GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
28, lo- 16 (1976)
Adrenal Steroid Biosynthesis by Two Species of South American Rodents: Octodon degus and Abrocoma benettil S. M. GALLI Department of Physiology 4, Chile, and Department
AND E. T. MARUSIC
and Biophysics, University of Chile, of Physiology and Biophysics, School of Chile, Santiago, Chile
Casilla
6524,
of Medicine,
Santiago University
Accepted August 8, 1975 The adrenal glands from two South American rodents, the degu (Octodon degus) and the chinchilla rat (Abfocoma benetti), were incubated in Krebs Ringer bicarbonate glucose with radioactive precursors. The major products obtained in both species were corticosterone and cortisol. Other corticoids identified include 18-hydroxycorticosterone, aldosterone, and HOC. The chinchilla rat is also able to produce in vitro several other unidentified corticoids that were not present in the degu adrenal gland. 18-Hydroxydeoxycorticosterone was tentatively identified in the Abrocoma.
A whole group of South American rodents, the Hystricomorpha (Simpson 1945), suddenly appeared in the Miocene. Many of these rodents, in slightly modified form, have continued to exist into the present. Some of the rodents of southern origin are particular to Chile (Osgood, 1943). We have selected two families of rodents that belong to the suborder Caviomorpha, Octodontidae (Octodon degus) and Abrocomidae (Abrocoma benetti), to study the steroidal pattern of the adrenal gland. These rodents are practically confined to Chile (Wood, 1955). Both rodents have the same habitat; however, the degu is a diurnal animal, while the Abrocoma is a nocturnal rodent. These species of rodents can be maintained in the animal room with the standard condition of the rats. The degu is completely adapted to captivity, and the reproductive cycle was maintained under laboratory conditons; six
to nine litters are obtained after a 3-month gestation period. This paper shows the results of the degu adrenal tissue when incubated with radioactive pregnenolone or progesterone. The results are compared with that of the Abrocoma benetti or chinchilla rat. The adrenal glands of both species are able to synthesize cortisol and corticosterone as the major corticoids. The adrenocorticoid pattern of these animals differs from that of the rat and offers new possibilities in the study of the adrenal cortex with particular reference to the mechanism controlling aldosterone production in rodents. MATERIALS Ten Abrocoma
1 This work was supported in part by Grant No. 1342 RI/RB from International Atomic Energy Agency and by Comisi6n Investigacibn Cientifica, Universidad de Chile. 10 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form resewed.
AND METHODS
(Chinchilla rat) and 20 (degu) were captured in the central part of Chile. These rodents were kept in the animal room until the beginning of the experiments, under the same conditions as white rat. They were fed with Jkina chow pellets and some alfalfa twice a week. The animals were kept in the animal room for 3 to 5 days. They were sacrificed by decapitation, and the adrenal glands were removed and dissected. Male animals were used in all the experiments. The degu and the Abrocoma have a relatively big adrenal gland as compared to the white laboratory rat of the same size. The average weight of the two adrenal glands of the degu was 104 f 14 mg/lOO Octodon
degus
benetti
benetti
CORTICOSTEROIDOGENESIS
IN
g of body weight (mean value and SD). The Abrocoma adrenal glands had a mean value of 85 * 21 mg/lOO g of body weight. Adrenal quarters from both species were preincubated for 60 min at 37” in 5 ml of Krebs Ringer bicarbonate glucose (KRBG) in an atmosphere of 95% 02 and 5% CO,. The medium was replaced and the incubation continued for 1-4 hr in the presence of radioactive precursors. The radioactive substrates were: pregnenolone 7[$H] (sp act 25 Ci/mM) and progesterone 4[‘*C] (sp act 53.3 mCi/ti). 0.5-l yCi of progesterone and 1.8&i of pregnenolone were added per incubation flask. The samples were counted in a Nuclear Chicago Room Temperature Counter. Approximately 230 mg of degu adrenal tissue and 200 mg of the Abrocoma adrenal gland were used per flask. The media was extracted with 10 vol. of dichloromethane and sequentially washed with 1110vol 0.2M NaOH, 0.1 N acetic acid, and water (Sandor and Idler, 1972). The extracts were evaporated to dryness under vacuum or by the use of an air stream at 35”, and the residue was run through different chromatographic systems in the presence of reference corticoid standards. The chromatograms were examined under uv hght to detect the added standards, and the radioactive peaks were located with a Packard Radiochromatogram Scanner. The solvent systems for the chromatographic separation and for the identification of the radioactive steroids are shown in Table 1. Washed Whatman No. 1 paper was used throughout (Neher, 1964). Al1 experiments involved the isolation and purification of labelled steroids to constant 3W14C ratio. Cortisol, aldosterone and corticosterone were purified in the presence of tracer amounts ofthe labelled standards (2.000 dpm of [“Cl steroid when the samples were obtained from the tritiated precursor, and 100.000 dpm of [H]steroid when [*C]progesterone was used in the incubation media). New England nuclear radioactive compounds were used, and the purity of the radioactive standards was checked by paper chromatography. The TABLE CHROMATOGRAPHIC
11
etiolactone of lg-hydroxycorticosterone was obtamed by periodic oxidation (Marusic and Mulrow, 1967). Silica Gel-G plates were used for al] tic separations. Radioactive zones corresponding to DOC standards in the paper chromatographic systems were run in tic (isooctane-tert-butanol 7: 1) and the extracted paper material showed r, values identical with those of authentic DOC . Figure 1 represents a flow chart for the identification of the radioactive steroid fractions. Acetyiation and chromic oxidation were performed by the procedure of Kliman and Peterson (1960). The method of Wilson (1953) was followed for periodic acid oxidation and he procedure of Bush and Willoughby (1952) for saponification. Isomorphicity of some compounds was achieved by recrystallization according to the method of Axelrod er al. (196.5).
