Comp. Biochem. Physiol. Vol. 85B, No. 2, pp. 497-501, 1986 0305-0491/86 $3.00 + 0.00 Printed in Great Britain Pergamon Journals Ltd ANDROSTENEDIONE METABOLISM IN THE SEA STAR ASTERIAS RUBENS L. STUDIED IN HOMOGENATES AND INTACT TISSUE: BIOSYNTHESIS OF THE NOVEL STEROID FATTY-ACYL TESTOSTERONE P. A. VOOGT and J. W. A. VAN RHEENEN Research group for Invertebrate Reproductive Physiology, Laboratory of Chemical Animal Physiology, State University of Utrecht, 8 Padualaan, Utrecht, The Netherlands (Tel: 030-532-578) (Received 23 December 1985) Abstract--1. Androstenedione metabolism in ovary and pyloric caeca of Asterias rubens was studied in incubations both of homogenates and pieces of tissue under different experimental conditions. 2. The major metabolites were testosterone and 5~-androstane-3,17-dione in homogenate incubations, and testosterone and a novel steroid conjugate: fatty-acyl testosterone in tissue incubations. 3. The results of androstenedione incubations with homogenates were strongly dependent on the experimental conditions: addition of ATP to the incubation medium stimulated the production of testosterone, but did not affect testosterone esterification. 4. Results were also dependent on the pretreatment of the animals. 5. The possible physiological function of the fatty-acyl testosterone formation will be discussed. INTRODUCTION The investigation of steroid metabolism in sea star was initiated about 15 years after Botticelli et aL (1960) reported the presence of progesterone and oestradiol-17fl in the ovaries of Pisaster ochraceus. Gaffney and Goad (1974) pioneered this field in studying progesterone metabolism in Asterias rubens, where they were followed soon by others (Colombo and Belvedere, 1976; Teshima et aL, 1977; Schoen- makers and Voogt, 1980). Androstenedione metabo- lism was studied by Colombo and Belvedere (1976), who showed the conversion of androstenedione into testosterone in the ovaries of Astropecten irregularis pentacanthus, and by Schoenmakers and Voogt (1981), who reported this conversion for the ovaries and pyloric caeca of Asterias rubens. Additional metabolites of androstenedione in A. rubens were 5~-androstane-3,17-dione and an unidentified com- pound (A) (Schoenmakers and Voogt, 1981). The formation of 5~-androstane-3,17-dione indicated the similarity between the metabolism of andro- stenedione and progesterone in the sea star. Recently Voogt et al. (submitted) showed the results of studies on progesterone metabolism in homogenates to be strongly dependent on the experi- mental conditions. They concluded that the meta- bolic pathways observed in homogenates need not be indicative for the natural situation. Therefore androstenedione metabolism in A. rubens was re- studied using both homogenates and pieces of tissue from ovaries and pyloric caeca, and by systematically varying the experimental conditions in homogenates. MATERIALS AND METHODS Animals Female sea stars, Asterias rubens, were collected from the Wadden Sea (The Netherlands) and maintained in the laboratory in aerated sea-water at a temperature of 12°C at natural daylength. Chemicals 7(n)-[3H]Androstenedione (sp. act. 303.4 GBq/mmol) was purchased from New England Nuclear Corporation (Bos- ton) and purified before use by thin-layer chromatography. Steroids were from Steraloids Incorporation (Pawling, USA) and Sigma Chemical Corporation (St. Louis, USA). Cofactors were obtained from Boehringer (Mannheim, W. Germany), and all other chemicals and organic solvents (analytical reagent grade) from Baker Chemicals (Deventer, The Netherlands). Incubations Intact tissue: pieces of ovary or pyloric caeca tissue (0.5 g) were incubated in a medium consisting of 0.5 ml propylene glycol containing the dissolved radioactive androstenedione, and 4 ml of a phosphate buffer (pH 7.4; 0.1 M) containing 0.25 M sucrose. No cofactors were added. Homogenates: ovaries and pyloric caeca were homogenized with a Potter-Elvehjem homogenizer at 0°C in a phosphate buffer (pH 7.4; 0.1 M) containing 0.25 M sucrose (w/v, 1:3). The homogenate was centrifuged at 800g and 4°C for 10min and the supernatant was used further. The incubation medium consisted of 0.5 ml propylene glycol containing about 37 kBq 7(n)-[ 3H]androstenedione, 