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Corticosteroids of the coelacanth Latimeria chalumnae smith: A provisional study on their identity
GENERAL
AND
COMPARATIVE
Corticosteroids
41, 287-295 (1980)
ENDOCRINOLOGY
of the Coelacanth Latimeria chalumnae A Provisional Study on Their Identity’
Smith:
B. TRUSCOTT Marine
Sciences
Research
Laborafory, Ne~cfoundland
Memorial
University
AIC
5S7,
of Newfoundland.
St. John’s,
Canada
Accepted October 23, 1979 An extract of frozen tissue cut from an area of the coelacanth kidney, presumed to be the site of adrenocortical cells, was analyzed by double isotope derivative assay for adrenocorticosteroids. Evidence for the presence of 11-deoxycorticosterone, cortisol, and corticosterone was obtained. No 1 I-deoxycortisol or cortisone was detected. Incubation of the tissue with exogenous radioactive steroid precursors and identification of some transformation products indicated the presence of active enzyme systems including S-ene-3P-hydroxysteroid dehydrogenase, 5-ene-4-ene-isomerase, 20a-hydroxysteroid dehydrogenase, liphydroxysteroid dehydrogenase, and pregnene-5or- and SP-hydrogenases.
The nature and biosynthesis of corticowas hoped that by incubation of the steroids have been described for representacoelacanth tissue with radioactive precurtives of most vertebrate classes (for a re- sors we might be able to identify steroidocent review, see Sandor et al., 1976); a no- genie enzyme systems. This paper relates table exception is the crossopterygian the details and results of our experiments Laiimeria. The corticosteroids of fish have based on the hopes and presumptions outbeen of special interest to us (Idler and lined above. Truscott, 1972), so we requested Dr. J. E. MATERIALS AND METHODS McCosker to obtain “interrenal tissue” for The tissue was derived from a male coelacanth, 110 us during dissection of a specimen obtained cm in length (No. 79, CAS 33111), caught November by the California Academy of Sciences 1975 27, 1973, and obtained as a frozen specimen by the Coelacanth Expedition, supported by the 1975 California Academy of Sciences expedition to the Charline H. Breeden Foundation. At the Comores. At the dissection a discrete elongate nodular time it was not known if the adrenocortical area was removed from the ventral surface of the kidtissue of the coelacanth would consist of ney and reserved as possible interrenal tissue (perDr. M. Lagios). As the diffuse clumps of cells or aggregated cells sonal communication, coelacanth kidney is a fused structure rotated and disforming a discrete interrenal body. In any placed ventroposteriorly, the surface from which the event we hoped that there might be sufti- tissue was removed corresponded to the most dorsal cient endogenous corticosteroid present in aspect of a more typical fish mesonephros. Subsequent the “interrenal” and surrounding renal tis- to dissection in California the tissue was refrozen, packed in dry ice, and shipped to us. It was still frozen sue to permit identification. Freezing and upon arrival and was held in a freezer at -80°C for 3 thawing of the tissue would not be expect- months. ed to change the structure of the corticoWhen the tissue was thawed to initiate the present steroids. Also steroidogenic enzymes in in- investigation it did not closely resemble the macroterrenal tissues of some fish have been scopic appearance of elasmobranch interrenal. The major portion was reddish pink with associated condemonstrated to remain active through the nective tissue. Overlying one side there was a yellowfreeze/thaw process (Truscott and Idler, brown nonencapsulated region less well defined after 1968; Simpson and Wright, 1970). Thus it thawing than it had appeared in the frozen state. The 1 M.S.R.L.
Contribution
No. 373.
tissue (wet weight, 815 mg) was divided into three parts: the yellow-brown segment (230 mg) was cut 287 00 16-6480/80/070287-09$0 1.0010 Copyright All rights
@ 1980 by Academic Press. Inc. of reproduction in any form reserved.
B. TRUSCOTT away from the remainder and a layer of tissue (170 mg) directly in contact with it was kept separate from the remainder (395 mg). In this text the three parts are referred to as “nodular,” “subnodular,” and “renal” tissue. Each mass of tissue was cut into small pieces, and representative pieces were removed for histology. These were fixed in Bouin’s fluid for 48 hr dehydrated in a graded series of ethanol, cleared in xylene, embedded in Paraplast, and sectioned at 5 pm. Stains used included hematoxylin and eosin and Cason’s modification of the Mallory-Heidenhain connective tissue stain. Materials required for the double isotope derivative assay (DIDA) of corticosteroids, and for the identification of transformation products of radioactive precursors, e.g., W-labeled indicator steroids, reference steroids, solvents, and chromatographic media, were purchased from suppliers and treated as described in earlier publications (Truscott et al.. 1978; Truscott, 1979). Methods of isolation, purification, derivative formation, and detection of radioactive steroids and their 3H-Iabeled acetates or reference compounds, e.g., silica thin layer chromatography (tic), paper chromatography (PC), recrystallization to constant isotope ratio or constant specific activity, measurement of radioactivity, and color reactions, were essentially those described for earlier similar experiments (Truscott and Idler, 1972; Truscott et al., 1978; Truscott, 1979). Tissue
incubation
and preparation
of steroidal
ex-
tracts. The three portions of coelacanth tissue were minced and suspended (100 mg/ml) in a Tris-buffered medium containing NADPH-generating factors as described earlier (Idler and Truscott, 1967). The temperature was maintained at 10” and the atmosphere was enriched with oxygen. After 4 hr the medium was removed, centrifuged, and the supernatant solutions combined for analysis of extracted corticosteroids. The tissue had disintegrated considerably during the preincubation. The remaining tissue in each flask plus the corresponding debris recovered by centrifugation were transferred with medium to another flask each containing three radioactive precursors: 1 &i (19 nmol) [“Clprogesterone, 10 $Zi (0.9 nmol) [3H]pregnenolone, and 10 j&i (0.1 nmol) [3H]corticosterone. The incubation with radioactive precursors was continued under conditions as described above for 16 hr. The medium from each flask was analyzed separately for radioactive conversion products. Neutral steroidal fractions were isolated from both the pooled preincubation and the individual radioactive incubation media by exhaustive extraction with ice-cold dichloromethane. The dichloromethane extracts were washed with dilute alkali, acid, and water before evaporation to dryness under nitrogen. Steroid identification. The extract from the preincubation medium was analyzed by DIDA. Measured
amounts, 2000-4500 dpm, of 4-‘W-labeled cortisol, cortisone, corticosterone, 1 I-deoxycortisol, 1 ldeoxycorticosterone, and la-hydroxycorticosterone were added to the extract before an initial fractionation by tic to remove both extremely polar and nonpolar material. To insure against any possibility of contamination of the sample with authentic steroid during tic, progesterone only was used as a guide for the elution of the corticosteroid area of the plate (Truscott and Idler, 1972). The total corticosteroid fraction and aliquots of each 4-“C-labeled ‘indicator steroid were acetylated with freshly distilled tritiated acetic anhydride (400 mCi/mmol). After acetylation and the addition of the appropriate radioinert carrier, the corticosteroid acetates prepared from the coelacanth extract were separated and purified by sequential chromatography (Truscott and Idler, 1972; Truscott et ul., 1978) and then mixed with approximately 8 mg authentic carrier for crystallization to constant isotope ratio. The Y-indicator steroids were carried through the chromatography and crystallization in identical fashion since they had to serve as procedural blanks. The extract prepared by incubation of the coelacanth tissue with radioactive precursors was mixed with approximately 5 pg each of authentic radioinert progesterone, 17a-hydroxyprogesterone, pregnenolone, 17a-hydroxypregnenolone, ll-deoxycorticosterone, corticosterone, 1l-dehydrocorticosterone, 11-deoxycortisol, cortisol, and la-hydroxycorticosterone. Silica tic of the total mixture in cyclohexane:ethyl acetate:toluene (10: 10: 1) and, after drying, irrigation of the plate in the same direction with chloroform:methanol:water (90: 10: 1) gave seven contiguous but discernible bands under uv light. The tic solvent system, cyclohexane:propanol-2 (7:3), separated lldeoxycortisol from 1 I-dehydrocorticosterone and 17a-hydroxypregnenolone from 1 l-deoxycorticosterone, and a mixture of benzene:ethyl acetate (4:l) separated 17a-hydroxyprogesterone from pregnenolone. Each of the nine fractions off tic was further fractionated by pc. Solvent systems for pc were selected according to the polarity of the fraction under examination; their composition and sequence of use were basically similar to that described by Idler and Sangalang (1970). Radioactive metabolites on paper chromatograms were detected by radioscan. Their initial identification was dependent upon their coincidence, or lack of it, with authentic steroids. The presumed identity of a metabolite was then confirmed or denied by further chromatography, derivative formation, and in a few instances by recrystallization to constant specific activity.
