Comp. Biochem. PhysioL, 1975, VoL 50B, pp. 211 to 213. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION STEROID FORMATION BY THE ADRENAL TISSUE OF THE POSSUM (TRICHOSURUS VULPECULA) MAGDA WEISS Depaxtment of Physiology, Monash University, Clayton, Victoria, Australia (Received 4 December 1973)
Abstract--1. Possum adrenal homogenates incubated with 14C-progesteroneyielded A-ring reduced metabolites, the major ones being 5fl-pregnane-3~-ol-20-one, 5fl-pregaane-3~,17,,20~-triol, 5flpregnane-3~,17~,21-triol-20-one and 5fl-androstane-3~-ol-17-one. 2. Similar experiments with adrenal slices yielded 17~-hydroxyprogesterone, cortisol, corticosterone and androstenedione. 3. A time-yield study indicated that reduction followed the usual synthetic pathways.
AN UNUSUALfeature of the adrenal venous secretion In order to establish the pathway of corticosteroid of Australian marsupials, not found in eutherians, biosynthesis in the possum a time-yield experiment is the presence of 21-deoxycortisol and 17~-hydroxy- was carded out (Table 1). Each section of the table 20fl-dihydro-progesterone in fairly large quantities illustrates, on top, the biologically active steroid and, (Weiss & Richards, 1971). The aim of this study below, the metabolites which are derived from it. therefore was to investigate the pathways of cortico- As indicated from the appearance of conversion steroid biogenesis in marsupials. products at the shorter time intervals, the reductive Whole possum adrenals were homogenized and processes followed the synthetic pathways, which incubated in Krebs--Ringer bicarbonate buffer in the were similar to the ones described by Hechter & presence of co-factors (Weiss, 1973) with 1 pCi 14C- Pincus (1954) for eutherians. Due to the difference progesterone (sp. act. 58 pCi/mM) as substrate. in the relative activities of the component enzymes, After 1 hr incubation about 30 labelled conversion each intermediate of the cortisol pathway has, howproducts were isolated but only the ones in highest ever, undergone subsequent reductions. The yields were positively identified. They were: 5fl- reduced metabolites accumulated and then formed pregnane-3ot-ol-20-one, 11.7+ 2.3~o; 5fl-pregnane- the major yields of the 1 and 2 hr incubates. Each' 3~,20ot-diol, 4"2+ 1.9%; 5fl-pregnane-30t,17,,20~- of the listed compounds were also produced in triol, 9.5 + 1-1%; 5fl-pregnane-3~,,17~,21-triol-20- smaller yields, in two or three other isomeric forms, one, 10.8 + 2.0~o; 5fl-androstane-3,~-ol- 17-one, but only tentative identifications were made. The 5.8+ l'7Yo; and 5fl-androstane-17fl-ol-3-one, 4.3 total androstane derivatives formed 22 per cent of + 1"2yo (mean + S.E.M., five experiments). The the yield indicating an active (C17,C20-desmolase identification of the isolated conversion products was system, in agreement with the findings of Vinson et based on the same criteria as described in previous al. (1971). To investigate if the low degree of llflpublications (Weiss & Richards, 1971; Weiss, 1973), hydroxylation (Table 1) was due to the limited preThe final identification was based on the abifity of the cursor pool, 0.5pCi 14C-11-deoxycortisol mixed labelled product when mixed with authentic inert with 100 