0.4 ml of a solution of NADPH in phosphate buffer (pH 7.4; 0.1 M; final concn of NADPH 2 mM), and 3.1 ml of the sucrose-phosphate buffer, to which 4 ml of the homogenate-supernatant was added. All incubations were performed continuously shaking at 18°C in an air atmosphere. Aliquots were taken from the incubation mixture at set times when homogenates were used; parallel incubations were carried out for set times when pieces of tissue were used. Incubations were terminated by addition of 5 ml dichloro- methane. Extraction and processing Homogenates were extracted directly; tissue pieces were first homogenized. Before extraction 50#g of each 497
Transcript
Comp. Biochem. Physiol. Vol. 85B, No. 2, pp. 497-501, 1986 0305-0491/86 $3.00 + 0.00 Printed in Great Britain Pergamon Journals Ltd
ANDROSTENEDIONE METABOLISM IN THE SEA STAR ASTERIAS RUBENS L. STUDIED IN HOMOGENATES AND
INTACT TISSUE: BIOSYNTHESIS OF THE NOVEL STEROID FATTY-ACYL TESTOSTERONE
P. A. VOOGT and J. W. A. VAN RHEENEN Research group for Invertebrate Reproductive Physiology, Laboratory of Chemical Animal Physiology,
State University of Utrecht, 8 Padualaan, Utrecht, The Netherlands (Tel: 030-532-578)
(Received 23 December 1985)
Abstract--1. Androstenedione metabolism in ovary and pyloric caeca of Asterias rubens was studied in incubations both of homogenates and pieces of tissue under different experimental conditions.
2. The major metabolites were testosterone and 5~-androstane-3,17-dione in homogenate incubations, and testosterone and a novel steroid conjugate: fatty-acyl testosterone in tissue incubations.
3. The results of androstenedione incubations with homogenates were strongly dependent on the experimental conditions: addition of ATP to the incubation medium stimulated the production of testosterone, but did not affect testosterone esterification.
4. Results were also dependent on the pretreatment of the animals. 5. The possible physiological function of the fatty-acyl testosterone formation will be discussed.
INTRODUCTION
The investigation of steroid metabolism in sea star was initiated about 15 years after Botticelli et aL (1960) reported the presence of progesterone and oestradiol-17fl in the ovaries of Pisaster ochraceus. Gaffney and Goad (1974) pioneered this field in studying progesterone metabolism in Asterias rubens, where they were followed soon by others (Colombo and Belvedere, 1976; Teshima et aL, 1977; Schoen- makers and Voogt, 1980). Androstenedione metabo- lism was studied by Colombo and Belvedere (1976), who showed the conversion of androstenedione into testosterone in the ovaries of Astropecten irregularis pentacanthus, and by Schoenmakers and Voogt (1981), who reported this conversion for the ovaries and pyloric caeca of Asterias rubens. Addit ional metabolites of androstenedione in A. rubens were 5~-androstane-3,17-dione and an unidentified com- pound (A) (Schoenmakers and Voogt, 1981). The formation of 5~-androstane-3,17-dione indicated the similarity between the metabolism of andro- stenedione and progesterone in the sea star.
Recently Voogt et al. (submitted) showed the results of studies on progesterone metabolism in homogenates to be strongly dependent on the experi- mental conditions. They concluded that the meta- bolic pathways observed in homogenates need not be indicative for the natural situation. Therefore androstenedione metabolism in A. rubens was re- studied using both homogenates and pieces of tissue from ovaries and pyloric caeca, and by systematically varying the experimental conditions in homogenates.
MATERIALS AND METHODS
Animals
Female sea stars, Asterias rubens, were collected from the Wadden Sea (The Netherlands) and maintained in the
laboratory in aerated sea-water at a temperature of 12°C at natural daylength.
Chemicals
7(n)-[3H]Androstenedione (sp. act. 303.4 GBq/mmol) was purchased from New England Nuclear Corporation (Bos- ton) and purified before use by thin-layer chromatography. Steroids were from Steraloids Incorporation (Pawling, USA) and Sigma Chemical Corporation (St. Louis, USA). Cofactors were obtained from Boehringer (Mannheim, W. Germany), and all other chemicals and organic solvents (analytical reagent grade) from Baker Chemicals (Deventer, The Netherlands).