Octodon degus
The results entwined w en degu adrenal quarters were incubated i radioactive progesterone ar 2. The figure re achieved in one p
pounds isolated after 1) 2,
of the radioinert standards adde aldosterone, and corticosterone.
1 SYSTEMS
System
Composition
Ti75 Bush A Bush B3
Toluene:methanol:water (4:3:1) Light petroleum:methanol:water (5:4:1) Light petroleum:benzene:methanol:water (3.33:1.66:4:1) Benzene:methanol:water (2: 1:1) Cyclohexane:benzene:methanol:water (4:4:2: 1) Chloroform saturated with formamide Toluene saturated with formamide Toluene saturated with propyleneglycol Isooctane:tert-butanol (7: 1)
Bush Bs CBM C/F T/F TPG TLC
RODENTS
Impregnation
20% Formamide in acetone 25% Formamide in acetone 30% Propylenegiycol in acetone
FIG. 1. Diagram of the purification
steps employed for the identification
bation increases up to 4 hr, there is an increase in the amount of the radioactive products. A radioactive zone between corticosterone and progesterone is clearly seen after
of radioactive metabolites.
4 hr of incubation. It might correspond to a metabolite of some of the newly synthesized steroids. The radioactive zones corresponding to
FIG. 2. In vitro steroid by the degu adrenal glands (4[‘4C]Progesterone was added to the incubation medium). Scannings of the radioactivity of the T/75 chromatograms are shown with the location of the uv absorbing zones on the same paper strip. Legend: 0, starting line of chromatograms; F, cortisol; ALDO, aldosterone; B, corticosterone; PROG, progesterone.
CORTICOSTEROIDQGENESIS
cortisol, aldosterone, and corticosterone were rechromatographed sequentially in the Bush B5 and ChloroformFormamide systems. A single radioactive zone correspondang to the added standards was observed in Further identification of these three corticoids was carried out by acetylation and/or oxidation, as indicated in Fig. I. The acetates obtained were chromatographed in the DCM and CBM systems. Almost pure compounds were obtained after the Bush B, system as indicated by the constant 3H/14C ratios for aldosterone, cortisol, and corticosterone (Table 2). The table also includes the 3H/14C for the oxidation products. Also, identification for cortisol and corticosterone was achieved by the estabhshment of isomorphicity. Crystallization was Carrie t in the presence of radioinert standards. e specific activity of the adrenosterone mother liquor was of 6.643 cpdmg and that of the crytals was of 6.088 cpm/mg. For the corticosterone acetate derivative the mother liquor was 7.583 cpm/mg and the specific activity of the crystals was 6.800 cpm/mg. The radioactive zone near the origin in the T/75 system was rechromatographed in the TPG system for 96 hr. A single radioactive
IN
13
RODENTS
peak with a (cortisol) value of 0.67 was observed corresponding again to 18hydr~xyco~ic~sterone. Further ~~e~.ti~cation of this cQrnp~u~d was carried out by oxidation; the etiolactone forme with corticosterone standa (c~rticoster~~~~ value ~olactone of 18-h corticosterone. In some experiments, trac DQC were found after the firs bation. The pre e of DQC as indicated in 1, but no were measured. Tritiated pregnenoione was also used as precursor for the biosynthesis of corticoids of the degu adrenal gland. A similar pattern to that observed with progesterone was obtained. However, after the first graphic system, a great spread oft tivity was observed in the rad~Qcb~oma~~gram scanner, and the main r-a ticoids obtained under this co greater percentage of impurities. A comparative table of the results obtalne 1 hr of incubation, with ~~Q~estero~e i&Fnenolone as precursors, is shown m Table 3. Cortisol is one of t ajar corn~~~~~s obtained with both precursors”
Chromatographic
systems
Acetate derivatives
Octodon degus
Abrocoma benetti
Steroids
T/l5
Bush B,
Aldosterone Corticosterone Cortisol Aldosterone Corticosterone Cortisol
2.36 2.05 2.54 2.12 2.95 4.26
3.10 2.40 3.05 3.53 3.39 4.17
3.50 2.52 3.30 4‘43 3.98 -
3.52 2.64 3.15 4.40 -
3.65 2.70 3.21 4.19 4.14 4.39
a Adrenal quarters were incubated with [‘“Cl progesterone, and tritiated standards were added before the fist chromatographic system. b The oxidation products measured were: aldosterone to monocecetate lactone (Bush B, system); corticosterone to 11-dehydrocorticosterone (TPG system); cortisol to adrenosterone (DCM system).
