not in the inner zones) steroids may be maintained in more than one pool and the metabolic end products of hormone synthesis are determined at least in part by this. Thus a free steroid pool gives rise to corticosterone and a bound pool to 18-0xygenated steroids including 1B-hydroxydeoxycorticosterone (1 B-OH DOC) and aldosterone. ACTH increases the proportion of free to bound steroid, and consequently increases the ratio of corticosterone to 18-OH DOC and aldosterone. These effects are only seen in products formed from endogenous precursors or an early labeled added precursor, such as [14C]acetate and not in products from late exogenous precursors, such as r3H]pregnenolone. In contrast to all of these findings, products from inner zones are homo-
AND WHITEHOUSE
geneous and apparently freely dialysable throughout such procedures (Whitehouse and Vinson, 1971, 1972; Vinson and Whitehouse, 1973a, b). It seemed worthwhile applying these techniques to a tissue from a reptile to give further information on the functions of the reptilian gland. This paper describes experiments on tissue from the South Australian skink, Tiliqua rugosa, in which a preliminary study of in vitro steroidogenesis indicates conformity with the normal reptilian pattern (Bourne and Seamark, 1973). MATERIALS
AND METHODS
Animals. Animals were collected in the wild and maintained on a mixed diet in the Department of Animal Physiology, Waite Agricultural Research lnstitute, South Australia, for brief periods. When required for collection of adrenal tissue, they were killed by decapitation and the adrenals were removed (approx 50 mg paired wt) cleaned of adhering fat and stored on ice for periods of up to 0.5 hr before incubation. Incubations. Three incubation experiments were performed. In all cases [7a - 3H]pregnenolone (Specific Activity 6.9 Cilmmole) and [ 1 - %]acetate (Specific Activity 61 mCi/mmole) were used as added precursors. Expt I. Eight pairs of adrenals were minced with a pair of scissors and incubated for 3.5 hr with 4 &i [3H]pregnenolone and 40 &i [“Clacetate at 37” in 10 ml Krebs-Ringer bicarbonate solution Steroids extracted from this incubation were used for the procedures for steroid identification (see below). Expt 2. Ten pairs of adrenals were incubated as in Expt 1, but in this case samples were withdrawn at 5, 15, 30, 60, 90, 120, and 240 min as previously described (Vinson, 1966; Whitehouse and Vinson, 1967) and the steroid extracts were fractionated separately. Expt 3. Twenty-four pairs of adrenals were subjected to incubation with dialysis as previously described (Whitehouse and Vinson, 197 1). In each case 1 &i [YH]pregnenolone and 10 PCi [%]acetate were used as added precursors and the tissue was placed in dialysis bags containing 2 ml Ringer solution with a further 15 ml bathing the outside. The 24 pairs of adrenals were in four experimental groups of 6 pairs each, viz., a control group (C), a group (A) with the addition of 2.5 U ACTH (Synacthen, Ciba) to the inside of the dialysis bag, a group (D) taken from animals pretreated with dexamethasone (0.4 mg.
SKINK ADRENOCORTICAL 12 hr previously), and a group (P) to which an extract of pituitary tissue taken from dexamethasone pretreated animals was added. Each flask received the equivalent off pituitary in 0.1 ml M acetic acid.
Extraction
and Quantitation
of Steroids
Steroids were extracted with ethyl acetate and chromatographed on paper in the systems T/70 and L/75 (Vinson and Whitehouse, 1969). Fractions corresponding to corticosterone and aldosterone were eluted and acetylated and oxidised to the y-lactone, respectively. DOC was also sought but not found. Steroids were then rechromatographed on paper and then quantitated using the glc techniques previously described (Vinson and Whitehouse, 1969). Aldosterone samples were subjected to glc twice using columns with different loadings, 1 and 3%, of XE-60 on gas Chrome Q and the mean values were used in calculation of results. Corticosterone samples were chromatographed on 3% XE-60. The isotope content was determined on remaining aliquots and treated as in previous publications (Vinson, 1966; Vinson and Whitehouse, 1969). Further identification procedures were carried out on the labeled extracted material and these are given in the Results.
RESULTS Identity of Compounds
Further identification was carried out on the isotopically labeled corticosterone acetate and aldosterone y-lactone obtained in Expt 1 by admixture of the extracted material with 5 pg authentic material. The mixtures were then chromatographed on thin layer in the systems: 1 -Light petroleum : benzene : ethyl acetate (1: 1:4); 2 -chloroform : methanol (9 : 1); and 3 -light petroleum : methanol (9 : 1). After each chromatogram, the specific activities were determined using the glc method for mass and scintillation counting for 3H and 14C content. Successive values for specific activity and 3H/14C ratios are given in Table 1. The area corresponding to deoxycorticosterone (DOC) in the chromatograms of this experiment was also eluted, acetylated, and rechromatographed (system L/75). Gas-liquid chromatography gave no indication of the presence of DOC
543
FUNCTION TABLE
1
IDENTITY OF Tiliqua ADRENAL INCUBATION PRODUCTS=
Sample Corticosterone acetate 1 2 3 Aldosterone-y-lactone 1 2 3
3H sp ac (cpdng)
3H/‘“C ratio
18.7 18.8 16.4
246 263 284
3.6 3.7 3.9
190 216 221
a Radioactive products were admixed with authentic unlabeled material and samples taken after three successive chromatograms on thin layer. Corticosterone was processed as the acetate and aldosterone after oxidation to the y-lactone. Specific activities were calculated following estimation of mass by glc and isotope content by liquid scintillation counting. Precursors were [aHlpregnenolone and [r4C]acetate.
(< 10 ng), and only trace amounts tope were detected (Fig. 1).
of iso-
Formation of Steroids with Time of Incubation (Expt 2)
Yields of corticosterone and aldosterone from [14C]acetate, [3H]pregnenolone, and from endogenous precursors are given in Figs. l-3. From [3H]-pregnenolone (Fig. 1) corticosterone was the major product and was formed very rapidly, reaching a maximum yield in about 15 min. The maximum yield of aldosterone was only about onetenth that of corticosterone. A very small
*
YlEtrl
1
2 HOURS
3
4
FIG. 1. Yield-time curves for products formed from [3H] pregnenolone during incubation of Tiliqua adrenal tissue. B = corticosterone, aldo = aldosterone, DOC = presumptive deoxycorticosterone.
544
VINSON,
BBAYSHER
AND
WHITEHOUSE
fore very different from Figs. 1 and 2. One point of similarity with Fig. 2 is that the maximum yield of aldosterone from endogenous precursors is about 60% that of corticosterone.