RESULTS The cytological condition of the tissue was variable, the peripheral regions showing more autolysis. It was not possible to
CORTICOSTEROIDS
OF
THE
was recovered from the extract. Since the indicator, la-[14C]hydroxycorticosterone, behaved normally through the purification procedures, we conclude that the conditions of 3H-acetylation were inadequate for the esterification of the la-OH group in the presence of the rather bulky residue obtained from the tissue extract. The rate of acetylation of the secondary la-OH group is known to be considerably slower than that of the primary hydroxyl groups (Idler and Truscott, 1967). The experimental data are summarized in Table 1. For crystallization to constant specific activity the amount of authentic carrier which could be used was limited by the amount of radioactivity recovered through the chromatographic purification. The first supernatant solution was aspirated off, but no crystals were removed for isotope measurement until the second crystallization. Thus in Table 1 the isotope ratios are recorded as crystallization Nos. 2, 3, and 4. The [3H]acetic anhydride was determined
determine cytological or nuclear detail even in the best-preserved regions. The connective tissue region contained blood vessels including an extensive longitudinal segment of a thick-walled vessel. The yellow-brown region and the tissue lying between it and the blood vessels did not have the microscopic appearance of the homogenous elasmobranch interrenal nor did it resemble hematopoietic tissue of teleosts. It consisted of lobules containing several different cell types including pigment cells and granular cells. The tissue which this region most closely resembled is the perirenal tissue of the African lung&h Protopterus as described by Janssens et al. (1965). No clearly defined interrenal cells could be located in the histological material. Assay of the total preincubation medium of the tissue indicated the presence of lldeoxycorticosterone, cortisol, and possibly corticosterone. Neither cortisone nor 1 ldeoxycortisol could be detected. No la[14C]hydroxycorticosterone [3H]diacetate TABLE COMPARISON TISSUE
OF ISOTOPE CUT
FROM
RATIOS THE
OBTAINED
COELACANTH
BY DOUBLE
1 ISOTOPE
DERIVATIVE
AND
OF THE
W-INDICATOR
TO THE
EXTRACT
KIDNEY ADDED
289
COELACANTH
ASSAY
aH/“C
Steroid
[3H]acetate
OF
INITIALLY
ratio
Crystallization Recovery” m’)
OF AN EXTRACT
STEROIDS
No.
PC0
2
3
4
SN’
Mass” (ng)
1 l-[‘4C]Deoxycorticosterone Tissue extract + 2330 dpm
(10 ng)’
42 39
4.59 15.6
4.55 11.2
4.58 10.5
4.57 10.6
4.55 11.1
[“C]Cortisol Tissue extract
33 25
3.27 4.27
3.23 4.44
3.28 4.29
3.26 4.31
3.28 4.51
-
(13 ng)
3.49 5.06
3.54 4.59
3.54 4.67
3.49 4.65
3.49 4.99
-
[YlCorticosterone Tissue extract
+ 3970 dpm
13 4
(7 ng)
43 32
1 I-[Y]Deoxycortisol Tissue extract + 4340 dpm
3.89 4.33
3.88 3.85
3.90 3.80
3.88 3.79
3.90 3.85
-
(16 ng)
48 23
[‘“C]Cortisone Tissue extract
40 17
3.28 3.65
3.22 3.40
3.22 3.41
3.19 3.30
3.20 3.26
-
(15 ng)
+ 2090 dpm
+ 4750 dpm
U Percentage of added W-indicator recovered through the assay procedure. b PC = final isotope ratio through chromatographic purification. c SN = final supernatant from crystallization. d Corrected for mass of added W-indicator. p Amount of Y-indicator steroid added to tissue extract before acetylation.
2 0 0
290
B.
TRUSCOTT
to have a specific activity of 330 mCi/mmol and this value was used to calculate the mass of steroid which would be represented by the differences in isotope ratio of the sample and 14C-indicator. Extracts of the three separate media resulting from the incubation of the coelacanth tissue with a mixture of radioactive precursors, [14C]progesterone, [3H]pregnenolone, and [3H]corticosterone, were separated by tic into nine fractions. Radioactivity isopolar with carrier progesterone, pregnenolone, and corticosterone accounted for approximately 95% of the total radioactivity recovered from each extract. Further examination by pc of the tic fractions indicated that several radioactive metabolites were present in each fraction, though qualitatively there were no major differences apparent among the extracts. All fractions from each extract were analyzed for the products of steroidogenic enzyme systems appropriately as denoted by the added carrier steroid. Failure to detect any 17a-hydroxyprogesterone or 17~ hydroxypregnenolone, 1 l-deoxycorticosterone, 1 I-deoxycortisol, or cortisol indicated that no 17-, 21-, or lip-hydroxylases were active in any part of the tissue at the time of incubation. No la-hydroxycorticosterone was found in any of the extracts. Some effort was made to identify, at least tentatively, major radioactive metabolites which could indicate the presence of a catabolic enzyme system, e.g., reductases and dehydrogenases. The radioactive metabolites isolated and identified as described below were present in each of the three extracts. Quantitative differences were not considered since corrections could not be made for procedural losses. A tritium-labeled compound, difficultly separable from the carrier la-hydroxycorticosterone, was isopolar with lip, 20a, 21-trihydroxy-4-pregnen-3-one on pc with Bush-type solvent systems but of insufficient quantity to permit a more definitive identification. Further evidence for 20~
hydroxysteroid dehydrogenase activity was the tentative identification of 5[3H]pregnene-3p, 20a-diol (20a-dihydropregnenolone). Radioactivity isopolar with 17a-hydroxypregnenolone in the tic fraction was separated by pc into four components, the major one isopolar with 20~ dihydropregnenolone. Acetylation of an aliquot of this fraction did not separate the radioactivity from authentic 20a-dihydropregnenolone diacetate. The remainder of the 3H-metabolite was recrystallized with carrier 20a-dihydropregnenolone. The specific activity of three successive crops of crystals was constant within limits of +6%. Chromatographic purification, both before and after acetylation, of the progesterone area did not deplete this fraction of tritium. The isotope ratio was low (0.046) but remained essentially constant through reduction with 20P-hydroxysteroid dehydrogenase and isolation of 20/3-hydroxy-4pregnene-3-one (20@dihydroprogesterone) by tic (0.044) and pc (0.047). Radioinert 20/3-dihydroprogesterone was then added and the mixture recrystallized with acetone:pentane. The isotope ratio through four successive crystallizations for the progesterone of the “nodular tissue” incubate was 0.046 k 4.9% and 0.034 t 2.9% for the “subnodular” tissue. The progesterone fraction recovered from incubation of the “renal” tissue was not recrystallized with radioinert carrier but the isotope ratio was constant, 0.051 2 3.3%, through derivative formation and chromatography as outlined above. A 14C-metabolite was isopolar with the pregnanediones. Addition of authentic 5~ and S/3-pregnane-3, 20-diones and tic with the solvent system cyclohexane:ethyl acetate (1.1) (Lisboa, 1965) indicated that although both pregnanediones were present, there was approximately three times more of the Sa-pregnane. Reduction of the 5aand Sp-pregnanediones with 20@hydroxysteroid dehydrogenase, isolation of the 20&dihydro derivatives by pc, and sub-
CORTICOSTEROIDS
OF
THE
COELACANTH
291
