GENERAL AND COMPARATIVE ENDOCRINOLOGY 41,408-416 (1980)

In Vitro Biosynthesis of from Progesterone by the Ovaries and Pyloric Ceca of the Starfish Aster& rubens

H.J.N. SCHOENMAKERS AND P.A. VOOGT

Laboratory of Chemical Animal Physiology and Section of Comparative Endocrinology of the Zoological Laboratory, State University of Utrecht, 8 Padualaan, 3508 TB Utrecht, The Netherlands Accepted January 16, 1980

In vitro biosynthesis of steroids from progesterone in ovaries and pyloric ceca of Asterias rabens has been investigated. The biosynthesis of 17a-hydroxyprogesterone, androstene- dione, , 20a-dihydroprogesterone, 1 I-desoxycorticosterone, and Sa-pregnane- 3,20-dione could be demonstrated to take place in the tissues of both organs by using [1,2-3H]progesterone as a precursor. The yields of intermediates of the A4-pathway and of 1 I-desoxycorticosterone are small, being higher in the ovaries than in the pyloric ceca. The yields of 20cz-dihydroprogesterone are low, those of 5a-pregnane-3,20-dione are high. In both cases the yields in the pyloric ceca exceed those in the ovaries. The results indicate the presence of the following biosynthetic enzyme systems in ovaries and pyloric ceca of As- terias rubens: 17a-hydroxylase, C,,-C,,,-lyase, 17P-HSD, 20p-HSD, 21-hydroxylase, and SLu-reductase. The importance of these enzymes for the metabolism of progesterone, i.e., the biosynthesis of C,,-steroids, 20a-dihydroprogesterone, and corticosteroids, will be dis- cussed. Steroidogenesis in invertebrates is less dition, ultrastructural evidence has been well known than in vertebrates. Only a few obtained for the presence of -syn- reports are available on the presence of thesizing cells in the ovaries of Aster& steroids and steroid biosynthesis in ar- rubens (Schoenmakers et al., 1977). Cho- thropods (Teshima and Kanazawa, 1971a, lesterol’ can be converted into pregnenolone b; Tcholakian and Eik-Nes, 1971) and in and progesterone in the ovaries and pyloric mollusks (Gottfried and Dorfman, 1970; De ceca of Asterias rubens (Schoenmakers, Longcamp et al., 1974; Lupo di Prisco et 1977, 1979). Therefore, the purpose of this al., 1973; Lupo di Prisco and Dessi’Ful- study is to examine the conversion of pro- gheri, 1975; Carreau and Drosdowsky, gesterone into other C,,-steroids and the 1977). For Echinodermata some data biosynthesis of &,-steroids from progester- suggest a steroid-synthesizing capacity. one. Since the pyloric ceca are assumed to Colombo and Belvedere (1976) reported be involved in the growth of the ovaries, steroid biosynthesis in the gonads of the the steroid biosynthesis of the ovaries will echinoid Paracentrotus lividus, the ophiu- be compared to that of the pyloric ceca. roid Ophiothrix fragilis, the holothuroid Progesterone was reported to be present Cucumaria planci, and the asteroid Astro- in the ovaries of the starfish Pisaster och- pecten irregularis pentacanthus. raceus (Botticelli et al., 1960) and Aster&

Some indications for the steroid-synthe- I Steroid nomenclature: cholesterol = 3P-hydroxy- sizing capacity in the gonads of Asterias cholest-5-ene; pregnenolone = 3/3-hydroxypregn-5- rubens have been obtained by demonstrat- ene-20-one; progesterone = pregn-4-ene-3,20-dione; ing the presence of the 3P-hydroxysteroid 17a-hydroxyprogesterone = 17a-hydroxypregn-4- dehydrogenase (3P-HSD) and the 17@hy- ene-3,20-dione: 20a-dihydroprogesterone = 20a- hydroxypregn-4-ene-3-one: 1 I-desoxycorticosterone droxysteroid dehydrogenase (17&HSD), = 21-hydroxypregn-4-ene-3,20-dione; androstene- two enzymes essential for steroid biosyn- dione = androst-4-ene-3,17-dione; testosterone = 17/3- thesis (Schoenmakers et al., 1976). In ad- hydroxyandrost-4-ene-3-one.