Incubations
Intact tissue: pieces of ovary or pyloric caeca tissue (0.5 g) were incubated in a medium consisting of 0.5 ml propylene glycol containing the dissolved radioactive androstenedione, and 4 ml of a phosphate buffer (pH 7.4; 0.1 M) containing 0.25 M sucrose. No cofactors were added. Homogenates: ovaries and pyloric caeca were homogenized with a Potter-Elvehjem homogenizer at 0°C in a phosphate buffer (pH 7.4; 0.1 M) containing 0.25 M sucrose (w/v, 1:3). The homogenate was centrifuged at 800g and 4°C for 10min and the supernatant was used further. The incubation medium consisted of 0.5 ml propylene glycol containing about 37 kBq 7(n)-[ 3H]androstenedione, 0.4 ml of a solution of NADPH in phosphate buffer (pH 7.4; 0.1 M; final concn of NADPH 2 mM), and 3.1 ml of the sucrose-phosphate buffer, to which 4 ml of the homogenate-supernatant was added.
All incubations were performed continuously shaking at 18°C in an air atmosphere. Aliquots were taken from the incubation mixture at set times when homogenates were used; parallel incubations were carried out for set times when pieces of tissue were used.
Incubations were terminated by addition of 5 ml dichloro- methane.
Extraction and processing
Homogenates were extracted directly; tissue pieces were first homogenized. Before extraction 50#g of each
497
498 P.A. VOOGT and J. W. A. VAN RHEENEN
of the steroids androstenedione, testosterone, oestrone and oestradiol-17fl was added as a carrier. Extraction of steroids and removal of non-steroidal lipids was performed as described previously (Voogt et al., submitted).
Thin-layer chromatography
Samples were subjected to TLC on Silicagel precoated plates (Merck, DC-Fertigplatten 60F 254). The following solvent systems were used. (1) Toluene/cyclohexane (I:1, v/v); used in some cases to remove most of the lipid materials; plates were developed three times. (2) Diisopropylether/chloroform/hexane (7: 2: I, by volume), three times developed. (3) Dichloromethane/methanol (97:3, v/v), once developed. Steroids were visualized with u.v.-light at 254 nm, or after spraying with a primuline solution at 366 nm (Wright, 1971).
Acetylation
Steroids were acetylated by dissolving them in some dry pyridine and adding an equal volume of acetic anhydride. The mixture was allowed to stand overnight at room temperature.
Saponification Steroid esters were dissolved in 0.2 ml methanol. Then
0.5 ml of a solution of 0.1 N NaOH in 70% methanol was added. After standing overnight under a N 2 atmosphere, 1.5 ml of distilled water was added and the steroid was extracted with diethylether (3 × 2 ml). The ether extract was "washed" with 0.01 N acetic acid and distilled water and then evaporated.
Purification and identification of steroids This was performed by means of TLC using several
solvent systems and comparing the r.f.-values with those of reference and carrier steroids. Finally radiolabelled steroids were recrystallized, after the addition of 10mg of the presumed steroids to constant specific radioactivity using aqueous methanol as the solvent.
Measurement of radioactivity and quantification of radio- active steroids after TLC
Radioactivity was measured by liquid-scintillation counting using either a Searle apparatus (type Analytic 92) with plastic vials and the ready to use Xylofluor (Baker Chemicals) or a Packard machine (type 2420) with plastic minivials and the ready to use emulsifier Scintillator 299 (Packard).
Radioactive areas on TLC plates were detected and recorded with a Berthold LB-2723 thin-layer radiogram scanner. Peak areas were measured with a digitizer 9864 a (Hewlett Packard). Percental distribution of radioactivity was determined by expressing the area of each peak as a percentage of the total area of all peaks.
RESULTS
Homogenates and pieces of tissue from (the same) ovaries and pyloric caeca of sea stars, collected in May and being in stage 4 (Schoenmakers et al., 1984), were incubated with radiolabelled androstenedione for times ranging from 1-240 min. After extraction with dichloromethane, the remaining radioactivity in the aqueous incubation medium of the 120 and 240min incubations of ovary homogenates was determined. It amounted to, respectively, 0.58 and 0.62% of the radioactivity recovered. Scannograms of the ovary incubations showed three peaks (Fig. 1), corresponding with the marker spots of testosterone, androstenedione and 5ct-androstane-3,17-dione.