sequent oxidation of these products, again underlying the “nodular” tissue and the with 20P-hydroxysteroid dehydrogenase, former accounted for over 70% of the total did not separate the radioactivity from the mass used in the incubations. carrier. There were insufficient counts to The results of our incubation studies can permit further identification of S/3- be considered in two parts: (i) the pre[ 14C]pregnanedione. The radioactivity sumptive identification of endogenous isopolar with Scr-pregnanedione was re- steroid in the preincubation medium and (ii) crystallized with authentic Sa-pregnanedithe transformation of radioactive precurone and through three successive crystallisors to products normally considered to be zations the specific activity was determined catabolites. to equal 270 dpm/mg (53%). The identification of 1 l-deoxycorticosterone, cortisol, and corticosterone in the A radioactive metabolite, isopolar with 1 l-dehydrocorticosterone, through chro- tissue extract is based on the demonstration of both isopolarity and isomorphism matographic purification, was acetylated acetates with the and the acetate purified by tic and pc. At of their 3H-labeled authentic steroid acetate. this point, the metabolite carried only the corresponding Table 1 records the data obtained by 3H-label. Authentic 1 1-[14C]dehydrocortimedium and costerone acetate was then added to give DIDA of the preincubation an isotope ratio of 3.69. Deacetylation with includes the calculated mass represented by acetylcholinesterase and reduction with the difference in final isotope ratios be20P-hydroxysteroid dehydrogenase with tween the sample and the indicator steroid. This calculation of mass assumes that purification by tic and pc of each derivative nonspecific tritium made no contribution to did not significantly change the isotope the final isotope ratio recorded for the samratio (av (4) = 3.46 ? 3%). ple. The assumption is supported by the reDISCUSSION sults obtained for 1I-deoxycortisol and Since at the time of the dissection in cortisone where there was no difference in California the anatomic nature of ad- the isotope ratios after recrystallization renocortical tissue of the coelacanth was between the 14C-indicator and the tissue not known, a discrete nodular body on the sample. Since the polarity of cortisol and surface of the kidney reminiscent of the in- corticosterone is not too different from that terrenal body of the elasmobranchs was of cortisone and 1 1-deoxycortisol, equally removed, along with some underlying tis- effective purification of the four acetates sue, as possible “interrenal” (Dr. M. D. could be expected. Thus, the difference in isotope ratios found for the cortisol and Lagios, personal communication). Recent corticosterone fractions of the tissue exwork by Lagios and Stasko-Concannon (1979) on kidney tissues from freshly fixed tract and the corresponding 14C-indicator is Latimeria indicates that the interrenal con- probably real and a measure of the corsists of numerous, small encapsulated cor- ticosteroid. puscles in close proximity to renal blood With due regard for their uncertain orivessels. The condition of the tissue used in gins, perhaps little significance can be atthe experiments described here was such tached to the quantitative differences in that no definitive histological studies could mass of 11-deoxycorticosterone, cortisol, be made but in the pieces of tissue actually and corticosterone. If active steriodogenic examined microscopically there were no enzymes were present during the preincuclearly defined interrenal cells. However, it bation of the tissue, then it is conceivable is possible that some small interrenal cor- that frozen storage, a double freeze/thaw puscles were present in the vascular tissue process, and the subsequent disintegration
292
B. TRUSCOTT
of tissue had more deleterious effects on ml. In the coelacanth tissue extract also some enzyme systems than on others. 1 1-deoxycorticosterone was quantitatively Simpson and Wright (1970), for example, the most important corticosteroid but correported that freezing and thawing de- tisol was present in a relatively significant stroyed the activity of a corticosteroid laamount, which brings us to the third hydroxylase in dogfish interrenal tissue but lungfish, the South American Lepidosiren. 1 lp- and 21-hydroxylase activities were still In this species, cortisol and aldosterone demonstrably present. However, it is were reported to be the principal plasmatic doubtful if any steroid hydroxylases were corticosteroids and though lesser amounts functional given the history of this particuof corticosterone were present, 1 I-delar tissue, and in fact by preincubation, we oxycorticosterone could not be detected probably extracted steroids present in the (Idler et al., 1972). tissue at the time of death of the animal. In Throughout the above discussion comthis case the quantity of any particular parisons have been drawn, almost by steroid at the time of analysis would depend necessity, between plasmatic steroids of not only on the initial concentrations but on the lungfishes and a tissue extract of the its chemical stability and perhaps to some coelacanth. There is little information extent upon its relative diffusibility. available on the synthesis in vitro of corHowever, if we accept the qualitative and ticosteroids by adrenocortical tissue of with reservation the quantitative validity of lungfishes: Janssens et al. (1965) identified the present work, the corticosteroids of the corticosterone only as a transformation coelacanth would appear to be a rather dis- product after incubation of “suspected adtinctive mixture comparable in certain fea- renocortical tissue” of the African lungfish tures to each of the three genera of Protopterus sp. with progesterone as exogenous precursor. Similarly our knowledge lungfishes. From a phylogenetic viewpoint, this is probably a valid comparison since of the plasmatic steroids of the coelacanth the coelacanth and lungfishes belong to is limited to an analysis of 1.4 ml of plasma the same subclass of vertebrates. Blair-West provided to us through the courtesy of Dr. et al. (1977) analyzed blood from the Grace Pickford and Dr. R. W. Griffith. We Australian lungfish Neocerutodusforsteri for analyzed this sample of plasma by DIDA and cortisol, corticosterone, aldosterone, and for cortisol, cortisone, aidosterone, 1I-deoxycorticosterone. In male Neocerutocorticosterone (Idler and Truscott, 1980) dus, the concentration of 1 l-deoxycorticobut unfortunately not for 1 l-deoxycortisterone (4.4 ng/lOO ml blood) was approxicosterone. We obtained no evidence for cortisone, or aldosterone, but mately four times higher than that of cortisol, the analytical data permitted the tentative aldosterone, and cortisol and corticosterone identification of corticosterone. were not present in detectable amounts. Two corticosteroids of interest are not A 50-ml blood sample from a male African listed in the table of results. Ia-Hydroxylungfish, Protopterus sp., was analyzed by DIDA for C-21 corticosteroids: ll-decorticosterone, the steroid unique to the oxycorticosterone was present (60 ng/lOO elasmobranchs (Truscott and Idler, 1968), was included in the initial planning of the ml) but cortisol, corticosterone, 1 l-deoxyexperiment since some characteristics of cortisol, aldosterone, and la-hydroxycorticosterone were not detected (Idler the coelacanth, e.g., pituitary organizaand Truscott, 1980). Aldosterone could not tion, rectal gland, urea retention, resemble those of the Chondrichthyes (Griffith et al., be detected in this particular blood sample from Protopterus by a radioimmunoassay 1974; Lagios, 1977). As stated earlier this sensitive to a concentration of 0.2 ng/lOO fraction was lost during acetylation.