0016-6480/80/070408-09$01.00/O 408 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. PROGESTERONE METABOLISM IN Asterias rubens 409

amurensis (Ikegami et al., 1971) and in the tion mixture and the enzyme reactions were brought to sea urchin Strongylocentrotus franciscanus stop by addition of 10 ml dichloromethane; three frac- (Botticelli et al., 1961). In studying the in tions were thus obtained for each tissue. Exfracfion and processing. To detect the steroids on vivo metabolism of progesterone in Asferius tic and to measure recovery, we added before extrac- rubens and Marthasterias glacialis Gaffney tion to each fraction 50 pg of each of the following and Goad (1974) found the conversion of steroids: progesterone, 17o-hydroxyprogesterone, progesterone into some intermediates, , testosterone, 20a-dihydroprogester- which are supposed to be possibly involved one, ll-desoxycorticosterone, and 5cu-pregnane-3, 20-dione. The extraction, tic, detection of steroids, in the biosynthesis of asterosaponins, derivative formation, recrystallizations, recovery mea- unique steroid-like compounds present in surements, measurement of radioactivity, and calcu- Asteroidea (Rio et al., 1965; Yasumoto et lation of steroid conversion were carried out as de- al., 1966; Grossert, 1972). scribed elsewhere (Schoenmakers, 1979). For tic the The present paper reports the results of following solvent systems were applied: System I-toluene-cyclohexane (l:l, v/v) to re- experiments in which ovaries and pyloric move most of the lipid materials. In this system the ceca of female specimens of Aster& ru- plates were developed three times; bens were incubated in vitro with tritiated System 2-benzene-ethylacetate (3: 1, v/v), three progesterone. times developed; System 3--dichloromethane-methanol (97:3, v/v), once developed. MATERIALS AND METHODS For recovery measurements with gas-liquid chroma- Animals. Specimens of Asterius rubens were col- tography (GLC), o-methyloximes were prepared of the lected in the Wadden Sea, east of the island of Texel steroids 17a-hydroxyprogesterone and 1 l-desoxy- (The Netherlands) on March 14, 1975. The animals corticosterone (Mambara and Iwata, 1973). were kept in aerated seawater at 6” for 3 days before being processed. Chemicals. All chemicals were of analytical grade; RESULTS organic solvents were redistilled once, just before use. The fresh weight of the ovaries is 52.00 g, NAD, NADH, NADP, NADPH, and ATP were ob- tained from Boehringer (Mannheim, West Germany). that of the pyloric ceca 30.50 g. This corre- [ 1 .2-3H]Progesterone (sp act 47.8 Ciimmol), purchased sponds to 20.39 and 11.96% of the total from New England Nuclear (Boston), was purified by fresh weight of the animal (255.2 g), re- thin-layer chromatography (tic), according to the spectively. The stage of the annual repro- method advised by manufacturer. ductive cycle of the ovaries was determined Dissrction of the animals. Dissection and sexing of starfishes, and checking parasitism, were carried out to be stage 4, the maturation stage. as described elsewhere (Schoenmakers, 1979). The After extraction of the different incuba- ovaries and pyloric ceca of one not-parasitized female tion mixtures triglycerides were removed animal were used for this study. The animal was his- from the extracts of pyloric ceca tissues by tologically examined to determine the stage of the an- precipitation with 8 ml aqueous methanol nual reproductive cycle (Schoenmakers and Goedhart, submitted for publication). (70%). The organic phases were evaporated lnctrbation. For incubation 2 g ovarian and 2 g in vacua and the residues subjected to tic pyloric ceca tissue were homogenized separately with following systems 1 and 2. Radioscans a Potter-Elvehjem homogenizer at 0” in 0.25 M su- proved that [ 1 ,2-3H]progesterone was con- crose (w/v, 1:3). verted into at least eight compounds by The incubation mixture consisted of 0.5 ml propyleneglycol containing about 5 &i [ 1,2- both ovarian and pyloric ceca tissues. Fig- 3H]progesterone, 3 ml of a solution of NAD, NADH, ure 1 shows radioscans of the steroids iso- NADP, NADPH, and ATP in phosphate buffer, and 12 lated from ovarian and pyloric ceca tissue, ml phosphate buffer (0.1 M, pH 7.4; final concentra- respectively, after an incubation of 120 min. tion of each cofactor 1 mM), to which 4 ml homogenate The radioactive areas corresponding to (corresponding to I.0 g tissue) was added. The incubations were carried out under continuous the added carriers were eluted. However, shaking at 25” in an air atmosphere. After 30, 60, and using tic system 2, 17a-hydroxyprogester- 120 min, 6-ml aliquots were pipetted from the incuba- one, testosterone, 20c+dihydroprogester- 410 SCHOENMAKERS AND VOOGT