14
GALL1 AND MARUSIC TABLE METABOLISMOFRADIOACTIVE
3
SUBSTRATES
BYTHE
DECU ADRENALGLAND
Percentage converted/100 mg of adrenal tissue/houp Final compounds Cortisol Aldosterone Corticosterone WHydroxycorticosterone
Pregnenolone 0.23 0.12 0.47 0.23
Progesterone 1.75 0.55 3.32 0.45
a Adrenal tissues were incubated with precursors during 1 hr. The results correspond to the values obtained after final purification of the steroids. Corrections for losses were achieved by adding tracer amounts of Y-standards (tritiated pregnenolone incubation) or 3H-standards [“‘Cl progesterone precursor), except for 1%OH B where no correction for losses was made.
andX, in different chromatographic systems did not allow us to draw any conclusions as compared with other well known corticoids (Fig. 3). The fraction X3 runs in the Bush B5 with two different peaks, one near the origin and Abrocoma benetti the other in front of aldosterone. It could be Radioactive progesterone was incubated a 19-hydroxyderivative. with the Abrocoma adrenal tissue. The reTable 4 presents a comparison of the sults obtained after the first chromatosteroidal pattern obtained in the chinchilla graphic system are shown in the upper part of rat and in the degu when radioactive progesFig. 3. terone was used as precursor. Both species A complex radioactive pattern is ob- of rodents were able to produce cortisol and served; there are at least seven radioactive corticosterone as the major corticoids. In zones. Identification and purification of the the chinchilla rat IS-hydroxyDOC was presradioactive areas corresponding to 18 ent. Several other unidentified compounds hydroxycorticosterone, cortisol, aldoappeared to be produced by this animal. sterone, and corticosterone were carried out Only tracer amounts of DOC were observed as indicated for the degu steroids. Table 2 in both species after the first hour of incubaalso includes the 3H/14C ratios for the Ab- tion. rocoma corticoids. DISCUSSION The crystals of adrenosterone had a specific activity of 18.733 cpm/mg and the Several studies have been carried out in mother liquor of 19.473 cpm/mg. The different species of rodents to establish the specific activity of the corticosterone de- corticoidal pattern in these animals. (Hof1965; rivative was of 9.547 cpm/mg and that of mann, 1956; Triller and Birmingham, Vinson, 1966; Ogunsuaet al., 1971). In genmother liquor was 10.833 cprn/mg. The compounds indicated as X,, X,, and eral, the rodents studied follow two different X, in Fig. 3 were run in the Bush B, system. patterns: (1) There is no production of corThe fraction X, moved with authentic 18 tisol in most species of the family Muroidea, hydroxydeoxycorticosterone in this system while in the Geomyoidea the major product and in the C/F and TPG systems. It probably of the adrenal cortex is cortisol; and (2) there is no corticosterone, or only traces are prescorresponds to IS-hydroxyDOC, according to the Rf obtained. The Rf obtained with X, ent (Ogunsua et al., 1971). To the concluThe results presented also indicate that there is no qualitative difference in steroidogenesis with progesterone or pregnenolone as precursors in the degu adrenal gland.
CQRTICOSTEROIDOGENESIS
A)
Tl75
!f
IN
RODENTS
SYSTEM
‘\ L
B)x 3 i ,
C)
BUSH
x, FRACTION
B, SYSTEM
BUSH
,- -1
8, SVSTEM
‘-,
FIG. 3. Scans of chromatograms of the Abrocoma adrenal gland. A) Methylene Chloride extract after i br of Incubation with 4[‘V]progesterone chromatographed in T/75 with radioinert F, ALDO, B. and PRQG. B) Ahquots of X, fraction of the T/75 system plus standard corticoids chromatographed in the Bush B;. C) Aliquots of the fraction X, of the T/75 system chromatographed in the Bush B5 system.
sions of these studies must now be added the results of this paper, which shows the results of experiments on other species of rodents, the Octodon degus and the Abrocoma be-
netti. These rodents have the capacity to synthesize cortisol and corticosterone as t major corticoids. Aldosterone an hydroxycorticosterone were also is
TABLE CQMPARISON
OF THE STEROIDAL CONVERSION
PATTERN VALUES
OBSERVED FOR
4
IN VITRO WITH
THE METABOLITES
DEGU OF
AND ABROCOMA
4 [“Cl
ADRENAL
Gums,
AND
PROGESTERONE
Percentage of progesterone converted per 100 mg of tissuea Steroids 1%OH corticosterone Cortisol Aldosterone Corticosterone 1%HydroxyDOC X, fraction
Octodon
degus
Abrocoma
0.6P i.6tY 0.39 4.WJ 0.57 8.36”
0.45 1.I50 0.55* 3.32b -
* The tissues were incubated during 1 hr. b The results are corrected for losses after final purification aldosterone, and corticosterone.