pg crystalline material was incubated with carrier to form crystals of constant specific activity, 50 mg of possum adrenal homogenate. About 8 per after each of three successive recrystallizations. cent of the added substrate remained unchanged, These results indicated that the possum adrenals 60 per cent of the yield was dihydro-ll-deoxypossess very active reductase systems, forming A- cortisol, 20 per cent tetrahydro-11-deoxycortisol ring and C-20 reduced metabolites from progester- and only 7 per cent was cortisol. A similar experione. This conversion of progesterone to reduced ment using cortisol as substrate produced 80 per products occurs also in a number of eutherian cent of A-ring reduced metabolites. In this respect species but only as a minor pathway (Levy & Saito, possum adrenal tissue re.~-mbles that of the guinea1966; Ertel & Ungar, 1964; Lantos et aL, 1965; pig which could also actively reduce cortisol (BrownCharreau et aL, 1968). Grant et al., 1960). On the other hand, homogenates 211
212
MAGDAWEISS Table I. Changes with time of percentage yield of conversion products, during incubation of possum adrenal homogenate with 14C-progesterone Incubation time (min) Products (~ yield)
3
6
9
12
15
60
120
84.0 4.7
76.8 5.0 0.1
56.5 7.3 0.1
53.0 7.8 0.2
46.2 8-2 0.2
3.4 10.5 2.7
2.0 I1.0 3.5
17 ~-Hydroxyprogesterone 53-Pregnane-3 ~, 17~-diol-20-one 5fl-Pregnane-3n,17%20a-triol
6.3
11.7 0.1
23.1 0.3 0.1
22.0 0.3 0.1
25-6 0.6 0-2
1.5 8.2
2.7 10.5
11-Deoxycortisol 53-Pregnane-3~,17~,21-triol-20-one
0"1
0'3 0'1
0'6 0"2
0"8 0"2
1-3 0-4
1'8 10"2
0"8 9'5
0-1
0'1
0'2 0"1
2"3 0"2
1'5 0"5
0"5 0"2 0"2
0"7 0"4 0"3
0"7 0"7 0'9
4"5 6'1
4'8 7"2
Progesterone 53-Pregnane-3-ol-20-one 53-Pregnane-3 n,20~-diol
Cortisol 53-Pregaane-3a,113,17~,21-tetrol-20-one Androstenedione 5fl-Androstane-173-ol-3-one 5fl-Androstane-3cx-ol-17-one
0'5 0"1
of the American opossum (Didelphis virginiana) when incubated with progesterone did not show a high reducing activity (Brownell et al., 1967), neither did adrenals of other Australian marsupials such as the kangaroo (Macropus canguru major), Tasmanian devil (Sarcophilus harisii) and native cat (Dasyurus viverrinus) (unpublished observations). Also, in studies with homogenates from the most primitive mammals, the monotreme A4-hydrogenation was not observed (Weiss, 1973). When slices of possum adrenals were used instead of homogenates and incubated in Krebs-Ringer bicarbonate with 14C-progesterone, 85 per cent of the substrate remained unchanged, 3.1~o was 17~OH-progesterone (the highest yield), cortisol was 0.8~o, corticosterone 0"1~o and androstenedione 0.4~ (average of three experiments). The cortisol/ corticosterone ratio was similar to that found in the adrenal venous blood in vivo (Weiss & McDonald, 1966). These observations are in contrast to the findings of Vinson et al. (1971) who reported corticosterone yields to be consistently higher than cortisol, when possum adrenal cortex was incubated with progesterone. The differences in metabolic end products with slices and homogenates emphasize the importance of cellular organization in the preferential availability of the particular enzyme system for the appropriate substrate. The functional role for the high reductase activity of possum adrenals is obscure as the possum liver, in contrast to the guinea-pig (Brown-Grant et al., 1960), is adequately equipped for A-ring reduction (McDonald & Weiss, 1967). The reductase activity in the adrenal tissue of the possum could have some regulatory action on steroid biosynthesis; however,