FIG. 1. Thin-layer radioscans of steroids extracted from ovarian (A) and from pyloric ceca tissue (B) ofAsterias rubens after 120 min incubation [1,2-3H]progesterone. References are: (1) testosterone; (2) 17whydroxyprogesterone; (3) 1 l-desoxycorticosterone; (4) 20wdihydroprogesterone; (5) andro- stenedione; (6) progesterone; (7) 5a-pregnane-3,20-dione.

one, and 1 1-desoxycorticosterone were in- authentic steroids were eluted and further adequately separated. Therefore, these purified by tic following system 3, formyla- compounds were eluted as one fraction and tion, and recrystallization. resubjected to tic, using system 3. This re- sulted into an effective separation of these 17wHydroxyprogesterone steroids. The 17a-hydroxyprogesterone areas The radioactive areas corresponding with were eluted and formylated. The radioac- PROGESTERONE METABOLISM IN Asferias rubens 411 tive compound of each fraction remained a constant specific radioactivity after re- unaffected and showed the same mobility in peated recrystallization. tic in system 3 as authentic 17a-hydroxy- progesterone. Furthermore, a constant Testosterone specific radioactivity was measured in most The testosterone areas were eluted and fractions after repeated recrystallization. formylated. Each fraction contained a radioactive compound with the same Rf value as authentic testosterone-formate. Androstenedione Repeated recrystallization of the individual The androstenedione areas were eluted. fractions to constant specific radioactivity Rechromatography in tic system 3 showed further confirmed the identity of the labeled that these areas are composed of a number substance. of radioactive compounds, one of which- The results of recrystallization and the accounting for only a relatively small part conversion percentages after correction for of the total metaboiites-corresponds with recovery are summarized in Table 1. authentic androstenedione. These andro- stenedione areas were eluted and the radio- 20wDihydroprogesterone active substance of each fraction resisted The 20a-dihydroprogesterone areas were formylation. The individual fractions showed eluted. Formylation of each fraction pro-

TABLE 1 SPECIFIC ACTIVITIES (dpm/mg) DURING THE LAST THREE RECRYSTALLIZATIONS OF ~~~~-~YDR~XYPR~GE~TER~NE,ANDROSTENEDIONE,ANDTESTOSTERONE-FORMATEANDTHECONVERSION PERCENTAGES(AFTER CORRECTION FORRECOVERY) OFTHESE STEROIDSISOLATED FROM OVARIAN AND PYLORIC CECA TISSUES OF Astrrias rubens AFTER INCUBATION WITH [1,'i?-3H]PROGEST~RONE

17wHydroxyprogesterone Androstenedione Testosterone-formate Incubation time Percentage Percentage Percentage Tissue (min) dpm/mg conversion dpmimg conversion dpmimg conversion 23 799 2541 Ovaries 30 24 789 2517 23 0.042 f_ 0.003 801 0.77 t- 0.03 2532 1.34 s_ 0.04 19 586 22 Ovaries 60 19 584 21 18 0.022 2 0.003 587 0.78 2 0.03 22 0.026 r 0.02 - 4495 687 Ovaries 120 - 4452 713 - 10.01 4382 2.6 2 0.1 707 0.49 2 0.02 13 616 30 Pyloric ceca 30 14 605 29 13 0.022 2 0.003 613 0.32 k 0.01 30 0.029 ‘-c0.002 26 174 21 Pyloric ceca 60 26 178 22 27 0.032 f 0.005 175 0.13 + 0.01 20 0.019 2 0.002 196 11 Pyloric ceca 120 - 187 10 CO.01 194 0.09 + 0.01 9 0.004 2 0.002 412 SCHOENMAKERS AND VOOGT