‘b YIELD x 104
Steroid Binding and Stimulation of Secretion (Expt 3)
FIG. 2. Yield-time curves for compounds from [‘%]acetate during the same incubation Fig. 1. B = corticosterone, aldo = aldosterone.
formed as for
trace of tritiated material corresponding to DOC was found in the earliest stages of incubation. From [14C]acetate (Fig. 2) maximum yields of both compounds were reached more slowly after about 1 hr. In contrast to the 3H results, the maximum yield of aldosterone was in this case about 60% of that of corticosterone. It is evident from the slopes of the curves that maximum rates of steroid formation from the radioactive precursors (Fig. 1 and 2) occur within the very earliest periods but Fig. 3 shows that this is not so in the case of endogenous precursor utilization. Here the production rates of both compounds appear to be very nearly zero for considerable periods, one-half hour in the case of corticosterone and 1.5 hr in the case of aldosterone. Maximum rates of formation occur between l-2 hr, immediately before the maximum yield is reached. These curves are there-
Characteristics of dialysibility of the compounds are shown in Figs. 4-6. Once again differences are apparent between the handling of the three types of precursor. In Fig. 4 it is clear that there is no difference in the dialysibility of tritiated corticosterone and tritiated aldosterone, which at approximately 60% seems to be indistinguishable from freely dialysable material under the conditions used (cf, Whitehouse and Vinson, 197 1). The various experimental treatments have no effect on this pattern. In contrast, in Fig. 5 a clear difference exists between 14C-labeled corticosterone and aldosterone and taken as a whole the [ 14C]aldosterone is significantly less dialysable than the [L4C]corticosterone and also than the [3H]aldosterone in the same incubations. This difference is consistent throughout all of the incubations. In further contrast, Fig. 6 shows that dialysibility of corticosterone and aldosterone from endogenous precursors is affected by experimental treatments, being 80
r
TILIQUA:
Tr
C D A P Coriicosierone
FIG. 3. Yield-time curves for products formed from endogenous precursors during the same incubations as for Figs. 1 and 2. B = corticosterone.
3H
C D A Aldarterone
P
FIG. 4. Dialysibility of products formed from [3H]pregnenolone during incubation of Tiliyua adrenal tissue. C = control, D = dexamethasone pretreated animals, A = dexamethasone pretreatment with the addition of Synacthen to the incubation medium inside the dialysis bag, P = dexamethasone pretreated, with the addition of Tiliqua pituitary extract to the incubation medium.
SKINK
TILIQUA:
ADRENOCORTICAL
54.5
FUNCTION
14C
80 60
70 60 76 Dialysable
50
50 4.
40
30
Dpm 1 n-3
20 10
20 C D
Dialysibility of products [‘Tlacetate in the same incubations Abbreviations as for Fig. 4. P value for dosterone vs [‘JH]aldosterone (cf. Fig. FIG.
30
5.
10
formed from as for Fig. 4. total [‘T]al4) < 0.05.
decreased with dexamethasone pretreatment, and increased with the addition of pituitary extract. Synthetic ACTH (Synacthen) had no effect. In the conditions of incubation used in these experiments absolute quantitative data are uncertain, since the dynamics of the conversions of precursors to steroid hormone changes with the effective changes in dilution consequent on the release of bound steroid which can then penetrate the outer compartment of the incubation flask (cf, Vinson and Whitehouse, 1973a). Furthermore, with assessment of two isotopes and mass of each steroid, rigorous determination of procedural losses is not possible. Determination of specific activities and of “H/14C ratios is not af-
II
1
P
Corticosterone
Aldoh?rore
FIG. 7. “H specific activities for products formed during dialysis incubation as for Figs. 4-6, abbreviations as for Fig. 4. P values for corticosterone C vs P < 0.05, for aldosterone C vs P i 0.01
fected by these losses, however, and reflects changes in stimulation of steroid forSpecific activities and “H/‘“C mation. ratios of the isolated compounds are given in Figs. 7-9. Figure 7 shows that in general there is a tenfold difference between the 3H specific activities of aldosterone and corticosterone. Specific activities of both steroids were decreased by pituitary extract and of aldosterone by ACTH. In contrast, Fig. 8 shows that the 14C specific activities of the two compounds were reasonably alike. The single exception is during stimulation by pituitary extract, when the Specific Activity for corticosTILIPUA
T I L IQ UA: ENDOCENOUS
: 14C S.A.‘s
0.7
% Dialysable
C_ DA P cOnlcaOerO”e
C
I:1
D A Aldvsmle
FIG. 6. Dialysibility of products formed from dogenous precursors in the same incubations as Figs. 4 and 5, abbreviations as for Fig. 5. P values corticosterone, C vs D < 0.05; D vs P < 0.001; aldosterone. C vs D,N S., D vs P < 0.05.
0.4 dpml
“g
0.3
P
enfor for for
CDAP Corticosterone
CDAP Aldosterone
FIG. 8. ‘T specific activities for products formed during dialysis incubation as for Figs. 4-7, abbreviations as in Fig. 4. P value, for corticosterone C vs P < 0.05.
546
VINSON,
BRAYSHER
TILIOUA
AND
WHITEHOUSE
1969; Mehdi and Sandor, 1974) but not in (Bourne and Seamark, 1973). Other 18-hydroxylated steroids such as 18-hydroxycorticosterone may also be produced, but the methods employed did not allow their characterisation. Tiliqua
ml Cortluaterone 3H/%
20D
ratio
1lIl l!oL
C
rl
A
P
20. Aldcsterone 3H/uC redo
C
rl
A
P
for products isolated following dialysis incubation as for Figs. 4-8, abbreviations as for Fig. 4. P values, for corticosterone, C vs D < 0.01; C vs P < 0.01; for aldosterone C vs P fi 0.05. FIG. 9. 3H/‘4C
ratios
terone was significantly increased. In Fig. 9, the 3H/14C ratios of the compounds differ by an order of magnitude. Dexamethasone pretreatment increased and pituitary extract decreased the value for corticosterone. Only the pituitary extract affects the 3H/14C ratio for aldosterone. DISCUSSION Identity of Steroid Products
The formation of corticosterone and aldosterone by Tiliqua adrenal tissue in vitro is consistent with the general literature on adrenal function in the reptiles. The lack of a significant accumulation of DOC may reflect simply its rapid conversion to corticosterone (Fig. 1). This steroid has been identified in incubations of other reptilian adrenals (Sandor et al., 1964; Huang et al.,
Pathway for Formation of Steroid Products
The yield time curves for tritiated products (Fig. 1) do not offer convincing evidence on the mode of formation of these steroids from pregnenolone. As noted above it is possible, but by no means certain, that DOC is very rapidly turned over to corticosterone, thus accounting for its very small yield and the very rapid rate of corticosterone formation. However, it is also possible that 1 lp-hydroxyprogesterone is an alternative intermediate (Boume and Seamark, 1973). More important, while corticosterone is clearly metabolised by the tissue and hence declines very rapidly in concentration, the evidence does not suggest that aldosterone is a major product of this metabolism. Incubation with labeled corticosterone would be required to clarify this point. In this respect the Tifiqua results contrast with those of other reptiles (e.g., Huang et al., 1969). However, although pregnenolone on this basis is a poor aldosterone precursor, it is important to emphasise that nevertheless the glands were extremely rich sources of aldosterone and that both from endogenous precursors and from [14C]acetate maximum yields of aldosterone were approximately 60% of those of corticosterone (Figs. 2,3). This difference between the poor utilisation of pregnenolone as an aldosterone precursor and the relatively extensive utilisation of acetate and endogenous precursors is also reflected in the order of magnitude difference between the 3H/‘4C ratios of corticosterone and aldosterone and also between their 3H but not their 14C specific