henetti
of the compounds
in the case of cortisof,
16
GALL1
AND
from the adrenal gland of both rodents. 18HydroxyDOC was also tentatively identified in the Abrocoma benetti. The Octodon degus is mainly a root- and seed-eater, and can be bred and raised in captivity without a source of drinking water. This led us to investigate if lPhydroxy-lldeoxycortisol was present. 19-Hydroxy11-deoxycortisol is the major steroid secreted by the adrenal gland of the Mongolean gerbil (Oliver and Peron, 1964). This animal ingests no water per se, and also excretes minimal quantities of urine. However, no compound with an Rf similar to 19-OH derivatives was detected in the Octodon; in fact, this animal has a very simple steroidal pattern. The water conservation mechanism in the degu remains to be investigated. Nevertheless, in the Abrocoma, which has the same habitat as the degu except that it is a nocturnal animal, some of the unidentified compounds could be 19-OH derivatives; for example, .& compound has in the Bush B5 system two peaks with an Rf similar to the 19-OH derivatives (Oliver and Peron 1964). No further identification was achieved because of the lack of authentic compounds. Further studies should be directed to establish the corticoids present in the blood of the degu and Abrocoma before final conclusions are drawn about the steroids produced in these South American species. Both rodents belong to the suborder Caviomorpha, family Abrocomidae and Octodontidae. Some other Chilean common species of this last family, such as Spalacopus cyanus (cururo) and Octodontornys gliroides (soco), have not been yet studied. The facts that the Octodon degus was adapted to captivity and that it can be reproduced in the laboratory offer the opportunity to utilize the degu as a model to study the regulatory mechanism of the adrenal gland in a rodent different from the laboratory rat or mouse. As mentioned above the degu differs from these two species in their capacity to synthesize cortisol. Since electrolyte levels are controlled mainly by aldosterone and the carbohydrate by corticosterone or
MARUSIC
cortisol, the degu is a potentially useful tool with which to study the mechanism controlling mineralocorticoids secretion in rodents. REFERENCES Axelrod et al. (1965). Definitive identification of microquantities of radioactive steroids by recrystallization to constant specific activity. Acta Endocrinol. (Copenhagen), Supplement 99, 49, l-77. Bush, 1. E., and Willoughby, M. (1952). Methods of paper chromatography of steroids applicable in the study of steroids in mammalian blood and tissues. Biochem. .I. 50, 37G-378. Hofmann, F. G. (1956). Observation onin vitro adrenal steroids synthesis in the albino mouse. Endocrinology 59, 712-715. Idler, D. R. (1972). Chapter 2. Steroid Methodology.Zn “Steroid in Nomammalian Vertebrates” (T. Sandor and D. R. Idler, eds.), Academic Press, New York. Kliman, B., and Peterson, R. E. (1960). Double isotope derivate assay of aldosterone in biological extracts. J. Biol. Chem. 235, 1639. Marusic, E. T., and Mulrow, P. (1967). In vitro convertion of corticosterone 4-C14 to 1%hydroxycorticosterone by zone fasciculate-reticularis of beef adrenal. Endocrinology 80, 214. Neher, R. (1964). “Steroid Chromatography.” Elsevier, Amsterdam. Ogunsua, A. O., De Nicola, A. F., Traikow, H., Birmingham, M., and Levine, S. (1971). Adrenal steroid biosynthesis by different species of mouselike rodents. Gen. Comp. Endocrinol. 16, 192-199. Oliver, J. T., and Peron F. G. (1964). 19 hydroxy-I 1 deoxicortisol, a major steroid secreted by adrenal gland of the Mongolean gerbil. Steroids 4,35 l-363. Osgood, W. H. (1943). “The Mammals ofchile.” Field Mus. Nat. Hist., Zool. Ser. 30, 32-41, 105-111. Sandor, T., and Idler, D. R. (1972). Steroid methodology. In “Steroids in Nonmammalian Vertebrates” (R. R. Idler, ed.), pp. 6-36. Academic Press, New York. Simpson, G. G. (1945). The principles of classification and classification of mammals. Bull. Amer. Mus. Natur. Hist. 85, l-350. Triller H., and Birmingham, M. (1965). Steroid production by incubated mouse adrenals. I. Characterization of steroid fractions. Gen. Comp. Endocrinol. 5, 618-623. Vinson, G. P. (1966). Pathways of corticoid biosynthesis from pregnenolone and progesterone in rat adrenal glands. J. Endocrinol. 34, 355-363. Wilson, H. (1953). Methods for the determination of formaldehydogenic steroids by diffusion and by direct reaction. J. Clin. Endocrinol. 13, 1465. Wood, A. E. (1955). A revised classification of the rodents. J. Mammals. 36, 165-187.