0 g Q Q g g z d d d d 6 6 tl fl tl tl $1 il PROGESTERONE METABOLISM IN Asrerias rrrbens 413 duced a compound with the same mobility dione, testosterone, 20a-dihydroprogester- in tic in system 3 as authentic 2Oc-u-dihydro- one, 5a-pregnane-3,20-dione, and 1 l- progesterone-formate. A constant specific desoxycorticosterone has been proved on radioactivity was obtained after repeated the basis of the following evidence: recrystallization of the individual fractions. -The tritiated free steroids have a chro- matographic mobility, similar to that of the 1 I-Desoxycorticosterone authentic materials. The 1 I-desoxycorticosterone areas were -The radioactive compounds, in case of eluted and formylated. The formylated testosterone, 20a-dihydroprogesterone, product of each fraction showed the same and 1 l-desoxycorticosterone, can be for- mobility in tic in system 3 as authentic ll- mylated and their radioactive derivatives desoxycorticosterone-formate. Finally, a have the same mobility in tic as the authen- constant specific radioactivity was reached tic steroid formates. The radioactive mate- after repeated recrystallization of the indi- rial, in case of 17a-hydroxyprogesterone, vidual fractions. androstenedione, and 5a-pregnane-3,20- dione, resisted formylation and therefore 5a-Pregnane-3,20-dione identification of these steroids can only be The 5a-pregnane-3,20-dione areas were considered as tentatively. eluted and subjected to formylation. The -After addition of carriers, recrystalli- radioactive compound of each fraction re- zation of the steroids and steroid formates mained unaffected and showed the same RI can be carried out up to constant specific value as authentic 5a-pregnane-3,20-dione. radioactivities (Tables 1 and 2). The individual fractions showed a constant The in vitro incubation experiments thus specific radioactivity after repeated re- demonstrate that the ovaries and the crystallization. pyloric ceca of female Asterias rubens are The results of recrystallization and the able to synthesize from progesterone the conversion percentages after correction for steroids just mentioned. This means that in recovery are summarized in Table 2. both organs the following biosynthetic After 30, 60, and 120 min incubation of enzymes are present: 17a-hydroxylase, ovarian tissue a large amount of progester- C,,-C,,-lyase, 17P-HSD, 20a-hydroxy- one had not been converted: 72, 69, and steroid dehydrogenase (20~HSD), LX- 54%, respectively. The total amount of reductase, and 21-hydroxylase. These en- compounds which were not further iden- zymes will be briefly discussed. tified increased to 19, 25, and 26%, respec- tively. 17wHydroxylase On the contrary, after 30,60, and 120 min From Table 1 it appears that the percent- incubation with pyloric ceca tissue a minor age conversion from progesterone into amount of unchanged progesterone re- 17a-hydroxyprogesterone is low (ovaries: mained: 23, 13, and lo%, respectively; the 0.04 to 0.02%; pyloric ceca: 0.03 to percentages conversion into nonidentified ~0.01%). The 17a-hydroxyprogesterone steroids were: 66, 82, and 83%, respec- yield in time decreases in the ovaries; the tively. decrease is irregular and less evident in the pyloric ceca. DISCUSSION After 30, 60, and 120 min incubation of C,,-C,,-Lyase and 17@HSD ovarian tissue and pyloric ceca tissue, the Table 1 shows that the conversion values conversion in vitro of progesterone into of androstenedione are higher in the ova- 17a-hydroxyprogesterone, androstene- ries (0.8-2.6%) than in the pyloric ceca 414 SCHOENMAKERS AND VOOGT