The material from the slowest moving peak was
S F • • • u 3 2 1
Fig. 1. Thin-layer radioscan of the steroids extracted from ovary homogenates of A. rubens after incubation with [3H]androstenedione for 30min (Exp. 1). Marker spots were: I, testosterone; 2, androstenedione; 3, 5ct-
androstane-3,17-dione.
rechromatographed in system 3 and again co- chromatographed with testosterone. After acety- lation and chromatography in the same system two peaks were visible. The largest peak corresponded with testosterone acetate, which was confirmed by recrystallization to constant specific radioactivity. The other component, being less polar in nature than testosterone acetate and which has been indicated by Schoenmakers and Voogt (1981) as compound A, was chromatographed in several solvent systems but could not be identified.
The materials corresponding with androstenedione and 5~-androstane-3,17-dione were subjected to TLC in system 3 and again co-chromatographed with the authentic steroids. They could not be acetylated and were recrystallized to constant specific radioactivity. The relative amounts of the radioactive steroids present at each incubation time were determined and are given in Table 1.
These data show that androstenedione metabolism is much higher in pieces o f tissue than in homo- genates, testosterone being the major metabolite in both systems. Compound " A " was only a minor component, not showing distinct changes in its rela- tive amount with incubation time. In the other incu- bations it was no longer separated from testosterone. 5ct-Androstane-3,17-dione was only found in the homogenate incubations.
Scannograms of the pyloric caeca incubations showed up to five peaks (Table 1). Three of them were the same as found in the ovary incubations and corresponded with testosterone, androstenedione and 5~-androstane-3,17-dione. The identity of these products was proved by TLC and by recrystallization to constant specific radioactivity. 5ct-Androstane- 3,17-dione was found in small amounts, only in the homogenate incubations.
The major metabolite in the caeca tissue incu- bations had a mobility slightly higher than that of
Androstenedione metabolism in sea star 499
Table 1. Relative amounts of steroids present after the incubation of pieces of tissue or homogenates of ovaries and pyloric caeca of Asterias rubens with [3H]androstenedione for various times. Amounts were calculated from TLC-radioscans and are expressed as the percentage
of the area of each peak from the total area of all peaks present Incubation
time Incubated 5,,-Androstane Compound* Fatty-acyl (mins) material Androstenedione -3,17-dione "A" Testosterone testosterone "X"* "Y"*
tes tosterone acetate. By alkaline hydrolysis a steroid was ob ta ined ~h !ch showed the mobil i ty of testo- s terone in severa-~VTLC systems and which, after acetylat ion, co -ch roma tog raphed with tes tos terone acetate. Wi th an excess of carr ier tes tosterone acetate the mater ia l was recrystallized to cons tan t specific radioactivity. F r o m these da ta it can be concluded tha t the original c o m p o u n d was fatty-acyl testos- terone.
Final ly there was a minor c o m p o n e n t with a mobil i ty in between those of 5~t-androstane- 3,17-dione and fatty-acyl testosterone, which was not identified and will be indicated with " X " .
The relative a m o u n t of the radioact ive steroids present at each incuba t ion t ime were de termined and are given in Table 1.
Fo r compar i son we also incuba ted in this experi- men t pieces of ovary tissue f rom sea stars which had been collected in May of the former year and had been main ta ined since then (for a year) in the labora- tory at a cons tan t water t empera ture of 12°C. Fo r convenience this incuba t ion will be indicated as ex- per iment lb.
Scannograms showed the same peaks as observed in the incubat ions of pyloric caeca described above (Table 1). Fat ty-acyl tes tosterone was the ma jo r metaboli te , whereas traces of 5~-androstane-3,17- dione were found only at one incuba t ion time. The unidentif ied c o m p o n e n t " X " was found in a ra ther high a m o u n t in the same incuba t ion (Fig. 2). A n o t h e r unidentif ied compound , indicated " Y " , with a mobil - ity even higher t han tha t of fatty-acyl tes tosterone was found after the longest incuba t ion time. The relative amoun t s of metabol i tes formed are given in Table 1.
The former exper iment showed considerable differ- ences between the incubat ions of pyloric caeca homo- genates and pyloric caeca intact tissue with reference to the fo rmat ion of fatty-acyl testosterone. Obviously condi t ions in the homogena te are no t favourable for the esterification of testosterone. Therefore we incu- ba ted pyloric caeca homogena tes (corresponding with 0.5 g of tissue) of sea stars collected in October , for 30 min with various combina t ions of the cofactors N A D P H , A T P and HSCoA, which were all used at final concent ra t ions of 2 mM. The results are shown in Table 2.