AND
COMPARATIVE
ENDOCRINOLOGY
28, lo- 16 (1976)
Adrenal Steroid Biosynthesis by Two Species of South American Rodents: Octodon degus and Abrocoma benettil S. M. GALLI Department of Physiology 4, Chile, and Department
AND E. T. MARUSIC
and Biophysics, University of Chile, of Physiology and Biophysics, School of Chile, Santiago, Chile
Casilla
6524,
of Medicine,
Santiago University
Accepted August 8, 1975 The adrenal glands from two South American rodents, the degu (Octodon degus) and the chinchilla rat (Abfocoma benetti), were incubated in Krebs Ringer bicarbonate glucose with radioactive precursors. The major products obtained in both species were corticosterone and cortisol. Other corticoids identified include 18-hydroxycorticosterone, aldosterone, and HOC. The chinchilla rat is also able to produce in vitro several other unidentified corticoids that were not present in the degu adrenal gland. 18-Hydroxydeoxycorticosterone was tentatively identified in the Abrocoma.
A whole group of South American rodents, the Hystricomorpha (Simpson 1945), suddenly appeared in the Miocene. Many of these rodents, in slightly modified form, have continued to exist into the present. Some of the rodents of southern origin are particular to Chile (Osgood, 1943). We have selected two families of rodents that belong to the suborder Caviomorpha, Octodontidae (Octodon degus) and Abrocomidae (Abrocoma benetti), to study the steroidal pattern of the adrenal gland. These rodents are practically confined to Chile (Wood, 1955). Both rodents have the same habitat; however, the degu is a diurnal animal, while the Abrocoma is a nocturnal rodent. These species of rodents can be maintained in the animal room with the standard condition of the rats. The degu is completely adapted to captivity, and the reproductive cycle was maintained under laboratory conditons; six
to nine litters are obtained after a 3-month gestation period. This paper shows the results of the degu adrenal tissue when incubated with radioactive pregnenolone or progesterone. The results are compared with that of the Abrocoma benetti or chinchilla rat. The adrenal glands of both species are able to synthesize cortisol and corticosterone as the major corticoids. The adrenocorticoid pattern of these animals differs from that of the rat and offers new possibilities in the study of the adrenal cortex with particular reference to the mechanism controlling aldosterone production in rodents. MATERIALS Ten Abrocoma
1 This work was supported in part by Grant No. 1342 RI/RB from International Atomic Energy Agency and by Comisi6n Investigacibn Cientifica, Universidad de Chile. 10 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form resewed.
AND METHODS
(Chinchilla rat) and 20 (degu) were captured in the central part of Chile. These rodents were kept in the animal room until the beginning of the experiments, under the same conditions as white rat. They were fed with Jkina chow pellets and some alfalfa twice a week. The animals were kept in the animal room for 3 to 5 days. They were sacrificed by decapitation, and the adrenal glands were removed and dissected. Male animals were used in all the experiments. The degu and the Abrocoma have a relatively big adrenal gland as compared to the white laboratory rat of the same size. The average weight of the two adrenal glands of the degu was 104 f 14 mg/lOO Octodon
degus
benetti
benetti
CORTICOSTEROIDOGENESIS
IN
g of body weight (mean value and SD). The Abrocoma adrenal glands had a mean value of 85 * 21 mg/lOO g of body weight. Adrenal quarters from both species were preincubated for 60 min at 37” in 5 ml of Krebs Ringer bicarbonate glucose (KRBG) in an atmosphere of 95% 02 and 5% CO,. The medium was replaced and the incubation continued for 1-4 hr in the presence of radioactive precursors. The radioactive substrates were: pregnenolone 7[$H] (sp act 25 Ci/mM) and progesterone 4[‘*C] (sp act 53.3 mCi/ti). 0.5-l yCi of progesterone and 1.8&i of pregnenolone were added per incubation flask. The samples were counted in a Nuclear Chicago Room Temperature Counter. Approximately 230 mg of degu adrenal tissue and 200 mg of the Abrocoma adrenal gland were used per flask. The media was extracted with 10 vol. of dichloromethane and sequentially washed with 1110vol 0.2M NaOH, 0.1 N acetic acid, and water (Sandor and Idler, 1972). The extracts were evaporated to dryness under vacuum or by the use of an air stream at 35”, and the residue was run through different chromatographic systems in the presence of reference corticoid standards. The chromatograms were examined under uv hght to detect the added standards, and the radioactive peaks were located with a Packard Radiochromatogram Scanner. The solvent systems for the chromatographic separation and for the identification of the radioactive steroids are shown in Table 1. Washed Whatman No. 1 paper was used throughout (Neher, 1964). Al1 experiments involved the isolation and purification of labelled steroids to constant 3W14C ratio. Cortisol, aldosterone and corticosterone were purified in the presence of tracer amounts ofthe labelled standards (2.000 dpm of [“Cl steroid when the samples were obtained from the tritiated precursor, and 100.000 dpm of [H]steroid when [*C]progesterone was used in the incubation media). New England nuclear radioactive compounds were used, and the purity of the radioactive standards was checked by paper chromatography. The TABLE CHROMATOGRAPHIC
11
etiolactone of lg-hydroxycorticosterone was obtamed by periodic oxidation (Marusic and Mulrow, 1967). Silica Gel-G plates were used for al] tic separations. Radioactive zones corresponding to DOC standards in the paper chromatographic systems were run in tic (isooctane-tert-butanol 7: 1) and the extracted paper material showed r, values identical with those of authentic DOC . Figure 1 represents a flow chart for the identification of the radioactive steroid fractions. Acetyiation and chromic oxidation were performed by the procedure of Kliman and Peterson (1960). The method of Wilson (1953) was followed for periodic acid oxidation and he procedure of Bush and Willoughby (1952) for saponification. Isomorphicity of some compounds was achieved by recrystallization according to the method of Axelrod er al. (196.5).