(0.3-o. 1%). Since the androstenedione The 20a-dihydroprogesterone yield in time yield in time increases in the ovaries and is irregular, without large fluctuations, in decreases in the pyloric ceca, the metab- the ovaries; the yield slightly decreases in olism of androstenedione in the ovaries the pyloric ceca. The enzyme was demon- may be different from that in the pyloric strated before by Colombo and Belvedere caeca. (1976) in the gonads of Astropecten ir- The yields of &-steroids are higher than regularis pentacanthus, Paracentrotus those of 17a-hydroxyprogesterone (Table lividus and Ophiothrix fragilis. 1). Thus, the C,,-steroids show a greater tendency to be accumulated. In the ovaries 21-Hydroxylase the yield of 17a-hydroxyprogesterone de- Table 2 shows that the yields of ll- creases in time, whereas that of andro- desoxycorticosterone are small (ovaries: stenedione increases. Thus, likely the bio- 0.31-0.01%; pyloric ceca: 0.18-0.01%). synthesis of 17a-hydroxyprogesterone pre- The enzyme seems to be more active in the ceded that of androstenedione, which is ovaries than in the pyloric ceca. The in accordance with the biosynthetic se- ovaries show decreasing 1 l-desoxycortico- quence in vertebrates. The testosterone sterone production in time; in the pyloric yield in the ovaries is irregular and allows ceca this decrease is less evident. There- no conclusions as to the biosynthetic se- fore, 1 1-desoxycorticosterone is probably quence. In the pyloric ceca the yield of not an end product. This enzyme was found 17a-hydroxyprogesterone is difficult to ex- before in Astropecten irregularis penta- plain, but most likely it decreases in time. canthus (Colombo and Belvedere, 1976). The yields of androstenedione and testos- terone distinctly decrease, which points to Sa-Reductase (5~R) at least some quantitative differences in As follows from Table 2, the percentage C,,-steroid metabolism in ovaries and conversion of progesterone into Sa-preg- pyloric ceca. In conclusion, though it has nane-3,20-dione is high in both organs, not been proved, it is likely that also in but the yields are higher in the pyloric ceca echinoderms the biosynthetic sequence will than in the ovaries (ovaries 2.7-6.4%; be: progesterone + 17a-hydroxyprogester- pyloric ceca 10.6-3.2%). The accumulation one * androstenedione. of Sa-pregnane-3,20-dione and probably its As follows from Table 1, the yields of formation are faster in the pyloric ceca than testosterone in the ovaries (1.3 -0.03%) are in the ovaries, since the yields are decreas- larger than those in the pyloric ceca ing in the former and increasing in the lat- (0.03-0.01%). The presence of 17/I-H?+ is ter. A Sa-reductase was also demonstrated in agreement with and confirms the earlier by Smith et al. (1972) for the conversion of observations of Schoenmakers et al. (1976). cholesterol into Sa-cholestan-3P-ol in As- Colombo and Belvedere (1976) demon- terias rubens. strated this enzyme in the ovaries of As- From the percentage unchanged proges- tropecten irregularis pentacanthus and in terone it appears that the metabolic capac- the gonads of Paracentrotus lividus. ity of the ovaries is different from that of the pyloric ceca. It may be possible that in 20~HSD this stage of the annual reproductive cycle, The yields of 20a-dihydroprogesterone i.e., the maturation stage, the capacity to are small in the ovaries (0.02-0.08%) and metabolize progesterone in the ovaries: is higher in the pyloric ceca (0.09-0.25%) reduced, or that this low capacity is inher- (Table 2). Thus the enzyme seems more ac- ent to the nature of the organ. Both organs, tive in the pyloric ceca than in the ovaries. however, are able to convert progesterone PROGESTERONE METABOLISM IN Asterias rubens 415 via different pathways. First, progesterone Therefore, a unique route leading from pro- can be metabolized into intermediates of gesterone to asterosaponins is supposed to the A4-pathways. In this experiment the be present in Aster& rubens. Gaffney and yields of these intermediates are in general Goad (1974) and Teshima et al. (1977), low but they are higher in the ovaries than studying the metabolism