SKINK
ADRENOCORTICAL
activities (Figs. 7-9). There are two possible interpretations of this data. One is that aldosterone and corticosterone are formed by entirely separate pathways which may originate with acetate but diverge before pregnenolone formation. The second is that the pathways may be similar, but occur in separate parts of the cell and utilise two discrete pools of steroid precursor. Compartmental
Arrangement
of Steroid
The view that separate pools of steroids and their precursors are involved in the formation of corticosterone and aldosterone is borne out by their characteristics of dialysibility. Tritiated corticosterone and aldosterone formed from pregnenolone are indistinguishable in this respect (Fig. 4), whereas [14C]aldosterone is significantly less dialysable either than corticosterone or C3H]aldosterone (Fig. 5). This provides clear evidence that [“HI and [‘4C]aldosterone exist in separate pools, whereas [3H] and [‘4C]corticosterone do not. Since [14C]acetate gives better yields of aldosterone relative to corticosterone than [“Hlpregnenolone and since [14C]acetate is similar to endogenous precursors in this respect, it is tempting to suppose that there exists a bound pool of [*“Cl steroid which is preferentially converted to aldosterone. This is consistent with findings obtained in similar studies with rat adrenal zona glomerulosa (Vinson and Whitehouse, 1973b). One point of difference however is that in the present experiments, [‘“Cl steroids and steroids from endogenous precursors behaved differently in terms of dialysibility (Figs. 5,6), whereas in the rat experiments they did not. Thus it was only in conditions of stimulation with Tiliqua pituitary extract that the dialysibility of corticosterone was significantly different from aldosterone. In contrast, with the 14C-labeled steroid, no changes in dialysibility occurred when the
FUNCTION
547
tissue was stimulated with pituitary extract, or at other times. This may suggest that endogenous precursors are contained in yet a third pool of steroid, penetrable by neither [14C]acetate nor by [3H]pregnenolone. It may be that this third pool is concerned not with biosynthesis of different steroid types but with storage and release of a steroid reserve. Further evidence for this interpretation is that both from [14C]acetate and from [“Hlpregnenolone, the most rapid rate of formation of the steroids occurs within the first few minutes of incubation (Figs. 1,2). However, from endogenous precursors the most rapid rate of steroid secretion occurs between 1 and 2 hr after the start of the incubation and for at least the first 30 min or so the rate is near zero (Fig. 3). Clearly the in vitro rate of secretion of steroids from endogenous precursors has at least in this species, no connection with the rate of biosynthesis from [14C]acetate. Efects of Stimulation The view that endogenous precursors may be maintained in a steroid pool conserved as a reserve for conditions of acute stimulation is borne out by the effects of dexamethasone pretreatment and stimulation with tropic factors. Dexamethasone pretreatment significantly increases the binding of both corticosterone and aldosterone (Fig. 6) and hence presumably inhibits secretion. ACTH (Synacthen) has little effect but the addition of a pituitary extract greatly increases the dialysibility of corticosterone (though not aldosterone) and hence presumably enhances secretion. The ambiguous results obtained with Synacthen may result simply from species variation in ACTH structure, since Tiliqua ACTH is known to differ from mammalian ACTH even in the 1-24 region (A. P. Scott, personal communication). Thus Synacthen may be an inappropriate stimulant for this species. The dilution of the
548
VINSON,
BRAYSHER
total steroid pool by a release of stored endogenously formed material is also reflected in the drastic decline in 3H specific activity of corticosterone (and aldosterone) (Fig. 7): biosynthesis is stimulated beyond this effect and the 14C specific activity of corticosterone is greatly increased (Fig. 8). Changes in “H/‘“C ratio (Fig. 9) appear to reflect the changes in “H specific activity in general, which suggests, as discussed above, that [14C]acetate is handled biosynthetically in the same manner as the endogenous precursors. Thus, once again the pituitary extract greatly diminishes the value for both compounds, reflecting a stimulation of acetate conversion and unchanged or reduced pregnenolone conversion. In this case a clear effect of dexamethasone pretreatment is seen, which as may be expected, is the reverse of pituitary extract stimulation. Some increase in 3H specific activity of corticosterone may also occur (Fig. 7) but obviously it is not so marked as the change in “HP4C ratio (Fig. 9). In all of these effects including changes in dialysibility in both 3H and 14C specific activities and in the isotope ratio the changes are generally more marked in corticosterone than aldosterone. This suggests that the mechanisms of pituitary stimulation may be concerned with enhancing the secretion of corticosterone relative to aldosterone as well as in increasing absolute secretion rates. Functions
of the Adrenal
of Tiliqua
The uniform unzoned adrenal of this lizard has some remarkable functional characteristics. It appears to possess at least three distinguishable pools of steroid, a free pool giving rise to corticosterone, a bound pool “biosynthetic pool” giving aldosterone, and a “secretory reserve” pool. In addition, it appears to respond to dexamethasone pretreatment and to pituitary stimulation by marked changes in the triosynthesis and secretion of corticosterone
AND
WHITEHOUSE
selectively with less marked effects on aldosterone. In these respects there is a close parallel with the functions of the zona glomerulosa of the rat adrenal, in which it is also possible to show the existence of a bound pool giving aldosterone (and 18-hydroxydeoxycorticosterone), a free pool giving corticosterone and a selective mechanism for stimulation by ACTH of corticosterone relative to the other products (Whitehouse and Vinson, 1972; Vinson and Whitehouse, 1973a, b). A “secretory reserve” pool is not suggested by the rat results but may well exist since the binding is affected by ACTH - this is a characteristic of the “secretory reserve” in Tiliqua. In contrast, the inner zones of the rat adrenal treated separately show none of these effects, neither sequestration of steroids and their precursors into bound and free pools, nor a selective effect of ACTH on corticosterone relative to the 18-oxygenated products, nor indeed do the inner zones produce aldosterone at all. ACKNOWLEDGMENTS We are most grateful to the Wellcome Trust for a research and travel grant (to GPV) and to Professor W. V. Macfarlane, University of Adelaide, for the generous provision of facilities. We are also grateful to Drs. D. Burley and V. G. Balmer, Ciba-Geigy. for the provision of Synacthen.
REFERENCES Bourne, A. R. and Seamark, R. F. (1973). The synthesis of corticosterone by the adrenal tissue of the lizard, Tiliqua ruyosa. Comp. Biochem. Physiol. 4.95, 275-277. Bradshaw, S. D. and Fontaine-Bertrand, E. (1970). Measurement of corticosteroids in reptilian and avian plasma by fluorometry and by competitive protein-binding radioassay. Comp. Biochem. Physiol. 36, 37-48. Callard, 1. P., Chan, S. W. C., and Callard, G. V. (1973). Hypothalamic-pituitary-adrenal relareptiles. In “Brain-Pituitarytionships in Adrenal Interrelationships” (A. Brodish and E. S. Redgate, eds.), pp. 270-292. Karger, Base]. Chester Jones, I. (1957). “The Adrenal Cortex.” University Press, Cambridge. Chester Jones, I., Phillips, J. G., and Holmes, W. N.