0'6 0'1 0"2
as the activity is so high it could perhaps be looked upon as some evolutionary remnant of an earlier metabolism. REFERENCES
BROWN-GRANT K., FORSCHIELLIE. & DOREMAN R. I. (1960) The Aa-hydrogenase of guinea-pig adrenal gland. J. biol. Chem. 235, 1317-1320. BROWNELL K. A., BECK R. R. & BESCH P. K. (1967) Steroid production by the normal opossum (Didelphis virginiana) adrenal in vitro. Gen. & compar. Endocr. 9, 214-216. CHARREAUE. H., DUFAUM. L., VLLLEED. B. & VILLEE C. A. (1968) Synthesis of 5~-pregnane-3,20-dione by fetal and adult human adrenals. J. Clin. endocr. Metab. 28, 629-632. ERTELR. J. & UNGARF. (1964) In vitro effect of 2-methyl1-2-bis(3-pyridyl)-l-propanone(SU-4885)and NADPH in the mouse adrenal. Endocrinol 75, 949-956. HECI-rrER O. & PrNcus G. (1954) Genesis of adrenocortical secretion. Physiol. Rev. 34, 459-496. LANTOSC. P., RAMANP. B., GRAVESJ. M. H., DORFMAN R. I. & FORCHIELLIE. (1965) The biosynthesis of 5apregnane-3,20-dione (allopregnanedione) by cow adrenal preparations. Steroids 6, 69-80. LEVY H. & SArro T. (1966) Conversion of 17-hydroxyprogesterone into 3~,17-dihydroxypregn-4-en-20-one, 17,203-dihydroxypregn-4-en-3-one and other substances by bovine adrenal perfusion. Steroids 7, 250-259. McDONALD I. R. & WEISS M. (1967) Turnover and excretion of cortisol in the Australian marsupial Trichosurus vulpecula. Acta. endocr., Copenh. Suppl. 119, 242. VINSONG. P., PHILLIPSJ. G., CHESTERJONESI. & TSANG W. N. (1971) Functional zonation of adrenocortical tissue in the brush possum (Trichosurus vulpecula). J. Endocr. 49, 131-140.
Steroid formation by possum adrenals Wwss M. (1973) Biosynthesis of adrenocortical steroids by monotremes echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). J. Endocr. 58, 251-262. WeLSS M. & McDoNALD I. R. (1966) Corticosteroid secretion in the Australian phalanger (Trichosurus vulpecula). Gen. & compar. Endocr. 7, 345-351.
213
WE~s M. & I~CHAltVS P. G. (1971) Adrenal steroid secretion in the Tasmanian devil (Sarcophilus harisii) and the Eastern native cat (Dasyurus viverrinus). A comparison of adrenocortical activity of different Australian marsupials. J. Endocr. 49, 263-275. Key Word Index--Steroid; adrenal; Trichosurus vulpecula; progesterone; cortisol.
SHORT COMMUNICATION STEROID FORMATION BY THE ADRENAL TISSUE OF THE POSSUM (TRICHOSURUS VULPECULA) MAGDA WEISS Depaxtment of Physiology, Monash University, Clayton, Victoria, Australia (Received 4 December 1973)
Abstract--1. Possum adrenal homogenates incubated with 14C-progesteroneyielded A-ring reduced metabolites, the major ones being 5fl-pregnane-3~-ol-20-one, 5fl-pregaane-3~,17,,20~-triol, 5flpregnane-3~,17~,21-triol-20-one and 5fl-androstane-3~-ol-17-one. 2. Similar experiments with adrenal slices yielded 17~-hydroxyprogesterone, cortisol, corticosterone and androstenedione. 3. A time-yield study indicated that reduction followed the usual synthetic pathways.