Octodon degus
The results entwined w en degu adrenal quarters were incubated i radioactive progesterone ar 2. The figure re achieved in one p
pounds isolated after 1) 2,
of the radioinert standards adde aldosterone, and corticosterone.
1 SYSTEMS
System
Composition
Ti75 Bush A Bush B3
Toluene:methanol:water (4:3:1) Light petroleum:methanol:water (5:4:1) Light petroleum:benzene:methanol:water (3.33:1.66:4:1) Benzene:methanol:water (2: 1:1) Cyclohexane:benzene:methanol:water (4:4:2: 1) Chloroform saturated with formamide Toluene saturated with formamide Toluene saturated with propyleneglycol Isooctane:tert-butanol (7: 1)
Bush Bs CBM C/F T/F TPG TLC
RODENTS
Impregnation
20% Formamide in acetone 25% Formamide in acetone 30% Propylenegiycol in acetone
FIG. 1. Diagram of the purification
steps employed for the identification
bation increases up to 4 hr, there is an increase in the amount of the radioactive products. A radioactive zone between corticosterone and progesterone is clearly seen after
of radioactive metabolites.
4 hr of incubation. It might correspond to a metabolite of some of the newly synthesized steroids. The radioactive zones corresponding to
FIG. 2. In vitro steroid by the degu adrenal glands (4[‘4C]Progesterone was added to the incubation medium). Scannings of the radioactivity of the T/75 chromatograms are shown with the location of the uv absorbing zones on the same paper strip. Legend: 0, starting line of chromatograms; F, cortisol; ALDO, aldosterone; B, corticosterone; PROG, progesterone.
CORTICOSTEROIDQGENESIS
cortisol, aldosterone, and corticosterone were rechromatographed sequentially in the Bush B5 and ChloroformFormamide systems. A single radioactive zone correspondang to the added standards was observed in Further identification of these three corticoids was carried out by acetylation and/or oxidation, as indicated in Fig. I. The acetates obtained were chromatographed in the DCM and CBM systems. Almost pure compounds were obtained after the Bush B, system as indicated by the constant 3H/14C ratios for aldosterone, cortisol, and corticosterone (Table 2). The table also includes the 3H/14C for the oxidation products. Also, identification for cortisol and corticosterone was achieved by the estabhshment of isomorphicity. Crystallization was Carrie t in the presence of radioinert standards. e specific activity of the adrenosterone mother liquor was of 6.643 cpdmg and that of the crytals was of 6.088 cpm/mg. For the corticosterone acetate derivative the mother liquor was 7.583 cpm/mg and the specific activity of the crystals was 6.800 cpm/mg. The radioactive zone near the origin in the T/75 system was rechromatographed in the TPG system for 96 hr. A single radioactive
IN
13
RODENTS
peak with a (cortisol) value of 0.67 was observed corresponding again to 18hydr~xyco~ic~sterone. Further ~~e~.ti~cation of this cQrnp~u~d was carried out by oxidation; the etiolactone forme with corticosterone standa (c~rticoster~~~~ value ~olactone of 18-h corticosterone. In some experiments, trac DQC were found after the firs bation. The pre e of DQC as indicated in 1, but no were measured. Tritiated pregnenoione was also used as precursor for the biosynthesis of corticoids of the degu adrenal gland. A similar pattern to that observed with progesterone was obtained. However, after the first graphic system, a great spread oft tivity was observed in the rad~Qcb~oma~~gram scanner, and the main r-a ticoids obtained under this co greater percentage of impurities. A comparative table of the results obtalne 1 hr of incubation, with ~~Q~estero~e i&Fnenolone as precursors, is shown m Table 3. Cortisol is one of t ajar corn~~~~~s obtained with both precursors”
Chromatographic
systems
Acetate derivatives
Octodon degus
Abrocoma benetti
Steroids
T/l5
Bush B,
Aldosterone Corticosterone Cortisol Aldosterone Corticosterone Cortisol
2.36 2.05 2.54 2.12 2.95 4.26
3.10 2.40 3.05 3.53 3.39 4.17
3.50 2.52 3.30 4‘43 3.98 -
3.52 2.64 3.15 4.40 -
3.65 2.70 3.21 4.19 4.14 4.39
a Adrenal quarters were incubated with [‘“Cl progesterone, and tritiated standards were added before the fist chromatographic system. b The oxidation products measured were: aldosterone to monocecetate lactone (Bush B, system); corticosterone to 11-dehydrocorticosterone (TPG system); cortisol to adrenosterone (DCM system).