SKINK
ADRENOCORTICAL
(1959). Comparative physiology ofthe adrenal cortex. In “Comparative Endocrinology” (A. Gorbman. ed.), pp. 582-612. Wiley, New York. Daugherty, D. R. and Callard, 1. P. (1972). Plasma corticosterone levels in the male iguanid lizard. Sceloporus cyanogenys under various physiological conditions. Gen. Camp. Endocrinol. 19, 69-79. Deane, H. W. (1962). The anatomy. chemistry and physiology of adrenocortical tissue. Handh. Exp. Pharmak. 14, l- 185. Gist, D. H. and DeRoos, R. (1966). Corticoids of the alligator adrenal gland and the effects of ACTH and progesterone on their production in vitro. Gen. Comp. Endocrinol. 8, 18-3 1. Huang. D. P., Vinson, G. P., and Phillips, J. G. ( 1969). The metabolism of pregnenolone and progesterone by cobra adrenal tissue in vitro and the effect of ACTH on product yield-time curves. Gen. Camp. Endocrinol. 12, 637-643. Leloup-Hatey. J. (1966). Etude in I&O de la corticosttroidog&&se effect&e par I’interrCnal de divers reptiles. J. de Physiol. (Paris) 58, 56 l-562. LeBrie, S. J. (1972). Endocrines and water and electrolyte balance in reptiles. Fed. Proc. 31, 1559-1603. Licht, P. and Bradshaw, S. D. (1969). A demonstration of corticotropic activity and its distribution in the para distalis of the reptile. Gen. Comp. Endocrinol. 13, 226-235. Macchi. 1. A. (1963). In vitro action of mammalian adrenocorticotropin and 5-hydroxytryptamine on adrenocortical secretion in the turtle, snake and bullfrog. Amer. Zooi. 3, 548. Macchi, I. A. (1964). In vifro corticoid biosynthesis by turtle and snake adrenals. 46th Meeting Endocrine Society 116. Macchi. I. A. and Phillips, J. G. (1966). In vitro effect of adrenocorticotropin on corticoid secretion in the turtle, snake and bullfrog. Cm. Camp. Endocrinol. 6, 170- 182. Mehdi, A. Z. and Sandor, T. (1974). Steroid biosynthesis by reptilian adrenals. Steroids, 24, 151-163. Phillips, J. G., Chester Jones, I., and Bellamy, D. (1962). Biosynthesis of adrenocortical hormones by adrenal glands of lizards and snakes. J. Endocrinol. 25,333-237.
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Sandor, T.. Lamoureux. J., and Lanthier. A. ( 1964). Adrenocortical function in reptiles. The in rsitro biosynthesis of adrenal cortical steroids by adrenal slices of two common North American turtles, the slider turtle I Pseudemys scripta elegans) and the painted turtle (Cl~rysemys picta picta). Steroids 4, 2 13-227. Symington. T. (1969). Adult adrenal cortex. In “Functional Pathology of the Human Adrenal Gland” (T. Symington. ed.), pp 3-181. Livingstone, Edinburgh. Tam. W.H., Phillips, J. G.. and Lofts, B.( 1972). Seasonal changes in the secretory activity of the adrenal gland of the cobra. (Nuja nuja L&m). Gen. Comp. Endocrinol. 19, 2 18-224. Vinson, G. P. (1966). Pathways of corticosteroid biosynthesis from pregnenolone and progesterone in rat adrenal glands. J. Endocrinol. 34, 355-363. Vinson, G. P. and Whitehouse, B. J. (1969). The relationship between corticosteroid biosynthesis from endogenous precursors and from added radioactive precursors by rat adrenal tissue in vitro. The effect of corticotrophin. Acta Endocrinol. (Copenhagen) 61. 695-708. Vinson. G. P. and Whitehouse, B. J. (1973a). Compartmental arrangement of steroids formed from [I - ‘lC]acetate by rat adrenal zona glomerulosa and the effect of corticotrophin. Acta Endocrinol. (Copenhagen) 72, 737-745. Vinson. G. P. and Whitehouse, B. J. (1973b). Biosynthesis and secretion of aldosterone by the rat adrenal zona glomerulosa and the significance of the compartmental arrangement of steroids. Acta Endocrinol. (Copenhagen) 72, 746-753. Whitehouse, B. J. and Vinson, G. P. (1967). Pathways of corticosteroid biosynthesis in avian adrenal glands. Gen. Comp. Endocrinol. 9, 161-169. Whitehouse, B. J. and Vinson, G. P. (197 1). Compartmental arrangement of steroid precursors and the control of steroid hormone secretion in vitro. Acra Endocrinol. (Copenhagen) 68, 467-476. Whitehouse, B. J. and Vinson, G. P. (1972). The effect of corticotrophin on compartmental arrangement of steroid precursors and the control of steroid hormone secretion by the rat adrenal cortex in vitro. Steroids Lipid Res. 3, I IO-1 17.
AND
COMPARATIVE
ENDOCRINOLOGY
26, 541-549 (1975)
In Vitro Steroidogenesis by the Nonzoned Adrenocortical Tissue of the Skink, Tiliqua rugosa G.P.
VINSON, M. BRAYSHER,
AND B.J.
WHITEHOUSE
Department
of Zoology, St Bartholomew’s Medical College, Charterhouse Square, London, EC1 M 6BQ, Department of Zoology, University of Adelaide, South Australia, and Department of Physiology, Queen Elizabeth College, Campden Hill Road, London, W8 7AH
5000
Accepted March 6, 1975 Steroid formation by adrenocortical tissue from the skink, Tiliqua rugosa, has been studied using established in vitro techniques. Both in conventional incubations, with timed sampling, and in incubations with dialysis, aldosterone, and corticosterone were major products. From endogenous precursors, and from [Ylacetate, yields of the two products were of the same order, whereas from [3H]pregnenolone maximal yields of corticosterone were at least tenfold greater than aldosterone. Maximal rates of steroid formation from the radioactive precursors occurred within the first few minutes of incubation, but maximal rates of steroid formation from endogenous precursors occurred significantly later, between l-2 hr. In incubations with dialysis [‘%]aldosterone was significantly less dialysable than [“Hlaldosterone under all conditions, whereas [“Cl and [3H]corticosterone were homogeneous. In contrast, neither aldosterone nor corticosterone formed from endogenous precursors were bound under control conditions, although binding was increased following dexamethasone pretreatment, and decreased following stimulation with Tiliqua pituitary extract (but not Synacthen), with concomitant changes in yields and specific activities. Inter alia the results suggest that products formed from [%]acetate and from [:‘H]pregnenolone may be maintained in separate pools within the tissue, and this accounts for their different metabolic fates. The bound pool, penetrated only by [3H]acetate, yields more aldosterone than the free, and may be termed a “biosynthetic pool.” In addition there exists a “secretory reserve pool.” This is suggested by the difference between rates of steroid secretion from endogenous and added precursors, and also from the changes in dialysibility seen in steroids formed from endogenous precursors under different conditions of stimulation. In both the compartmental arrangement of steroids, and the production of large yields of aldosterone the adrenocortical tissue of Tiliqua shows similarities to the zona glomerulosa, but not the inner zones of the rat adrenal cortex.