AN UNUSUALfeature of the adrenal venous secretion In order to establish the pathway of corticosteroid of Australian marsupials, not found in eutherians, biosynthesis in the possum a time-yield experiment is the presence of 21-deoxycortisol and 17~-hydroxy- was carded out (Table 1). Each section of the table 20fl-dihydro-progesterone in fairly large quantities illustrates, on top, the biologically active steroid and, (Weiss & Richards, 1971). The aim of this study below, the metabolites which are derived from it. therefore was to investigate the pathways of cortico- As indicated from the appearance of conversion steroid biogenesis in marsupials. products at the shorter time intervals, the reductive Whole possum adrenals were homogenized and processes followed the synthetic pathways, which incubated in Krebs--Ringer bicarbonate buffer in the were similar to the ones described by Hechter & presence of co-factors (Weiss, 1973) with 1 pCi 14C- Pincus (1954) for eutherians. Due to the difference progesterone (sp. act. 58 pCi/mM) as substrate. in the relative activities of the component enzymes, After 1 hr incubation about 30 labelled conversion each intermediate of the cortisol pathway has, howproducts were isolated but only the ones in highest ever, undergone subsequent reductions. The yields were positively identified. They were: 5fl- reduced metabolites accumulated and then formed pregnane-3ot-ol-20-one, 11.7+ 2.3~o; 5fl-pregnane- the major yields of the 1 and 2 hr incubates. Each' 3~,20ot-diol, 4"2+ 1.9%; 5fl-pregnane-30t,17,,20~- of the listed compounds were also produced in triol, 9.5 + 1-1%; 5fl-pregnane-3~,,17~,21-triol-20- smaller yields, in two or three other isomeric forms, one, 10.8 + 2.0~o; 5fl-androstane-3,~-ol- 17-one, but only tentative identifications were made. The 5.8+ l'7Yo; and 5fl-androstane-17fl-ol-3-one, 4.3 total androstane derivatives formed 22 per cent of + 1"2yo (mean + S.E.M., five experiments). The the yield indicating an active (C17,C20-desmolase identification of the isolated conversion products was system, in agreement with the findings of Vinson et based on the same criteria as described in previous al. (1971). To investigate if the low degree of llflpublications (Weiss & Richards, 1971; Weiss, 1973), hydroxylation (Table 1) was due to the limited preThe final identification was based on the abifity of the cursor pool, 0.5pCi 14C-11-deoxycortisol mixed labelled product when mixed with authentic inert with 100 pg crystalline material was incubated with carrier to form crystals of constant specific activity, 50 mg of possum adrenal homogenate. About 8 per after each of three successive recrystallizations. cent of the added substrate remained unchanged, These results indicated that the possum adrenals 60 per cent of the yield was dihydro-ll-deoxypossess very active reductase systems, forming A- cortisol, 20 per cent tetrahydro-11-deoxycortisol ring and C-20 reduced metabolites from progester- and only 7 per cent was cortisol. A similar experione. This conversion of progesterone to reduced ment using cortisol as substrate produced 80 per products occurs also in a number of eutherian cent of A-ring reduced metabolites. In this respect species but only as a minor pathway (Levy & Saito, possum adrenal tissue re.~-mbles that of the guinea1966; Ertel & Ungar, 1964; Lantos et aL, 1965; pig which could also actively reduce cortisol (BrownCharreau et aL, 1968). Grant et al., 1960). On the other hand, homogenates 211
212
MAGDAWEISS Table I. Changes with time of percentage yield of conversion products, during incubation of possum adrenal homogenate with 14C-progesterone Incubation time (min) Products (~ yield)
3
6
9
12
15
60
120
84.0 4.7
76.8 5.0 0.1
56.5 7.3 0.1
53.0 7.8 0.2
46.2 8-2 0.2
3.4 10.5 2.7
2.0 I1.0 3.5
17 ~-Hydroxyprogesterone 53-Pregnane-3 ~, 17~-diol-20-one 5fl-Pregnane-3n,17%20a-triol
6.3
11.7 0.1
23.1 0.3 0.1
22.0 0.3 0.1
25-6 0.6 0-2
1.5 8.2
2.7 10.5
11-Deoxycortisol 53-Pregnane-3~,17~,21-triol-20-one
0"1
0'3 0'1
0'6 0"2
0"8 0"2
1-3 0-4
1'8 10"2
0"8 9'5
0-1
0'1
0'2 0"1
2"3 0"2
1'5 0"5
0"5 0"2 0"2
0"7 0"4 0"3
0"7 0"7 0'9
4"5 6'1
4'8 7"2