S F • • • 0 3 2 t
Fig. 2. Thin-layer radioscans of the steroids extracted from pieces of ovary tissue of A. rubens after incubation with [3H]androstenedione for 4 hr (exp. lb). Marker spots were: 1, testosterone; 2, androstenedione; 3, 5ct-androstane-3,17-
dione.
500 P.A. VOOGT and J. W. A. VAN RH~NEN
Table 2. Relative amounts of radioactive steroids present after the incubation of pyloric caeca homogenate of Asterias rubens in the presence of various cofactors with [~H]androstenedione for 30 rain. Amounts were calculated from scannograms as
the percentage of the area of each peak from the total area of the peaks present Cofactor(s) 5~-Androstane- Fatty-acyl
Tables 1 and 2 show that the main metabolic pathway in androstenedione metabolism is the con- version into testosterone, which was identified by TLC and recrystallization. The activity of the enzyme involved, 17//-HSD, exceeds by far that of 5a-reductase, which converts androstenedione into 5~-androstane-3,17-dione. The latter enzyme is the most active in progesterone metabolism, funnelling progesterone nearly quantitatively into the direction of 5c¢-pregnane-3,20-dione and 3/~-hydroxy-5c~- pregnan-20-one (Gaffney and Goad, 1974; Teshima e t al. , 1977; Schoenmakers and Voogt, 1981; Voogt e t al. , 1986). In androstenedione metabolism, 5~- reductase seems to catalyze only a siding, which apparently is more often followed in homogenates than in tissue pieces. In experiment 2 (Table 2) 5~-androstane-3,17-dione was formed in only one case, whereas in the parallel incubation of pyloric caeca tissue it had not been formed at all. This again demonstrates that metabolism in homogenates may distinctly differ from that in intact tissue (Voogt et al. , 1986). Schoenmakers and Voogt (1981) found 5~-reductase activity in ovary homogenates to be considerably higher than in pyloric caeca homo- genates. This phenomenon was not observed in the present study (Table 1).
Compound A (Schoenmakers and Voogt, 1981), distinguished in the ovary incubations, was a minor component both in homogenates and tissue pieces. Its chromatographic behaviour in several TLC systems was rather similar to that of ethiocholanolone and androsterone. However, recrystallization definitely proved that it was not identical with one of these two steroids.
Androstenedione metabolism was higher in the homogenate incubations of pyloric caeca than of ovary (Table 1), which is in agreement with earlier observations (Schoenmakers and Voogt, 1981).
Table 1 also shows that androstenedione metabo- lism in tissue pieces is much higher than in homo- genates. This holds good also for experiment 2. In the incubation of pyloric caeca tissue, which was per- formed parallel with those of the homogenates listed in Table 2, only 11.3% of the radioactivity was associated with androstenedione after 30 rain. The much higher activity in intact tissue strongly con- trasts with the earlier findings with respect to pro- gesterone metabolism, which was higher in homo- genates than in tissue pieces (Voogt e t al. , submitted). The latter phenomenon may result from limitations of transport by diffusion of progesterone into the tissue, whereas the opposite situation observed for androstenedione metabolism must be due to condi- tions in the homogenates suboptimal for 17/~-HSD.
Differences between the results of homogenate and intact tissue incubations of the ovaries were only quantitative, for pyloric caeca they were also qual- itative.
The main product of androstenedione metabolism in pyloric caeca tissue turned out to be fatty-acyl testosterone. The identity of the steroid moiety was determined by TLC and recrystallization. The iden- tity of the other part of the conjugate was deduced from the following observations: The conjugate showed a low polarity (lower than 5~-androstane- 3,17-dione) and was easily hydrolysed under alkaline conditions. Further its chromatographic behaviour resembled that of testosterone acetate.
The nearly complete absence of fatty-acyl testo- sterone formation in the pyloric caeca homogenate indicates that conditions in that medium were not only suboptimal for testosterone production but also for its esterification.