Like other nonmammalian vertebrates, reptiles show no differentiation of their adrenal cortical tissue into zones on the mammalian pattern (Chester Jones, 1957; Deane, 1962; Symington, 1969). Despite this, investigations of adrenocortical functions among the reptiles have shown that the hormonal products are identical with those found among mammalian secretions and include both corticosterone and aldosterone. These are formed in vitro from both added radioactive precursors such as 541 Copyright All rights
@ 1975 by Academic Press. Inc. of reproduction in any form reserved.
pregnenolone or progesterone and also from endogenous precursors (Phillips, Chester Jones, and Bellamy, 1962; Macchi, 1963: 1964; Macchi and Phillips, 1966; Sandor Lamoureux and Lanthier, 1964; Gist and deRoos, 1966; LeloupHatey, 1966; Huang, Vinson, and Phillips, 1969; Tam, Phillips, and Lofts, 1972; Bourne and Seamark, 1973; Mehdi and Sandor, 1974). 17a-Hydroxycorticosteroids have not been reported in reptiles. In rive, corticosterone and aldosterone
542
VINSON,
BRAYSHER
may be present in circulating plasma (Chester Jones, Phillips, and Holmes, 1959; Bradshaw and Fontaine-Bertrand, 1970; Licht and Bradshaw, 1969; Daugherty and Callard, 1972; Vinson, Braysher, and V. H. T. James, unpublished observations) and although early work failed to show stimulatory effects of ACTH added to incubation media in vitro (Gist and deRoos, 1966) more recently good stimulation of corticosteroid formation and secretion by mammalian ACTH has been shown in reptilian preparations both in vitro and in vivo (Huang et al., 1969; Licht and Bradshaw, 1969; Daugherty and Callard, 1972; Callard, Chan, and Callard, 1973). In addition, it is probable that changes in electrolyte balance may also elicit stimulation of adrenal steroidogenesis and secretion (Bradshaw, 1972; IeBrie, 1972). Thus, the adrenocortical tissue of the reptile, undifferentiated into zones, shows many of the functions of the structurally much more complex mammalian gland. In recent work with rat adrenal glands, another functional characteristic of the mammalian gland has emerged. Using a technique of incubation with dialysis it can be shown that in the zona glomerulosa (but not in the inner zones) steroids may be maintained in more than one pool and the metabolic end products of hormone synthesis are determined at least in part by this. Thus a free steroid pool gives rise to corticosterone and a bound pool to 18-0xygenated steroids including 1B-hydroxydeoxycorticosterone (1 B-OH DOC) and aldosterone. ACTH increases the proportion of free to bound steroid, and consequently increases the ratio of corticosterone to 18-OH DOC and aldosterone. These effects are only seen in products formed from endogenous precursors or an early labeled added precursor, such as [14C]acetate and not in products from late exogenous precursors, such as r3H]pregnenolone. In contrast to all of these findings, products from inner zones are homo-
AND WHITEHOUSE
geneous and apparently freely dialysable throughout such procedures (Whitehouse and Vinson, 1971, 1972; Vinson and Whitehouse, 1973a, b). It seemed worthwhile applying these techniques to a tissue from a reptile to give further information on the functions of the reptilian gland. This paper describes experiments on tissue from the South Australian skink, Tiliqua rugosa, in which a preliminary study of in vitro steroidogenesis indicates conformity with the normal reptilian pattern (Bourne and Seamark, 1973). MATERIALS
AND METHODS
Animals. Animals were collected in the wild and maintained on a mixed diet in the Department of Animal Physiology, Waite Agricultural Research lnstitute, South Australia, for brief periods. When required for collection of adrenal tissue, they were killed by decapitation and the adrenals were removed (approx 50 mg paired wt) cleaned of adhering fat and stored on ice for periods of up to 0.5 hr before incubation. Incubations. Three incubation experiments were performed. In all cases [7a - 3H]pregnenolone (Specific Activity 6.9 Cilmmole) and [ 1 - %]acetate (Specific Activity 61 mCi/mmole) were used as added precursors. Expt I. Eight pairs of adrenals were minced with a pair of scissors and incubated for 3.5 hr with 4 &i [3H]pregnenolone and 40 &i [“Clacetate at 37” in 10 ml Krebs-Ringer bicarbonate solution Steroids extracted from this incubation were used for the procedures for steroid identification (see below). Expt 2. Ten pairs of adrenals were incubated as in Expt 1, but in this case samples were withdrawn at 5, 15, 30, 60, 90, 120, and 240 min as previously described (Vinson, 1966; Whitehouse and Vinson, 1967) and the steroid extracts were fractionated separately. Expt 3. Twenty-four pairs of adrenals were subjected to incubation with dialysis as previously described (Whitehouse and Vinson, 197 1). In each case 1 &i [YH]pregnenolone and 10 PCi [%]acetate were used as added precursors and the tissue was placed in dialysis bags containing 2 ml Ringer solution with a further 15 ml bathing the outside. The 24 pairs of adrenals were in four experimental groups of 6 pairs each, viz., a control group (C), a group (A) with the addition of 2.5 U ACTH (Synacthen, Ciba) to the inside of the dialysis bag, a group (D) taken from animals pretreated with dexamethasone (0.4 mg.
SKINK ADRENOCORTICAL 12 hr previously), and a group (P) to which an extract of pituitary tissue taken from dexamethasone pretreated animals was added. Each flask received the equivalent off pituitary in 0.1 ml M acetic acid.
Extraction
and Quantitation
of Steroids
Steroids were extracted with ethyl acetate and chromatographed on paper in the systems T/70 and L/75 (Vinson and Whitehouse, 1969). Fractions corresponding to corticosterone and aldosterone were eluted and acetylated and oxidised to the y-lactone, respectively. DOC was also sought but not found. Steroids were then rechromatographed on paper and then quantitated using the glc techniques previously described (Vinson and Whitehouse, 1969). Aldosterone samples were subjected to glc twice using columns with different loadings, 1 and 3%, of XE-60 on gas Chrome Q and the mean values were used in calculation of results. Corticosterone samples were chromatographed on 3% XE-60. The isotope content was determined on remaining aliquots and treated as in previous publications (Vinson, 1966; Vinson and Whitehouse, 1969). Further identification procedures were carried out on the labeled extracted material and these are given in the Results.
RESULTS Identity of Compounds
Further identification was carried out on the isotopically labeled corticosterone acetate and aldosterone y-lactone obtained in Expt 1 by admixture of the extracted material with 5 pg authentic material. The mixtures were then chromatographed on thin layer in the systems: 1 -Light petroleum : benzene : ethyl acetate (1: 1:4); 2 -chloroform : methanol (9 : 1); and 3 -light petroleum : methanol (9 : 1). After each chromatogram, the specific activities were determined using the glc method for mass and scintillation counting for 3H and 14C content. Successive values for specific activity and 3H/14C ratios are given in Table 1. The area corresponding to deoxycorticosterone (DOC) in the chromatograms of this experiment was also eluted, acetylated, and rechromatographed (system L/75). Gas-liquid chromatography gave no indication of the presence of DOC
543
FUNCTION TABLE
1
IDENTITY OF Tiliqua ADRENAL INCUBATION PRODUCTS=
Sample Corticosterone acetate 1 2 3 Aldosterone-y-lactone 1 2 3
3H sp ac (cpdng)
3H/‘“C ratio
18.7 18.8 16.4
246 263 284
3.6 3.7 3.9
190 216 221
a Radioactive products were admixed with authentic unlabeled material and samples taken after three successive chromatograms on thin layer. Corticosterone was processed as the acetate and aldosterone after oxidation to the y-lactone. Specific activities were calculated following estimation of mass by glc and isotope content by liquid scintillation counting. Precursors were [aHlpregnenolone and [r4C]acetate.