CORTICOSTEROIDS
OF
[14C]Aldosterone was not added as an indicator steroid before 3H-acetylation, but radioinert aldosterone diacetate was added to the total acetylated extract before fractionation. Further purification of the aldosterone diacetate fraction removed all 3H counts, indicating no aldosterone was present in the original extract. However, since recovery through the total procedure could not be calculated, the negative result is not as meaningful as those for 1 1-deoxycortisol and cortisone, and is therefore not recorded in Table 1. There of course also remains the possibility that the principal steroid in the original extract was one of uncommon structure and not included in the assay. Incubation of the tissue (debris) with radioactive precursors indicated that the only enzyme system, still active, that could be classified as steroidogenic was a 5-ene3/3-hydroxysteroid dehydrogenase:5eneGene isomerase (5ene-3p OHSD:isomerase). The radioactivity in the progesterone fraction recovered from the incubation media contained approximately 5% tritium that was not separable from [ 14C]progesterone through chromatography, derivative formation, and recrystallization. Although no blank incubation was carried out, previous work has not demonstrated the spontaneous conversion of pregnenolone to progesterone under the conditions of incubation used here. Counting error as an explanation for the apparent residual tritium was discounted (Kobayashi and Maudsley, 1970). Thus, within the limits of tracer methodology where the identification of radioactivity is perforce indirect, it would appear that [3H]progesterone had been formed from [3H] pregnenolone as a result of 5-ene-3P-0HSD:isomerase activity. Tissues other than adrenocortical have been shown by tracer methodology to contain steroidogenic enzyme systems: 5-ene-3P-0HSD:isomerase and 21-hydroxylase in the teleost corpuscles of Stannius and body kidney (Idler and Freeman, 1966; Colombo et al., 1971) and
THE
COELACANTH
293
in bovine medullary tissue, 17~ and liphydroxylases were active as well (Carballeira et al., 1965). In the teleost tissues the yield of product from the radioactive exogenous precursor was generally low as it was in the present study. Thus, whether adrenocortical corpuscles (Lagios and StaskoConcannon, 1979) were actually present in the incubated coelacanth tissue is still open to question. Also we must appreciate that the work on the teleost and bovine tissues was done on freshly excised tissue whereas the coelacanth tissue had been in frozen storage for several years. All other enzyme systems that were presumed to be present by virtue of the identity of radioactive transformation products could be classified as catabolic. Since it was impossible to attempt to identify all of the transformation products, the number of enzyme systems demonstrated to be actively present was limited to the following: 2Ophydroxysteroid dehydrogenase, 1 lp-hydroxysteroid dehydrogenase, and pregnene-5a- and (probably) S/3-hydrogenases. These catabolic enzyme systems are usually concentrated in the liver but also are present in normal adrenocortical and kidney tissue (Kime, 1978). 1 lp-Hydroxysteroid dehydrogenase activity has been reported in the interrenal gland of elasmobranchs (Truscott and Idler, 1968) and head kidney of teleosts (see Idler and Truscott, 1972). The pregnanediones, products of ~CX- and SP-hydrogenases, were identified from incubation of body kidney of trout with progesterone (Arai et al., 1969; Colombo et al., 1971). In summary, the coelacanth tissue, in spite of its history of frozen storage, had maintained at least some of the steroid catabolic enzyme activity previously reported for other species of fish. Although, as the title implies, the present study of the corticosteroids of the coelacanth is not considered to be definitive, the results should provide useful information to any investigator interested in
294
B. TRUSCOTT
comparative endocrinology who, in the future, is fortunate enough to obtain body tissues or fluids from the coelacanth. ACKNOWLEDGMENTS The author is indebted to Dr. M. P. Burton for the histological examination of the tissue and expresses gratitude to her for advice and assistance in describing the nature of the tissue. Thanks are due to Dr. J. E. McCosker, Steinhart Aquarium, San Francisco, California, for making available and arranging shipment of the coelacanth tissue: to Dr. M. D. Lagios, Children’s Hospital of San Francisco, for providing a description of the tissue and its anatomic position as observed at the dissection of the California Academy of Sciences specimen, Latimeria No. 79, CAS 33111; to Dr. D. Duncan, B. C. Research, Vancouver, British Columbia, for intercepting the shipment of tissue from California and replenishing the dry ice in the package; to Dr. D. R. Idler for initiating the request for coelacanth tissue and for his constructive criticism of the work and of this report. The competent technical assistance of Mrs. V. Boland is gratefully acknowledged. This work was supported in part by a grant, A9752, from the Natural Sciences and Engineering Research Council of Canada. Nore added in proof: Analysis by DIDA of 4 ml gall bladder bile from this specimen of coelacanth indicated a compound, isopolar and isomorphous with cortisol, was released by hydrolysis with sulfatase from Helix pornaria. Neither cortisone, corticosterone, Il-deoxycorticosterone nor their tetrahydro derivatives, nor tetrahydrocortisol, could be detected in the bile.
REFERENCES Arai, R., Tajima, H., and Tamaoki, B. (1969). In vitro transformation of steroids by the head kidney, the body kidney, and the Corpuscles of Stannius of the rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 12, 99- 109. Blair-West, J. R., Coghlan, J. P., Denton, D. A., Gibson, A. P., Oddie, C. J., Sawyer, W. H., and Scoggins, B. A. (1977). Plasma renin activity and blood corticosteroids in the Australian lungfish Neoceratodus
f‘orsteri.
J.
Endocrinol.
74,
137- 142. Carballeira, A., Mehdi, A., and Venning, E. H. (1965). Metabolism of labelled steroid precursors by normal bovine adrenal medulla in vitro. Proc. Sm. Exp. Biol. Med. 119, 751-756. Colombo, L., Bern, H. A., and Pieprzyk, J. (1971). Steroid transformations by the Corpuscles of Stannius and the body kidney of Salmo gairdnerii (Teleostei). Gen. Comp. Endocrinol. 16, 74-84. Griffith, R. W., Umminger, B. L., Grant, B. F., Pang, P. K. T., and Pickford, G. E. (1974). Serum composition of the coelacanth, Latimeria chalumnae Smith. J. Exp. Zoo/. 187, 87- 102.
Idler,
D. R., and Freeman, H. C. (1966). Steroid transformations by Corpuscles of Stannius of the Atlantic cod (Gadus morhua L.). J. Fish. Res. Board Canad. 23, 1249- 1255. Idler, D. R., and Sangalang, G. B. (1970). Steroids of a Chondrostean: In vitro steroidogenesis in yellow bodies isolated from kidneys and along the posterior cardinal veins of the American Atlantic sturgeon, Acipenser oxyrhynchus Mitchill. J. Endocrinol.
48, 627-637.