Results of experiment 1 suggest that esterification is specific for pyloric caeca tissue. However, in the parallel incubation of ovaries from sea stars main- tained in the laboratory for a year (exp. lb), fatty- acyl testosterone was the main product of an- drostenedione metabolism which in its turn is higher than that of the ovary tissue in experiment 1. In general, androstenedione metabolism in ovary tissue from experiment 1 b strongly resembles that in pyloric caeca tissue of experiment 1. This holds good also with reference to the presence of metabolite "X", which was not identified.
The different results of the incubations with ovary tissue are not easily explained. However, one can easily imagine that animals held in captivity for a year under much more stable environmental conditions than encountered in na ture , are no longer synchronic with their free living fellows and are in a (slightly) different stage of the reproductive cycle. How this may be, this experiment shows that one can reach quite different results depending on the pretreatment of the animals.
Since conditions in pyloric caeca homogenates might be suboptimal for the formation and esterification of testosterone as a result of loss of structure and compartmentation or of inadequate (too low) concentrations of cofactors, we performed a series of incubations of homogenate~ which differed in cofactors added (Table 2). The parallel incubation of tissue pieces showed the fqllowing percental distri- bution of radioactivity: androstenedione (11.3%), testosterone (20.9%) and fatty-acyl testosterone (67.8%). Comparison of this distribution with those of Table 2 shows that androstenedione metabolism in none of the homogenates reached the level of that in intact tissue. Table 2 shows that the addition of ATP
Androstenedione metabolism in sea star 501
raises androstenedione metabolism, leading particu- larly to an increased production of testosterone. The addition of HSCoA seems to have hardly any effect. The effect of ATP is hard to explain, since no mechanism is known in which the action of 17fl-HSD is ATP dependent.
Whereas it seems possible to stimulate testosterone production by adding ATP, none of the additions showed a distinct effect on testosterone esterification. Since the latter process is thought to imply the reaction between testosterone and fatty-acyl SCoA, ATP and HSCoA were added to stimulate fatty-acyl SCoA formation, which may lead to an increased esterification, However, the expected result was not obtained.
As far as we know the formation of fatty-acid esters of testosterone has not been reported before. Liebermann and coworkers reported the presence of fatty-acid esters of pregnenolone, 17ct-hydroxy- pregnenolone, dehydroepiandrosterone, and 3fl- hydroxy-5~t-pregnan-20-one in bovine adrenal glands and corpora lutea (Hochberg et al., 1977; Millon- Nussbaum et al., 1979; Albert et al., 1980). Esters of 3fl-hydroxy-5~-pregnan-20-one have also been found in Aster ias rubens (Teshima et al., 1977). All these esters have in common that esterification has taken place at the 3fl-position of a A s or saturated steroid. These steroids are, perhaps with the exception of 3fl-hydroxy-5~-pregnan-20-one (Albert et al., 1980), biologically inactive. The esterification reported here has taken place at the 17fl-position of a, in vertebrate animals, potent steroid. Fatty-acid esters of 5fl-androstane-3~,17fl-diol were reported for the sea water turtle (Breuer et al., 1981). It is not completely clear from that paper whether esterification had taken place at one or at both of the hydroxyl groups. It was supposed that the physiological function of the esterification of the 3fl-hydroxysteroids of the so- called AS-pathway was to protect them against the activity of 3fl-hydroxysteroid dehydrogenase (Hoch- berg et al., 1977; Millon-Nussbaum et al., 1979). In other words esterification might prevent the for- mation of biologically active steroids and thus might have a regulatory function. When 3fl-hydroxy-5ct- pregnan-20-one is biologically active (as was sug- gested by Albert et al., 1980), esterification might inactivate this steroid. Maybe this is also the function of the esterification of testosterone in female sea star, but more information is needed as to the distinct relationship of esterification with the reproductive cycle.
In conclusion: this study confirmed the results of Schoenmakers and Voogt (1981) obtained with incu- bations of homogenates of ovary and pyloric caeca of Aster ias rubens with androstenedione. It also demon- strated that results obtained with homogenates may be strongly different from those obtained with intact tissue, as had been found earlier with respect to
progesterone metabolism (Voogt et al., 1986), and that results depend on the experimental conditions (compositions of incubation medium or pretreatment of the animals). Further it showed the production of a novel fatty-acyl steroid.
Acknowledgement--The authors thank Miss M. H. van Hattum for typewriting the manuscript and Mr D. Smit and his colleagues for reproducing the illustrations.
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