(< 10 ng), and only trace amounts tope were detected (Fig. 1).
of iso-
Formation of Steroids with Time of Incubation (Expt 2)
Yields of corticosterone and aldosterone from [14C]acetate, [3H]pregnenolone, and from endogenous precursors are given in Figs. l-3. From [3H]-pregnenolone (Fig. 1) corticosterone was the major product and was formed very rapidly, reaching a maximum yield in about 15 min. The maximum yield of aldosterone was only about onetenth that of corticosterone. A very small
*
YlEtrl
1
2 HOURS
3
4
FIG. 1. Yield-time curves for products formed from [3H] pregnenolone during incubation of Tiliqua adrenal tissue. B = corticosterone, aldo = aldosterone, DOC = presumptive deoxycorticosterone.
544
VINSON,
BBAYSHER
AND
WHITEHOUSE
fore very different from Figs. 1 and 2. One point of similarity with Fig. 2 is that the maximum yield of aldosterone from endogenous precursors is about 60% that of corticosterone.
‘b YIELD x 104
Steroid Binding and Stimulation of Secretion (Expt 3)
FIG. 2. Yield-time curves for compounds from [‘%]acetate during the same incubation Fig. 1. B = corticosterone, aldo = aldosterone.
formed as for
trace of tritiated material corresponding to DOC was found in the earliest stages of incubation. From [14C]acetate (Fig. 2) maximum yields of both compounds were reached more slowly after about 1 hr. In contrast to the 3H results, the maximum yield of aldosterone was in this case about 60% of that of corticosterone. It is evident from the slopes of the curves that maximum rates of steroid formation from the radioactive precursors (Fig. 1 and 2) occur within the very earliest periods but Fig. 3 shows that this is not so in the case of endogenous precursor utilization. Here the production rates of both compounds appear to be very nearly zero for considerable periods, one-half hour in the case of corticosterone and 1.5 hr in the case of aldosterone. Maximum rates of formation occur between l-2 hr, immediately before the maximum yield is reached. These curves are there-
Characteristics of dialysibility of the compounds are shown in Figs. 4-6. Once again differences are apparent between the handling of the three types of precursor. In Fig. 4 it is clear that there is no difference in the dialysibility of tritiated corticosterone and tritiated aldosterone, which at approximately 60% seems to be indistinguishable from freely dialysable material under the conditions used (cf, Whitehouse and Vinson, 197 1). The various experimental treatments have no effect on this pattern. In contrast, in Fig. 5 a clear difference exists between 14C-labeled corticosterone and aldosterone and taken as a whole the [ 14C]aldosterone is significantly less dialysable than the [L4C]corticosterone and also than the [3H]aldosterone in the same incubations. This difference is consistent throughout all of the incubations. In further contrast, Fig. 6 shows that dialysibility of corticosterone and aldosterone from endogenous precursors is affected by experimental treatments, being 80
r
TILIQUA:
Tr
C D A P Coriicosierone
FIG. 3. Yield-time curves for products formed from endogenous precursors during the same incubations as for Figs. 1 and 2. B = corticosterone.
3H
C D A Aldarterone
P
FIG. 4. Dialysibility of products formed from [3H]pregnenolone during incubation of Tiliyua adrenal tissue. C = control, D = dexamethasone pretreated animals, A = dexamethasone pretreatment with the addition of Synacthen to the incubation medium inside the dialysis bag, P = dexamethasone pretreated, with the addition of Tiliqua pituitary extract to the incubation medium.
SKINK
TILIQUA:
ADRENOCORTICAL
54.5
FUNCTION
14C
80 60
70 60 76 Dialysable
50
50 4.
40
30
Dpm 1 n-3
20 10
20 C D
Dialysibility of products [‘Tlacetate in the same incubations Abbreviations as for Fig. 4. P value for dosterone vs [‘JH]aldosterone (cf. Fig. FIG.
30
5.
10
formed from as for Fig. 4. total [‘T]al4) < 0.05.
decreased with dexamethasone pretreatment, and increased with the addition of pituitary extract. Synthetic ACTH (Synacthen) had no effect. In the conditions of incubation used in these experiments absolute quantitative data are uncertain, since the dynamics of the conversions of precursors to steroid hormone changes with the effective changes in dilution consequent on the release of bound steroid which can then penetrate the outer compartment of the incubation flask (cf, Vinson and Whitehouse, 1973a). Furthermore, with assessment of two isotopes and mass of each steroid, rigorous determination of procedural losses is not possible. Determination of specific activities and of “H/14C ratios is not af-
II
1
P
Corticosterone
Aldoh?rore
FIG. 7. “H specific activities for products formed during dialysis incubation as for Figs. 4-6, abbreviations as for Fig. 4. P values for corticosterone C vs P < 0.05, for aldosterone C vs P i 0.01
fected by these losses, however, and reflects changes in stimulation of steroid forSpecific activities and “H/‘“C mation. ratios of the isolated compounds are given in Figs. 7-9. Figure 7 shows that in general there is a tenfold difference between the 3H specific activities of aldosterone and corticosterone. Specific activities of both steroids were decreased by pituitary extract and of aldosterone by ACTH. In contrast, Fig. 8 shows that the 14C specific activities of the two compounds were reasonably alike. The single exception is during stimulation by pituitary extract, when the Specific Activity for corticosTILIPUA
T I L IQ UA: ENDOCENOUS
: 14C S.A.‘s
0.7
% Dialysable
C_ DA P cOnlcaOerO”e
C
I:1
D A Aldvsmle
FIG. 6. Dialysibility of products formed from dogenous precursors in the same incubations as Figs. 4 and 5, abbreviations as for Fig. 5. P values corticosterone, C vs D < 0.05; D vs P < 0.001; aldosterone. C vs D,N S., D vs P < 0.05.
0.4 dpml
“g
0.3
P
enfor for for
CDAP Corticosterone
CDAP Aldosterone
FIG. 8. ‘T specific activities for products formed during dialysis incubation as for Figs. 4-7, abbreviations as in Fig. 4. P value, for corticosterone C vs P < 0.05.