Idler. D. R., and Truscott, B. (1967). la-Hydroxycorticosterone: Synthesis in vitro and properties of an interrenal steroid in the blood of cartilaginous fish (GenusRaja). Steroids 9,457-477. Idler, D. R., and Truscott, B. (1972). Corticosteroids in fish. In “Steroids in Non-mammalian Vertebrates” (D. R. Idler, ed.), pp. 127-252. Academic Press, New York. Idler, D. R., and Truscott, B. (1980). Phylogeny of adrenal corticosteroids of vertebrates. In “Evolution of Vertebrate Endocrine Systems” (P. K. T. Pang and A. Epple, eds.). Texas Tech. Univ. Press, Lubbock. Idler, D. R., Sangalang, G. B., and Truscott, B. (1972). Corticosteroids in the South American lungfish. Gen. Comp. Endocrinol. Suppl. 3, 238-244. Janssens, P. A., Vinson, G. P., Chester Jones, I., and Mosley, W. (1965). Amphibian characteristics of the adrenal cortex of the African lunglish (Protopterus sp.). J. Enducrinol. 32, 373-382. Kime, D. E. (1978). Catabolism of the adrenocortical hormones. In “General, Comparative and Clinical Endocrinology of the Adrenal Cortex” (I. Chester Jones and I. W. Henderson, eds.), Vol. 2, pp. 265-290. Academic Press, New York. Kobayashi, Y., and Maudsley, D. V. (1970). Practical aspects of double isotope counting. In “The Current Status of Liquid Scintillation Counting” (E. D. Bransome, Jr., ed.), pp. 76-85. Grune & Stratton, New York. Lagios, M. D. (1977). The coelacanth and the Chondrichthyes as sister groups. A review of shared apomorph characters and a cladistic analysis and reinterpretation. In “Abstracts, 58th Annual Meeting, Pacific division,” A.A.A.S., San Francisco. Lagios, M. D., and Stasko-Concannon, S. (1979). Presumptive interrenal tissue (adrenocortical homolog) of the coelacanth Larimeria chalumnae. Gen.
Comp.
Endocrinol.
37, 404-406.
Lisboa, B. P. (1965). Application of thin-layer chromatography to the separation of saturated 21deoxy-pregnane steroids. Steroids 6, 605-625. Sandor, T., Fazekas, A. G., and Robinson, B. H. (1976). The biosynthesis of corticosteroids throughout the vertebrates. In “General, Comparative and Clinical Endocrinology of the Adrenal cortex” (I. Chester Jones and I. W. Hender-
CORTICOSTEROIDS
OF THE COELACANTH
son, eds.) Vol. 1, pp. 25-142. Academic Press, New York. Simpson, T. H., and Wright, R. S. (1970). Synthesis of corticosteroids by the interrenal gland of selachian elasmobranch fish. J. Endocrinol. 46, 261-268. Truscott, B. (1979). Steroid metabolism in fish. Identification of steroid moieties of hydrolyzable conjugates of cortisol in the bile of trout Salmo gairdncrii. Gen. Camp. Endocrinol. 38, 196-206. Truscott, B.. and Idler, D. R. (1968). The widespread
295
occurrence of a corticosteroid la-hydroxylase in the interrenals of Elasmobranchii. J. Endocrinol. 40, 515-526. Truscott, B., and Idler, D. R. (1972). Corticosteroids in plasma of elasmobranchs. Camp. Biochrm. Physiol. 42A, 41-50. Truscott, B., Idler, D. R., Sundararaj, B. I., and Goswami, S. V. (1978). Effects of gonadotropins and adrenocorticotropin on plasmatic steroids of the catfish, Hetrropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 34, 149- 157.
AND
COMPARATIVE
Corticosteroids
41, 287-295 (1980)
ENDOCRINOLOGY
of the Coelacanth Latimeria chalumnae A Provisional Study on Their Identity’
Smith:
B. TRUSCOTT Marine
Sciences
Research
Laborafory, Ne~cfoundland
Memorial
University
AIC
5S7,
of Newfoundland.
St. John’s,
Canada
Accepted October 23, 1979 An extract of frozen tissue cut from an area of the coelacanth kidney, presumed to be the site of adrenocortical cells, was analyzed by double isotope derivative assay for adrenocorticosteroids. Evidence for the presence of 11-deoxycorticosterone, cortisol, and corticosterone was obtained. No 1 I-deoxycortisol or cortisone was detected. Incubation of the tissue with exogenous radioactive steroid precursors and identification of some transformation products indicated the presence of active enzyme systems including S-ene-3P-hydroxysteroid dehydrogenase, 5-ene-4-ene-isomerase, 20a-hydroxysteroid dehydrogenase, liphydroxysteroid dehydrogenase, and pregnene-5or- and SP-hydrogenases.
The nature and biosynthesis of corticowas hoped that by incubation of the steroids have been described for representacoelacanth tissue with radioactive precurtives of most vertebrate classes (for a re- sors we might be able to identify steroidocent review, see Sandor et al., 1976); a no- genie enzyme systems. This paper relates table exception is the crossopterygian the details and results of our experiments Laiimeria. The corticosteroids of fish have based on the hopes and presumptions outbeen of special interest to us (Idler and lined above. Truscott, 1972), so we requested Dr. J. E. MATERIALS AND METHODS McCosker to obtain “interrenal tissue” for The tissue was derived from a male coelacanth, 110 us during dissection of a specimen obtained cm in length (No. 79, CAS 33111), caught November by the California Academy of Sciences 1975 27, 1973, and obtained as a frozen specimen by the Coelacanth Expedition, supported by the 1975 California Academy of Sciences expedition to the Charline H. Breeden Foundation. At the Comores. At the dissection a discrete elongate nodular time it was not known if the adrenocortical area was removed from the ventral surface of the kidtissue of the coelacanth would consist of ney and reserved as possible interrenal tissue (perDr. M. Lagios). As the diffuse clumps of cells or aggregated cells sonal communication, coelacanth kidney is a fused structure rotated and disforming a discrete interrenal body. In any placed ventroposteriorly, the surface from which the event we hoped that there might be sufti- tissue was removed corresponded to the most dorsal cient endogenous corticosteroid present in aspect of a more typical fish mesonephros. Subsequent the “interrenal” and surrounding renal tis- to dissection in California the tissue was refrozen, packed in dry ice, and shipped to us. It was still frozen sue to permit identification. Freezing and upon arrival and was held in a freezer at -80°C for 3 thawing of the tissue would not be expect- months. ed to change the structure of the corticoWhen the tissue was thawed to initiate the present steroids. Also steroidogenic enzymes in in- investigation it did not closely resemble the macroterrenal tissues of some fish have been scopic appearance of elasmobranch interrenal. The major portion was reddish pink with associated condemonstrated to remain active through the nective tissue. Overlying one side there was a yellowfreeze/thaw process (Truscott and Idler, brown nonencapsulated region less well defined after 1968; Simpson and Wright, 1970). Thus it thawing than it had appeared in the frozen state. The 1 M.S.R.L.
Contribution
No. 373.
tissue (wet weight, 815 mg) was divided into three parts: the yellow-brown segment (230 mg) was cut 287 00 16-6480/80/070287-09$0 1.0010 Copyright All rights
@ 1980 by Academic Press. Inc. of reproduction in any form reserved.