14
GALL1 AND MARUSIC TABLE METABOLISMOFRADIOACTIVE
3
SUBSTRATES
BYTHE
DECU ADRENALGLAND
Percentage converted/100 mg of adrenal tissue/houp Final compounds Cortisol Aldosterone Corticosterone WHydroxycorticosterone
Pregnenolone 0.23 0.12 0.47 0.23
Progesterone 1.75 0.55 3.32 0.45
a Adrenal tissues were incubated with precursors during 1 hr. The results correspond to the values obtained after final purification of the steroids. Corrections for losses were achieved by adding tracer amounts of Y-standards (tritiated pregnenolone incubation) or 3H-standards [“‘Cl progesterone precursor), except for 1%OH B where no correction for losses was made.
andX, in different chromatographic systems did not allow us to draw any conclusions as compared with other well known corticoids (Fig. 3). The fraction X3 runs in the Bush B5 with two different peaks, one near the origin and Abrocoma benetti the other in front of aldosterone. It could be Radioactive progesterone was incubated a 19-hydroxyderivative. with the Abrocoma adrenal tissue. The reTable 4 presents a comparison of the sults obtained after the first chromatosteroidal pattern obtained in the chinchilla graphic system are shown in the upper part of rat and in the degu when radioactive progesFig. 3. terone was used as precursor. Both species A complex radioactive pattern is ob- of rodents were able to produce cortisol and served; there are at least seven radioactive corticosterone as the major corticoids. In zones. Identification and purification of the the chinchilla rat IS-hydroxyDOC was presradioactive areas corresponding to 18 ent. Several other unidentified compounds hydroxycorticosterone, cortisol, aldoappeared to be produced by this animal. sterone, and corticosterone were carried out Only tracer amounts of DOC were observed as indicated for the degu steroids. Table 2 in both species after the first hour of incubaalso includes the 3H/14C ratios for the Ab- tion. rocoma corticoids. DISCUSSION The crystals of adrenosterone had a specific activity of 18.733 cpm/mg and the Several studies have been carried out in mother liquor of 19.473 cpm/mg. The different species of rodents to establish the specific activity of the corticosterone de- corticoidal pattern in these animals. (Hof1965; rivative was of 9.547 cpm/mg and that of mann, 1956; Triller and Birmingham, Vinson, 1966; Ogunsuaet al., 1971). In genmother liquor was 10.833 cprn/mg. The compounds indicated as X,, X,, and eral, the rodents studied follow two different X, in Fig. 3 were run in the Bush B, system. patterns: (1) There is no production of corThe fraction X, moved with authentic 18 tisol in most species of the family Muroidea, hydroxydeoxycorticosterone in this system while in the Geomyoidea the major product and in the C/F and TPG systems. It probably of the adrenal cortex is cortisol; and (2) there is no corticosterone, or only traces are prescorresponds to IS-hydroxyDOC, according to the Rf obtained. The Rf obtained with X, ent (Ogunsua et al., 1971). To the concluThe results presented also indicate that there is no qualitative difference in steroidogenesis with progesterone or pregnenolone as precursors in the degu adrenal gland.
CQRTICOSTEROIDOGENESIS
A)
Tl75
!f
IN
RODENTS
SYSTEM
‘\ L
B)x 3 i ,
C)
BUSH
x, FRACTION
B, SYSTEM
BUSH
,- -1
8, SVSTEM
‘-,
FIG. 3. Scans of chromatograms of the Abrocoma adrenal gland. A) Methylene Chloride extract after i br of Incubation with 4[‘V]progesterone chromatographed in T/75 with radioinert F, ALDO, B. and PRQG. B) Ahquots of X, fraction of the T/75 system plus standard corticoids chromatographed in the Bush B;. C) Aliquots of the fraction X, of the T/75 system chromatographed in the Bush B5 system.
sions of these studies must now be added the results of this paper, which shows the results of experiments on other species of rodents, the Octodon degus and the Abrocoma be-
netti. These rodents have the capacity to synthesize cortisol and corticosterone as t major corticoids. Aldosterone an hydroxycorticosterone were also is
TABLE CQMPARISON
OF THE STEROIDAL CONVERSION
PATTERN VALUES
OBSERVED FOR
4
IN VITRO WITH
THE METABOLITES
DEGU OF
AND ABROCOMA
4 [“Cl
ADRENAL
Gums,
AND
PROGESTERONE
Percentage of progesterone converted per 100 mg of tissuea Steroids 1%OH corticosterone Cortisol Aldosterone Corticosterone 1%HydroxyDOC X, fraction
Octodon
degus
Abrocoma
0.6P i.6tY 0.39 4.WJ 0.57 8.36”
0.45 1.I50 0.55* 3.32b -
* The tissues were incubated during 1 hr. b The results are corrected for losses after final purification aldosterone, and corticosterone.