546
VINSON,
BRAYSHER
TILIOUA
AND
WHITEHOUSE
1969; Mehdi and Sandor, 1974) but not in (Bourne and Seamark, 1973). Other 18-hydroxylated steroids such as 18-hydroxycorticosterone may also be produced, but the methods employed did not allow their characterisation. Tiliqua
ml Cortluaterone 3H/%
20D
ratio
1lIl l!oL
C
rl
A
P
20. Aldcsterone 3H/uC redo
C
rl
A
P
for products isolated following dialysis incubation as for Figs. 4-8, abbreviations as for Fig. 4. P values, for corticosterone, C vs D < 0.01; C vs P < 0.01; for aldosterone C vs P fi 0.05. FIG. 9. 3H/‘4C
ratios
terone was significantly increased. In Fig. 9, the 3H/14C ratios of the compounds differ by an order of magnitude. Dexamethasone pretreatment increased and pituitary extract decreased the value for corticosterone. Only the pituitary extract affects the 3H/14C ratio for aldosterone. DISCUSSION Identity of Steroid Products
The formation of corticosterone and aldosterone by Tiliqua adrenal tissue in vitro is consistent with the general literature on adrenal function in the reptiles. The lack of a significant accumulation of DOC may reflect simply its rapid conversion to corticosterone (Fig. 1). This steroid has been identified in incubations of other reptilian adrenals (Sandor et al., 1964; Huang et al.,
Pathway for Formation of Steroid Products
The yield time curves for tritiated products (Fig. 1) do not offer convincing evidence on the mode of formation of these steroids from pregnenolone. As noted above it is possible, but by no means certain, that DOC is very rapidly turned over to corticosterone, thus accounting for its very small yield and the very rapid rate of corticosterone formation. However, it is also possible that 1 lp-hydroxyprogesterone is an alternative intermediate (Boume and Seamark, 1973). More important, while corticosterone is clearly metabolised by the tissue and hence declines very rapidly in concentration, the evidence does not suggest that aldosterone is a major product of this metabolism. Incubation with labeled corticosterone would be required to clarify this point. In this respect the Tifiqua results contrast with those of other reptiles (e.g., Huang et al., 1969). However, although pregnenolone on this basis is a poor aldosterone precursor, it is important to emphasise that nevertheless the glands were extremely rich sources of aldosterone and that both from endogenous precursors and from [14C]acetate maximum yields of aldosterone were approximately 60% of those of corticosterone (Figs. 2,3). This difference between the poor utilisation of pregnenolone as an aldosterone precursor and the relatively extensive utilisation of acetate and endogenous precursors is also reflected in the order of magnitude difference between the 3H/‘4C ratios of corticosterone and aldosterone and also between their 3H but not their 14C specific
SKINK
ADRENOCORTICAL
activities (Figs. 7-9). There are two possible interpretations of this data. One is that aldosterone and corticosterone are formed by entirely separate pathways which may originate with acetate but diverge before pregnenolone formation. The second is that the pathways may be similar, but occur in separate parts of the cell and utilise two discrete pools of steroid precursor. Compartmental
Arrangement
of Steroid
The view that separate pools of steroids and their precursors are involved in the formation of corticosterone and aldosterone is borne out by their characteristics of dialysibility. Tritiated corticosterone and aldosterone formed from pregnenolone are indistinguishable in this respect (Fig. 4), whereas [14C]aldosterone is significantly less dialysable either than corticosterone or C3H]aldosterone (Fig. 5). This provides clear evidence that [“HI and [‘4C]aldosterone exist in separate pools, whereas [3H] and [‘4C]corticosterone do not. Since [14C]acetate gives better yields of aldosterone relative to corticosterone than [“Hlpregnenolone and since [14C]acetate is similar to endogenous precursors in this respect, it is tempting to suppose that there exists a bound pool of [*“Cl steroid which is preferentially converted to aldosterone. This is consistent with findings obtained in similar studies with rat adrenal zona glomerulosa (Vinson and Whitehouse, 1973b). One point of difference however is that in the present experiments, [‘“Cl steroids and steroids from endogenous precursors behaved differently in terms of dialysibility (Figs. 5,6), whereas in the rat experiments they did not. Thus it was only in conditions of stimulation with Tiliqua pituitary extract that the dialysibility of corticosterone was significantly different from aldosterone. In contrast, with the 14C-labeled steroid, no changes in dialysibility occurred when the
FUNCTION
547
tissue was stimulated with pituitary extract, or at other times. This may suggest that endogenous precursors are contained in yet a third pool of steroid, penetrable by neither [14C]acetate nor by [3H]pregnenolone. It may be that this third pool is concerned not with biosynthesis of different steroid types but with storage and release of a steroid reserve. Further evidence for this interpretation is that both from [14C]acetate and from [“Hlpregnenolone, the most rapid rate of formation of the steroids occurs within the first few minutes of incubation (Figs. 1,2). However, from endogenous precursors the most rapid rate of steroid secretion occurs between 1 and 2 hr after the start of the incubation and for at least the first 30 min or so the rate is near zero (Fig. 3). Clearly the in vitro rate of secretion of steroids from endogenous precursors has at least in this species, no connection with the rate of biosynthesis from [14C]acetate. Efects of Stimulation The view that endogenous precursors may be maintained in a steroid pool conserved as a reserve for conditions of acute stimulation is borne out by the effects of dexamethasone pretreatment and stimulation with tropic factors. Dexamethasone pretreatment significantly increases the binding of both corticosterone and aldosterone (Fig. 6) and hence presumably inhibits secretion. ACTH (Synacthen) has little effect but the addition of a pituitary extract greatly increases the dialysibility of corticosterone (though not aldosterone) and hence presumably enhances secretion. The ambiguous results obtained with Synacthen may result simply from species variation in ACTH structure, since Tiliqua ACTH is known to differ from mammalian ACTH even in the 1-24 region (A. P. Scott, personal communication). Thus Synacthen may be an inappropriate stimulant for this species. The dilution of the
548
VINSON,
BRAYSHER
total steroid pool by a release of stored endogenously formed material is also reflected in the drastic decline in 3H specific activity of corticosterone (and aldosterone) (Fig. 7): biosynthesis is stimulated beyond this effect and the 14C specific activity of corticosterone is greatly increased (Fig. 8). Changes in “H/‘“C ratio (Fig. 9) appear to reflect the changes in “H specific activity in general, which suggests, as discussed above, that [14C]acetate is handled biosynthetically in the same manner as the endogenous precursors. Thus, once again the pituitary extract greatly diminishes the value for both compounds, reflecting a stimulation of acetate conversion and unchanged or reduced pregnenolone conversion. In this case a clear effect of dexamethasone pretreatment is seen, which as may be expected, is the reverse of pituitary extract stimulation. Some increase in 3H specific activity of corticosterone may also occur (Fig. 7) but obviously it is not so marked as the change in “HP4C ratio (Fig. 9). In all of these effects including changes in dialysibility in both 3H and 14C specific activities and in the isotope ratio the changes are generally more marked in corticosterone than aldosterone. This suggests that the mechanisms of pituitary stimulation may be concerned with enhancing the secretion of corticosterone relative to aldosterone as well as in increasing absolute secretion rates. Functions
of the Adrenal
of Tiliqua
The uniform unzoned adrenal of this lizard has some remarkable functional characteristics. It appears to possess at least three distinguishable pools of steroid, a free pool giving rise to corticosterone, a bound pool “biosynthetic pool” giving aldosterone, and a “secretory reserve” pool. In addition, it appears to respond to dexamethasone pretreatment and to pituitary stimulation by marked changes in the triosynthesis and secretion of corticosterone
AND
WHITEHOUSE
selectively with less marked effects on aldosterone. In these respects there is a close parallel with the functions of the zona glomerulosa of the rat adrenal, in which it is also possible to show the existence of a bound pool giving aldosterone (and 18-hydroxydeoxycorticosterone), a free pool giving corticosterone and a selective mechanism for stimulation by ACTH of corticosterone relative to the other products (Whitehouse and Vinson, 1972; Vinson and Whitehouse, 1973a, b). A “secretory reserve” pool is not suggested by the rat results but may well exist since the binding is affected by ACTH - this is a characteristic of the “secretory reserve” in Tiliqua. In contrast, the inner zones of the rat adrenal treated separately show none of these effects, neither sequestration of steroids and their precursors into bound and free pools, nor a selective effect of ACTH on corticosterone relative to the 18-oxygenated products, nor indeed do the inner zones produce aldosterone at all. ACKNOWLEDGMENTS We are most grateful to the Wellcome Trust for a research and travel grant (to GPV) and to Professor W. V. Macfarlane, University of Adelaide, for the generous provision of facilities. We are also grateful to Drs. D. Burley and V. G. Balmer, Ciba-Geigy. for the provision of Synacthen.