B. TRUSCOTT away from the remainder and a layer of tissue (170 mg) directly in contact with it was kept separate from the remainder (395 mg). In this text the three parts are referred to as “nodular,” “subnodular,” and “renal” tissue. Each mass of tissue was cut into small pieces, and representative pieces were removed for histology. These were fixed in Bouin’s fluid for 48 hr dehydrated in a graded series of ethanol, cleared in xylene, embedded in Paraplast, and sectioned at 5 pm. Stains used included hematoxylin and eosin and Cason’s modification of the Mallory-Heidenhain connective tissue stain. Materials required for the double isotope derivative assay (DIDA) of corticosteroids, and for the identification of transformation products of radioactive precursors, e.g., W-labeled indicator steroids, reference steroids, solvents, and chromatographic media, were purchased from suppliers and treated as described in earlier publications (Truscott et al.. 1978; Truscott, 1979). Methods of isolation, purification, derivative formation, and detection of radioactive steroids and their 3H-Iabeled acetates or reference compounds, e.g., silica thin layer chromatography (tic), paper chromatography (PC), recrystallization to constant isotope ratio or constant specific activity, measurement of radioactivity, and color reactions, were essentially those described for earlier similar experiments (Truscott and Idler, 1972; Truscott et al., 1978; Truscott, 1979). Tissue
incubation
and preparation
of steroidal
ex-
tracts. The three portions of coelacanth tissue were minced and suspended (100 mg/ml) in a Tris-buffered medium containing NADPH-generating factors as described earlier (Idler and Truscott, 1967). The temperature was maintained at 10” and the atmosphere was enriched with oxygen. After 4 hr the medium was removed, centrifuged, and the supernatant solutions combined for analysis of extracted corticosteroids. The tissue had disintegrated considerably during the preincubation. The remaining tissue in each flask plus the corresponding debris recovered by centrifugation were transferred with medium to another flask each containing three radioactive precursors: 1 &i (19 nmol) [“Clprogesterone, 10 $Zi (0.9 nmol) [3H]pregnenolone, and 10 j&i (0.1 nmol) [3H]corticosterone. The incubation with radioactive precursors was continued under conditions as described above for 16 hr. The medium from each flask was analyzed separately for radioactive conversion products. Neutral steroidal fractions were isolated from both the pooled preincubation and the individual radioactive incubation media by exhaustive extraction with ice-cold dichloromethane. The dichloromethane extracts were washed with dilute alkali, acid, and water before evaporation to dryness under nitrogen. Steroid identification. The extract from the preincubation medium was analyzed by DIDA. Measured
amounts, 2000-4500 dpm, of 4-‘W-labeled cortisol, cortisone, corticosterone, 1 I-deoxycortisol, 1 ldeoxycorticosterone, and la-hydroxycorticosterone were added to the extract before an initial fractionation by tic to remove both extremely polar and nonpolar material. To insure against any possibility of contamination of the sample with authentic steroid during tic, progesterone only was used as a guide for the elution of the corticosteroid area of the plate (Truscott and Idler, 1972). The total corticosteroid fraction and aliquots of each 4-“C-labeled ‘indicator steroid were acetylated with freshly distilled tritiated acetic anhydride (400 mCi/mmol). After acetylation and the addition of the appropriate radioinert carrier, the corticosteroid acetates prepared from the coelacanth extract were separated and purified by sequential chromatography (Truscott and Idler, 1972; Truscott et ul., 1978) and then mixed with approximately 8 mg authentic carrier for crystallization to constant isotope ratio. The Y-indicator steroids were carried through the chromatography and crystallization in identical fashion since they had to serve as procedural blanks. The extract prepared by incubation of the coelacanth tissue with radioactive precursors was mixed with approximately 5 pg each of authentic radioinert progesterone, 17a-hydroxyprogesterone, pregnenolone, 17a-hydroxypregnenolone, ll-deoxycorticosterone, corticosterone, 1l-dehydrocorticosterone, 11-deoxycortisol, cortisol, and la-hydroxycorticosterone. Silica tic of the total mixture in cyclohexane:ethyl acetate:toluene (10: 10: 1) and, after drying, irrigation of the plate in the same direction with chloroform:methanol:water (90: 10: 1) gave seven contiguous but discernible bands under uv light. The tic solvent system, cyclohexane:propanol-2 (7:3), separated lldeoxycortisol from 1 I-dehydrocorticosterone and 17a-hydroxypregnenolone from 1 l-deoxycorticosterone, and a mixture of benzene:ethyl acetate (4:l) separated 17a-hydroxyprogesterone from pregnenolone. Each of the nine fractions off tic was further fractionated by pc. Solvent systems for pc were selected according to the polarity of the fraction under examination; their composition and sequence of use were basically similar to that described by Idler and Sangalang (1970). Radioactive metabolites on paper chromatograms were detected by radioscan. Their initial identification was dependent upon their coincidence, or lack of it, with authentic steroids. The presumed identity of a metabolite was then confirmed or denied by further chromatography, derivative formation, and in a few instances by recrystallization to constant specific activity.
RESULTS The cytological condition of the tissue was variable, the peripheral regions showing more autolysis. It was not possible to
CORTICOSTEROIDS
OF
THE
was recovered from the extract. Since the indicator, la-[14C]hydroxycorticosterone, behaved normally through the purification procedures, we conclude that the conditions of 3H-acetylation were inadequate for the esterification of the la-OH group in the presence of the rather bulky residue obtained from the tissue extract. The rate of acetylation of the secondary la-OH group is known to be considerably slower than that of the primary hydroxyl groups (Idler and Truscott, 1967). The experimental data are summarized in Table 1. For crystallization to constant specific activity the amount of authentic carrier which could be used was limited by the amount of radioactivity recovered through the chromatographic purification. The first supernatant solution was aspirated off, but no crystals were removed for isotope measurement until the second crystallization. Thus in Table 1 the isotope ratios are recorded as crystallization Nos. 2, 3, and 4. The [3H]acetic anhydride was determined
determine cytological or nuclear detail even in the best-preserved regions. The connective tissue region contained blood vessels including an extensive longitudinal segment of a thick-walled vessel. The yellow-brown region and the tissue lying between it and the blood vessels did not have the microscopic appearance of the homogenous elasmobranch interrenal nor did it resemble hematopoietic tissue of teleosts. It consisted of lobules containing several different cell types including pigment cells and granular cells. The tissue which this region most closely resembled is the perirenal tissue of the African lung&h Protopterus as described by Janssens et al. (1965). No clearly defined interrenal cells could be located in the histological material. Assay of the total preincubation medium of the tissue indicated the presence of lldeoxycorticosterone, cortisol, and possibly corticosterone. Neither cortisone nor 1 ldeoxycortisol could be detected. No la[14C]hydroxycorticosterone [3H]diacetate TABLE COMPARISON TISSUE
OF ISOTOPE CUT
FROM
RATIOS THE
OBTAINED
COELACANTH
BY DOUBLE
1 ISOTOPE
DERIVATIVE
AND
OF THE
W-INDICATOR
TO THE
EXTRACT
KIDNEY ADDED
289
COELACANTH
ASSAY
aH/“C
Steroid
[3H]acetate
OF
INITIALLY
ratio
Crystallization Recovery” m’)
OF AN EXTRACT
STEROIDS
No.
PC0
2
3
4
SN’
Mass” (ng)
1 l-[‘4C]Deoxycorticosterone Tissue extract + 2330 dpm
(10 ng)’
42 39
4.59 15.6
4.55 11.2
4.58 10.5
4.57 10.6
4.55 11.1
[“C]Cortisol Tissue extract
33 25
3.27 4.27
3.23 4.44
3.28 4.29
3.26 4.31
3.28 4.51
-
(13 ng)
3.49 5.06
3.54 4.59
3.54 4.67
3.49 4.65
3.49 4.99
-
[YlCorticosterone Tissue extract
+ 3970 dpm
13 4
(7 ng)
43 32
1 I-[Y]Deoxycortisol Tissue extract + 4340 dpm
3.89 4.33
3.88 3.85
3.90 3.80
3.88 3.79
3.90 3.85
-
(16 ng)
48 23
[‘“C]Cortisone Tissue extract
40 17
3.28 3.65
3.22 3.40
3.22 3.41
3.19 3.30
3.20 3.26
-
(15 ng)
+ 2090 dpm
+ 4750 dpm
U Percentage of added W-indicator recovered through the assay procedure. b PC = final isotope ratio through chromatographic purification. c SN = final supernatant from crystallization. d Corrected for mass of added W-indicator. p Amount of Y-indicator steroid added to tissue extract before acetylation.
2 0 0
290
B.