henetti
of the compounds
in the case of cortisof,
16
GALL1
AND
from the adrenal gland of both rodents. 18HydroxyDOC was also tentatively identified in the Abrocoma benetti. The Octodon degus is mainly a root- and seed-eater, and can be bred and raised in captivity without a source of drinking water. This led us to investigate if lPhydroxy-lldeoxycortisol was present. 19-Hydroxy11-deoxycortisol is the major steroid secreted by the adrenal gland of the Mongolean gerbil (Oliver and Peron, 1964). This animal ingests no water per se, and also excretes minimal quantities of urine. However, no compound with an Rf similar to 19-OH derivatives was detected in the Octodon; in fact, this animal has a very simple steroidal pattern. The water conservation mechanism in the degu remains to be investigated. Nevertheless, in the Abrocoma, which has the same habitat as the degu except that it is a nocturnal animal, some of the unidentified compounds could be 19-OH derivatives; for example, .& compound has in the Bush B5 system two peaks with an Rf similar to the 19-OH derivatives (Oliver and Peron 1964). No further identification was achieved because of the lack of authentic compounds. Further studies should be directed to establish the corticoids present in the blood of the degu and Abrocoma before final conclusions are drawn about the steroids produced in these South American species. Both rodents belong to the suborder Caviomorpha, family Abrocomidae and Octodontidae. Some other Chilean common species of this last family, such as Spalacopus cyanus (cururo) and Octodontornys gliroides (soco), have not been yet studied. The facts that the Octodon degus was adapted to captivity and that it can be reproduced in the laboratory offer the opportunity to utilize the degu as a model to study the regulatory mechanism of the adrenal gland in a rodent different from the laboratory rat or mouse. As mentioned above the degu differs from these two species in their capacity to synthesize cortisol. Since electrolyte levels are controlled mainly by aldosterone and the carbohydrate by corticosterone or
MARUSIC
cortisol, the degu is a potentially useful tool with which to study the mechanism controlling mineralocorticoids secretion in rodents. REFERENCES Axelrod et al. (1965). Definitive identification of microquantities of radioactive steroids by recrystallization to constant specific activity. Acta Endocrinol. (Copenhagen), Supplement 99, 49, l-77. Bush, 1. E., and Willoughby, M. (1952). Methods of paper chromatography of steroids applicable in the study of steroids in mammalian blood and tissues. Biochem. .I. 50, 37G-378. Hofmann, F. G. (1956). Observation onin vitro adrenal steroids synthesis in the albino mouse. Endocrinology 59, 712-715. Idler, D. R. (1972). Chapter 2. Steroid Methodology.Zn “Steroid in Nomammalian Vertebrates” (T. Sandor and D. R. Idler, eds.), Academic Press, New York. Kliman, B., and Peterson, R. E. (1960). Double isotope derivate assay of aldosterone in biological extracts. J. Biol. Chem. 235, 1639. Marusic, E. T., and Mulrow, P. (1967). In vitro convertion of corticosterone 4-C14 to 1%hydroxycorticosterone by zone fasciculate-reticularis of beef adrenal. Endocrinology 80, 214. Neher, R. (1964). “Steroid Chromatography.” Elsevier, Amsterdam. Ogunsua, A. O., De Nicola, A. F., Traikow, H., Birmingham, M., and Levine, S. (1971). Adrenal steroid biosynthesis by different species of mouselike rodents. Gen. Comp. Endocrinol. 16, 192-199. Oliver, J. T., and Peron F. G. (1964). 19 hydroxy-I 1 deoxicortisol, a major steroid secreted by adrenal gland of the Mongolean gerbil. Steroids 4,35 l-363. Osgood, W. H. (1943). “The Mammals ofchile.” Field Mus. Nat. Hist., Zool. Ser. 30, 32-41, 105-111. Sandor, T., and Idler, D. R. (1972). Steroid methodology. In “Steroids in Nonmammalian Vertebrates” (R. R. Idler, ed.), pp. 6-36. Academic Press, New York. Simpson, G. G. (1945). The principles of classification and classification of mammals. Bull. Amer. Mus. Natur. Hist. 85, l-350. Triller H., and Birmingham, M. (1965). Steroid production by incubated mouse adrenals. I. Characterization of steroid fractions. Gen. Comp. Endocrinol. 5, 618-623. Vinson, G. P. (1966). Pathways of corticoid biosynthesis from pregnenolone and progesterone in rat adrenal glands. J. Endocrinol. 34, 355-363. Wilson, H. (1953). Methods for the determination of formaldehydogenic steroids by diffusion and by direct reaction. J. Clin. Endocrinol. 13, 1465. Wood, A. E. (1955). A revised classification of the rodents. J. Mammals. 36, 165-187.