Progesterone 53-Pregnane-3-ol-20-one 53-Pregnane-3 n,20~-diol
Cortisol 53-Pregaane-3a,113,17~,21-tetrol-20-one Androstenedione 5fl-Androstane-173-ol-3-one 5fl-Androstane-3cx-ol-17-one
0'5 0"1
of the American opossum (Didelphis virginiana) when incubated with progesterone did not show a high reducing activity (Brownell et al., 1967), neither did adrenals of other Australian marsupials such as the kangaroo (Macropus canguru major), Tasmanian devil (Sarcophilus harisii) and native cat (Dasyurus viverrinus) (unpublished observations). Also, in studies with homogenates from the most primitive mammals, the monotreme A4-hydrogenation was not observed (Weiss, 1973). When slices of possum adrenals were used instead of homogenates and incubated in Krebs-Ringer bicarbonate with 14C-progesterone, 85 per cent of the substrate remained unchanged, 3.1~o was 17~OH-progesterone (the highest yield), cortisol was 0.8~o, corticosterone 0"1~o and androstenedione 0.4~ (average of three experiments). The cortisol/ corticosterone ratio was similar to that found in the adrenal venous blood in vivo (Weiss & McDonald, 1966). These observations are in contrast to the findings of Vinson et al. (1971) who reported corticosterone yields to be consistently higher than cortisol, when possum adrenal cortex was incubated with progesterone. The differences in metabolic end products with slices and homogenates emphasize the importance of cellular organization in the preferential availability of the particular enzyme system for the appropriate substrate. The functional role for the high reductase activity of possum adrenals is obscure as the possum liver, in contrast to the guinea-pig (Brown-Grant et al., 1960), is adequately equipped for A-ring reduction (McDonald & Weiss, 1967). The reductase activity in the adrenal tissue of the possum could have some regulatory action on steroid biosynthesis; however,
0'6 0'1 0"2
as the activity is so high it could perhaps be looked upon as some evolutionary remnant of an earlier metabolism. REFERENCES
BROWN-GRANT K., FORSCHIELLIE. & DOREMAN R. I. (1960) The Aa-hydrogenase of guinea-pig adrenal gland. J. biol. Chem. 235, 1317-1320. BROWNELL K. A., BECK R. R. & BESCH P. K. (1967) Steroid production by the normal opossum (Didelphis virginiana) adrenal in vitro. Gen. & compar. Endocr. 9, 214-216. CHARREAUE. H., DUFAUM. L., VLLLEED. B. & VILLEE C. A. (1968) Synthesis of 5~-pregnane-3,20-dione by fetal and adult human adrenals. J. Clin. endocr. Metab. 28, 629-632. ERTELR. J. & UNGARF. (1964) In vitro effect of 2-methyl1-2-bis(3-pyridyl)-l-propanone(SU-4885)and NADPH in the mouse adrenal. Endocrinol 75, 949-956. HECI-rrER O. & PrNcus G. (1954) Genesis of adrenocortical secretion. Physiol. Rev. 34, 459-496. LANTOSC. P., RAMANP. B., GRAVESJ. M. H., DORFMAN R. I. & FORCHIELLIE. (1965) The biosynthesis of 5apregnane-3,20-dione (allopregnanedione) by cow adrenal preparations. Steroids 6, 69-80. LEVY H. & SArro T. (1966) Conversion of 17-hydroxyprogesterone into 3~,17-dihydroxypregn-4-en-20-one, 17,203-dihydroxypregn-4-en-3-one and other substances by bovine adrenal perfusion. Steroids 7, 250-259. McDONALD I. R. & WEISS M. (1967) Turnover and excretion of cortisol in the Australian marsupial Trichosurus vulpecula. Acta. endocr., Copenh. Suppl. 119, 242. VINSONG. P., PHILLIPSJ. G., CHESTERJONESI. & TSANG W. N. (1971) Functional zonation of adrenocortical tissue in the brush possum (Trichosurus vulpecula). J. Endocr. 49, 131-140.
Steroid formation by possum adrenals Wwss M. (1973) Biosynthesis of adrenocortical steroids by monotremes echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). J. Endocr. 58, 251-262. WeLSS M. & McDoNALD I. R. (1966) Corticosteroid secretion in the Australian phalanger (Trichosurus vulpecula). Gen. & compar. Endocr. 7, 345-351.
213
WE~s M. & I~CHAltVS P. G. (1971) Adrenal steroid secretion in the Tasmanian devil (Sarcophilus harisii) and the Eastern native cat (Dasyurus viverrinus). A comparison of adrenocortical activity of different Australian marsupials. J. Endocr. 49, 263-275. Key Word Index--Steroid; adrenal; Trichosurus vulpecula; progesterone; cortisol.