TRUSCOTT
to have a specific activity of 330 mCi/mmol and this value was used to calculate the mass of steroid which would be represented by the differences in isotope ratio of the sample and 14C-indicator. Extracts of the three separate media resulting from the incubation of the coelacanth tissue with a mixture of radioactive precursors, [14C]progesterone, [3H]pregnenolone, and [3H]corticosterone, were separated by tic into nine fractions. Radioactivity isopolar with carrier progesterone, pregnenolone, and corticosterone accounted for approximately 95% of the total radioactivity recovered from each extract. Further examination by pc of the tic fractions indicated that several radioactive metabolites were present in each fraction, though qualitatively there were no major differences apparent among the extracts. All fractions from each extract were analyzed for the products of steroidogenic enzyme systems appropriately as denoted by the added carrier steroid. Failure to detect any 17a-hydroxyprogesterone or 17~ hydroxypregnenolone, 1 l-deoxycorticosterone, 1 I-deoxycortisol, or cortisol indicated that no 17-, 21-, or lip-hydroxylases were active in any part of the tissue at the time of incubation. No la-hydroxycorticosterone was found in any of the extracts. Some effort was made to identify, at least tentatively, major radioactive metabolites which could indicate the presence of a catabolic enzyme system, e.g., reductases and dehydrogenases. The radioactive metabolites isolated and identified as described below were present in each of the three extracts. Quantitative differences were not considered since corrections could not be made for procedural losses. A tritium-labeled compound, difficultly separable from the carrier la-hydroxycorticosterone, was isopolar with lip, 20a, 21-trihydroxy-4-pregnen-3-one on pc with Bush-type solvent systems but of insufficient quantity to permit a more definitive identification. Further evidence for 20~
hydroxysteroid dehydrogenase activity was the tentative identification of 5[3H]pregnene-3p, 20a-diol (20a-dihydropregnenolone). Radioactivity isopolar with 17a-hydroxypregnenolone in the tic fraction was separated by pc into four components, the major one isopolar with 20~ dihydropregnenolone. Acetylation of an aliquot of this fraction did not separate the radioactivity from authentic 20a-dihydropregnenolone diacetate. The remainder of the 3H-metabolite was recrystallized with carrier 20a-dihydropregnenolone. The specific activity of three successive crops of crystals was constant within limits of +6%. Chromatographic purification, both before and after acetylation, of the progesterone area did not deplete this fraction of tritium. The isotope ratio was low (0.046) but remained essentially constant through reduction with 20P-hydroxysteroid dehydrogenase and isolation of 20/3-hydroxy-4pregnene-3-one (20@dihydroprogesterone) by tic (0.044) and pc (0.047). Radioinert 20/3-dihydroprogesterone was then added and the mixture recrystallized with acetone:pentane. The isotope ratio through four successive crystallizations for the progesterone of the “nodular tissue” incubate was 0.046 k 4.9% and 0.034 t 2.9% for the “subnodular” tissue. The progesterone fraction recovered from incubation of the “renal” tissue was not recrystallized with radioinert carrier but the isotope ratio was constant, 0.051 2 3.3%, through derivative formation and chromatography as outlined above. A 14C-metabolite was isopolar with the pregnanediones. Addition of authentic 5~ and S/3-pregnane-3, 20-diones and tic with the solvent system cyclohexane:ethyl acetate (1.1) (Lisboa, 1965) indicated that although both pregnanediones were present, there was approximately three times more of the Sa-pregnane. Reduction of the 5aand Sp-pregnanediones with 20@hydroxysteroid dehydrogenase, isolation of the 20&dihydro derivatives by pc, and sub-
CORTICOSTEROIDS
OF
THE
COELACANTH
291
sequent oxidation of these products, again underlying the “nodular” tissue and the with 20P-hydroxysteroid dehydrogenase, former accounted for over 70% of the total did not separate the radioactivity from the mass used in the incubations. carrier. There were insufficient counts to The results of our incubation studies can permit further identification of S/3- be considered in two parts: (i) the pre[ 14C]pregnanedione. The radioactivity sumptive identification of endogenous isopolar with Scr-pregnanedione was re- steroid in the preincubation medium and (ii) crystallized with authentic Sa-pregnanedithe transformation of radioactive precurone and through three successive crystallisors to products normally considered to be zations the specific activity was determined catabolites. to equal 270 dpm/mg (53%). The identification of 1 l-deoxycorticosterone, cortisol, and corticosterone in the A radioactive metabolite, isopolar with 1 l-dehydrocorticosterone, through chro- tissue extract is based on the demonstration of both isopolarity and isomorphism matographic purification, was acetylated acetates with the and the acetate purified by tic and pc. At of their 3H-labeled authentic steroid acetate. this point, the metabolite carried only the corresponding Table 1 records the data obtained by 3H-label. Authentic 1 1-[14C]dehydrocortimedium and costerone acetate was then added to give DIDA of the preincubation an isotope ratio of 3.69. Deacetylation with includes the calculated mass represented by acetylcholinesterase and reduction with the difference in final isotope ratios be20P-hydroxysteroid dehydrogenase with tween the sample and the indicator steroid. This calculation of mass assumes that purification by tic and pc of each derivative nonspecific tritium made no contribution to did not significantly change the isotope the final isotope ratio recorded for the samratio (av (4) = 3.46 ? 3%). ple. The assumption is supported by the reDISCUSSION sults obtained for 1I-deoxycortisol and Since at the time of the dissection in cortisone where there was no difference in California the anatomic nature of ad- the isotope ratios after recrystallization renocortical tissue of the coelacanth was between the 14C-indicator and the tissue not known, a discrete nodular body on the sample. Since the polarity of cortisol and surface of the kidney reminiscent of the in- corticosterone is not too different from that terrenal body of the elasmobranchs was of cortisone and 1 1-deoxycortisol, equally removed, along with some underlying tis- effective purification of the four acetates sue, as possible “interrenal” (Dr. M. D. could be expected. Thus, the difference in isotope ratios found for the cortisol and Lagios, personal communication). Recent corticosterone fractions of the tissue exwork by Lagios and Stasko-Concannon (1979) on kidney tissues from freshly fixed tract and the corresponding 14C-indicator is Latimeria indicates that the interrenal con- probably real and a measure of the corsists of numerous, small encapsulated cor- ticosteroid. puscles in close proximity to renal blood With due regard for their uncertain orivessels. The condition of the tissue used in gins, perhaps little significance can be atthe experiments described here was such tached to the quantitative differences in that no definitive histological studies could mass of 11-deoxycorticosterone, cortisol, be made but in the pieces of tissue actually and corticosterone. If active steriodogenic examined microscopically there were no enzymes were present during the preincuclearly defined interrenal cells. However, it bation of the tissue, then it is conceivable is possible that some small interrenal cor- that frozen storage, a double freeze/thaw puscles were present in the vascular tissue process, and the subsequent disintegration
292
B. TRUSCOTT