REFERENCES Bourne, A. R. and Seamark, R. F. (1973). The synthesis of corticosterone by the adrenal tissue of the lizard, Tiliqua ruyosa. Comp. Biochem. Physiol. 4.95, 275-277. Bradshaw, S. D. and Fontaine-Bertrand, E. (1970). Measurement of corticosteroids in reptilian and avian plasma by fluorometry and by competitive protein-binding radioassay. Comp. Biochem. Physiol. 36, 37-48. Callard, 1. P., Chan, S. W. C., and Callard, G. V. (1973). Hypothalamic-pituitary-adrenal relareptiles. In “Brain-Pituitarytionships in Adrenal Interrelationships” (A. Brodish and E. S. Redgate, eds.), pp. 270-292. Karger, Base]. Chester Jones, I. (1957). “The Adrenal Cortex.” University Press, Cambridge. Chester Jones, I., Phillips, J. G., and Holmes, W. N.
SKINK
ADRENOCORTICAL
(1959). Comparative physiology ofthe adrenal cortex. In “Comparative Endocrinology” (A. Gorbman. ed.), pp. 582-612. Wiley, New York. Daugherty, D. R. and Callard, 1. P. (1972). Plasma corticosterone levels in the male iguanid lizard. Sceloporus cyanogenys under various physiological conditions. Gen. Camp. Endocrinol. 19, 69-79. Deane, H. W. (1962). The anatomy. chemistry and physiology of adrenocortical tissue. Handh. Exp. Pharmak. 14, l- 185. Gist, D. H. and DeRoos, R. (1966). Corticoids of the alligator adrenal gland and the effects of ACTH and progesterone on their production in vitro. Gen. Comp. Endocrinol. 8, 18-3 1. Huang. D. P., Vinson, G. P., and Phillips, J. G. ( 1969). The metabolism of pregnenolone and progesterone by cobra adrenal tissue in vitro and the effect of ACTH on product yield-time curves. Gen. Camp. Endocrinol. 12, 637-643. Leloup-Hatey. J. (1966). Etude in I&O de la corticosttroidog&&se effect&e par I’interrCnal de divers reptiles. J. de Physiol. (Paris) 58, 56 l-562. LeBrie, S. J. (1972). Endocrines and water and electrolyte balance in reptiles. Fed. Proc. 31, 1559-1603. Licht, P. and Bradshaw, S. D. (1969). A demonstration of corticotropic activity and its distribution in the para distalis of the reptile. Gen. Comp. Endocrinol. 13, 226-235. Macchi. 1. A. (1963). In vitro action of mammalian adrenocorticotropin and 5-hydroxytryptamine on adrenocortical secretion in the turtle, snake and bullfrog. Amer. Zooi. 3, 548. Macchi, I. A. (1964). In vifro corticoid biosynthesis by turtle and snake adrenals. 46th Meeting Endocrine Society 116. Macchi. I. A. and Phillips, J. G. (1966). In vitro effect of adrenocorticotropin on corticoid secretion in the turtle, snake and bullfrog. Cm. Camp. Endocrinol. 6, 170- 182. Mehdi, A. Z. and Sandor, T. (1974). Steroid biosynthesis by reptilian adrenals. Steroids, 24, 151-163. Phillips, J. G., Chester Jones, I., and Bellamy, D. (1962). Biosynthesis of adrenocortical hormones by adrenal glands of lizards and snakes. J. Endocrinol. 25,333-237.
FUNCTION
549
Sandor, T.. Lamoureux. J., and Lanthier. A. ( 1964). Adrenocortical function in reptiles. The in rsitro biosynthesis of adrenal cortical steroids by adrenal slices of two common North American turtles, the slider turtle I Pseudemys scripta elegans) and the painted turtle (Cl~rysemys picta picta). Steroids 4, 2 13-227. Symington. T. (1969). Adult adrenal cortex. In “Functional Pathology of the Human Adrenal Gland” (T. Symington. ed.), pp 3-181. Livingstone, Edinburgh. Tam. W.H., Phillips, J. G.. and Lofts, B.( 1972). Seasonal changes in the secretory activity of the adrenal gland of the cobra. (Nuja nuja L&m). Gen. Comp. Endocrinol. 19, 2 18-224. Vinson, G. P. (1966). Pathways of corticosteroid biosynthesis from pregnenolone and progesterone in rat adrenal glands. J. Endocrinol. 34, 355-363. Vinson, G. P. and Whitehouse, B. J. (1969). The relationship between corticosteroid biosynthesis from endogenous precursors and from added radioactive precursors by rat adrenal tissue in vitro. The effect of corticotrophin. Acta Endocrinol. (Copenhagen) 61. 695-708. Vinson. G. P. and Whitehouse, B. J. (1973a). Compartmental arrangement of steroids formed from [I - ‘lC]acetate by rat adrenal zona glomerulosa and the effect of corticotrophin. Acta Endocrinol. (Copenhagen) 72, 737-745. Vinson. G. P. and Whitehouse, B. J. (1973b). Biosynthesis and secretion of aldosterone by the rat adrenal zona glomerulosa and the significance of the compartmental arrangement of steroids. Acta Endocrinol. (Copenhagen) 72, 746-753. Whitehouse, B. J. and Vinson, G. P. (1967). Pathways of corticosteroid biosynthesis in avian adrenal glands. Gen. Comp. Endocrinol. 9, 161-169. Whitehouse, B. J. and Vinson, G. P. (197 1). Compartmental arrangement of steroid precursors and the control of steroid hormone secretion in vitro. Acra Endocrinol. (Copenhagen) 68, 467-476. Whitehouse, B. J. and Vinson, G. P. (1972). The effect of corticotrophin on compartmental arrangement of steroid precursors and the control of steroid hormone secretion by the rat adrenal cortex in vitro. Steroids Lipid Res. 3, I IO-1 17.