of tissue had more deleterious effects on ml. In the coelacanth tissue extract also some enzyme systems than on others. 1 1-deoxycorticosterone was quantitatively Simpson and Wright (1970), for example, the most important corticosteroid but correported that freezing and thawing de- tisol was present in a relatively significant stroyed the activity of a corticosteroid laamount, which brings us to the third hydroxylase in dogfish interrenal tissue but lungfish, the South American Lepidosiren. 1 lp- and 21-hydroxylase activities were still In this species, cortisol and aldosterone demonstrably present. However, it is were reported to be the principal plasmatic doubtful if any steroid hydroxylases were corticosteroids and though lesser amounts functional given the history of this particuof corticosterone were present, 1 I-delar tissue, and in fact by preincubation, we oxycorticosterone could not be detected probably extracted steroids present in the (Idler et al., 1972). tissue at the time of death of the animal. In Throughout the above discussion comthis case the quantity of any particular parisons have been drawn, almost by steroid at the time of analysis would depend necessity, between plasmatic steroids of not only on the initial concentrations but on the lungfishes and a tissue extract of the its chemical stability and perhaps to some coelacanth. There is little information extent upon its relative diffusibility. available on the synthesis in vitro of corHowever, if we accept the qualitative and ticosteroids by adrenocortical tissue of with reservation the quantitative validity of lungfishes: Janssens et al. (1965) identified the present work, the corticosteroids of the corticosterone only as a transformation coelacanth would appear to be a rather dis- product after incubation of “suspected adtinctive mixture comparable in certain fea- renocortical tissue” of the African lungfish tures to each of the three genera of Protopterus sp. with progesterone as exogenous precursor. Similarly our knowledge lungfishes. From a phylogenetic viewpoint, this is probably a valid comparison since of the plasmatic steroids of the coelacanth the coelacanth and lungfishes belong to is limited to an analysis of 1.4 ml of plasma the same subclass of vertebrates. Blair-West provided to us through the courtesy of Dr. et al. (1977) analyzed blood from the Grace Pickford and Dr. R. W. Griffith. We Australian lungfish Neocerutodusforsteri for analyzed this sample of plasma by DIDA and cortisol, corticosterone, aldosterone, and for cortisol, cortisone, aidosterone, 1I-deoxycorticosterone. In male Neocerutocorticosterone (Idler and Truscott, 1980) dus, the concentration of 1 l-deoxycorticobut unfortunately not for 1 l-deoxycortisterone (4.4 ng/lOO ml blood) was approxicosterone. We obtained no evidence for cortisone, or aldosterone, but mately four times higher than that of cortisol, the analytical data permitted the tentative aldosterone, and cortisol and corticosterone identification of corticosterone. were not present in detectable amounts. Two corticosteroids of interest are not A 50-ml blood sample from a male African listed in the table of results. Ia-Hydroxylungfish, Protopterus sp., was analyzed by DIDA for C-21 corticosteroids: ll-decorticosterone, the steroid unique to the oxycorticosterone was present (60 ng/lOO elasmobranchs (Truscott and Idler, 1968), was included in the initial planning of the ml) but cortisol, corticosterone, 1 l-deoxyexperiment since some characteristics of cortisol, aldosterone, and la-hydroxycorticosterone were not detected (Idler the coelacanth, e.g., pituitary organizaand Truscott, 1980). Aldosterone could not tion, rectal gland, urea retention, resemble those of the Chondrichthyes (Griffith et al., be detected in this particular blood sample from Protopterus by a radioimmunoassay 1974; Lagios, 1977). As stated earlier this sensitive to a concentration of 0.2 ng/lOO fraction was lost during acetylation.
CORTICOSTEROIDS
OF
[14C]Aldosterone was not added as an indicator steroid before 3H-acetylation, but radioinert aldosterone diacetate was added to the total acetylated extract before fractionation. Further purification of the aldosterone diacetate fraction removed all 3H counts, indicating no aldosterone was present in the original extract. However, since recovery through the total procedure could not be calculated, the negative result is not as meaningful as those for 1 1-deoxycortisol and cortisone, and is therefore not recorded in Table 1. There of course also remains the possibility that the principal steroid in the original extract was one of uncommon structure and not included in the assay. Incubation of the tissue (debris) with radioactive precursors indicated that the only enzyme system, still active, that could be classified as steroidogenic was a 5-ene3/3-hydroxysteroid dehydrogenase:5eneGene isomerase (5ene-3p OHSD:isomerase). The radioactivity in the progesterone fraction recovered from the incubation media contained approximately 5% tritium that was not separable from [ 14C]progesterone through chromatography, derivative formation, and recrystallization. Although no blank incubation was carried out, previous work has not demonstrated the spontaneous conversion of pregnenolone to progesterone under the conditions of incubation used here. Counting error as an explanation for the apparent residual tritium was discounted (Kobayashi and Maudsley, 1970). Thus, within the limits of tracer methodology where the identification of radioactivity is perforce indirect, it would appear that [3H]progesterone had been formed from [3H] pregnenolone as a result of 5-ene-3P-0HSD:isomerase activity. Tissues other than adrenocortical have been shown by tracer methodology to contain steroidogenic enzyme systems: 5-ene-3P-0HSD:isomerase and 21-hydroxylase in the teleost corpuscles of Stannius and body kidney (Idler and Freeman, 1966; Colombo et al., 1971) and
THE
COELACANTH
293
in bovine medullary tissue, 17~ and liphydroxylases were active as well (Carballeira et al., 1965). In the teleost tissues the yield of product from the radioactive exogenous precursor was generally low as it was in the present study. Thus, whether adrenocortical corpuscles (Lagios and StaskoConcannon, 1979) were actually present in the incubated coelacanth tissue is still open to question. Also we must appreciate that the work on the teleost and bovine tissues was done on freshly excised tissue whereas the coelacanth tissue had been in frozen storage for several years. All other enzyme systems that were presumed to be present by virtue of the identity of radioactive transformation products could be classified as catabolic. Since it was impossible to attempt to identify all of the transformation products, the number of enzyme systems demonstrated to be actively present was limited to the following: 2Ophydroxysteroid dehydrogenase, 1 lp-hydroxysteroid dehydrogenase, and pregnene-5a- and (probably) S/3-hydrogenases. These catabolic enzyme systems are usually concentrated in the liver but also are present in normal adrenocortical and kidney tissue (Kime, 1978). 1 lp-Hydroxysteroid dehydrogenase activity has been reported in the interrenal gland of elasmobranchs (Truscott and Idler, 1968) and head kidney of teleosts (see Idler and Truscott, 1972). The pregnanediones, products of ~CX- and SP-hydrogenases, were identified from incubation of body kidney of trout with progesterone (Arai et al., 1969; Colombo et al., 1971). In summary, the coelacanth tissue, in spite of its history of frozen storage, had maintained at least some of the steroid catabolic enzyme activity previously reported for other species of fish. Although, as the title implies, the present study of the corticosteroids of the coelacanth is not considered to be definitive, the results should provide useful information to any investigator interested in
294
B. TRUSCOTT
comparative endocrinology who, in the future, is fortunate enough to obtain body tissues or fluids from the coelacanth. ACKNOWLEDGMENTS The author is indebted to Dr. M. P. Burton for the histological examination of the tissue and expresses gratitude to her for advice and assistance in describing the nature of the tissue. Thanks are due to Dr. J. E. McCosker, Steinhart Aquarium, San Francisco, California, for making available and arranging shipment of the coelacanth tissue: to Dr. M. D. Lagios, Children’s Hospital of San Francisco, for providing a description of the tissue and its anatomic position as observed at the dissection of the California Academy of Sciences specimen, Latimeria No. 79, CAS 33111; to Dr. D. Duncan, B. C. Research, Vancouver, British Columbia, for intercepting the shipment of tissue from California and replenishing the dry ice in the package; to Dr. D. R. Idler for initiating the request for coelacanth tissue and for his constructive criticism of the work and of this report. The competent technical assistance of Mrs. V. Boland is gratefully acknowledged. This work was supported in part by a grant, A9752, from the Natural Sciences and Engineering Research Council of Canada. Nore added in proof: Analysis by DIDA of 4 ml gall bladder bile from this specimen of coelacanth indicated a compound, isopolar and isomorphous with cortisol, was released by hydrolysis with sulfatase from Helix pornaria. Neither cortisone, corticosterone, Il-deoxycorticosterone nor their tetrahydro derivatives, nor tetrahydrocortisol, could be detected in the bile.
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