of progesterone in in the pyloric ceca. The organs show de- Asterias rubens. found some intermediates creasing yields of these steroids in time for the biosynthesis of asterosaponins, with the exception of an increasing yield of namely 3P-hydroxy-5a-pregnane-20-one androstenedione in the ovaries. Therefore, and 3P,6a-dihydroxy-Sa-pregnane-20-one. the identified steroids probably do not rep- Structurally it does not seem impossible resent end products. that 5a-pregnane-3,20-dione is another in- These results can be compared with pre- termediate for the formation of asterosapo- viously obtained data on the ability of nins. The 5a-pregnane-3,20-dione yield in steroid biosynthesis of the starfish Asterias time decreases in the pyloric ceca and in- rubens, i.e., the presence of side-chain creases in the ovaries; the amount un- cleaving enzyme complex, A5-A4-isomer- changed progesterone is much smaller in ase, and 3@HSD (Schoenmakers, 1979) the pyloric ceca than in the ovaries; the and of steroid-synthesizing cells in the yield of nonidentified steroids which might ovaries (Schoenmakers et al., 1977). It be intermediates in the synthesis of as- may then be concluded that Asterias rubens terosaponins is much higher in the pyloric is able to synthesize in the ovaries and the ceca than in the ovaries. Therefore, it may pyloric ceca C,,-steroids from cholesterol. be supposed that the pyloric ceca are more Formation of these steroids via the A4- active in the biosynthesis of the as- pathway, just as in vertebrates, seems to be terosaponins than the ovaries. not impossible. Moreover, the ovaries ap- No information is available on the func- pear to be more active in the biosynthesis of tion of progesterone and its metabolites these steroids than the pyloric ceca. No re- presently identified in Asterias rubens. ports are available on the biosynthesis of Studies on the biosynthesis and functions of C,,-steroids in Echinodermata. steroids with regard to the reproduction of A second metabolic route of progester- Aster& rubens will be continued. one is the conversion into 20a-dihydropro- ACKNOWLEDGMENTS gesterone, also a common pathway in ver- tebrates. The formation of ll-desoxy- The authors wish to thank Prof. Dr. P. G. W. J. van Oordt, Dr. R. C. H. M. Oudejans, and Dr. J. G. D. corticosterone from progesterone indicates Lambert for their critical comments. Appreciation is a progesterone metabolism leading to cor- expressed to Dr. R. J. Burer and Mr. T. K. F. Schulz ticosteroids. for their skilful technical assistance. However, the metabolic pathways of REFERENCES progesterone in Asterias ruberzs mentioned so far and comparable with those found in Botticelh, C. R., Hisaw, F. L., Jr., and Wotiz, H. H. (1960). -17P and progesterone in ovaries other animals are not intensively used when of the starfish (Pisastrr ochraceous) Proc. Sot. compared with the conversion of proges- Exp. Biol. Med. 103, 875-877. terone into 5a-pregnane-3,20-dione. In the Botticelli, C. R., Hisaw, F. L. Jr., and Wotiz, H. H. present experiments the highest yields are (1961). Estrogens and progesterone in the sea ur- found for Sa-pregnane-3,20-dione in ovaries chin (Stron~ylo~entrottrs ,franciscanus) and pec- ten (Pectrn hrricitts). Proc. Sm. Exp. Biol. Med. and pyloric ceca. Moreover, in both organs, 106, 887-889. especially in the pyloric ceca, the produc- Carreau, S., and Drosdowsky, M. (1977). The in vitro tion of unidentified steroids is important. biosynthesis of steroids by the gonad of the 416 SCHOENMAKERS AND VOOGT

cuttlefish (Sepia officinalis L.). Gen. Comp. En- Asterias rubens. Proc. Int. Union Physiol. Sci. docrinol. 33, 554-565. 13, 673. Colombo, L., and Belvedere, P. (1976). Gonadal Schoenmakers, H. J. N. (1979). In vitro biosynthesis steroidogenesis in echinoderms. Gen. Comp. En- of steroids from cholesterol by the ovaries and docrinol. 29, 255-256. pyloric caeca of the starfish Asterias rubens. De Longcamp, D., Lubet, P., and Drosdowsky, M. Comp. Biochem. Physiol. 63B, 179-184. (1974). The in vitro biosynthesis of steroids by the Schoenmakers, H. J. N., Colenbrander, P. H. J. M., gonad of the mussel (Mytilus edulis). Gen. Comp. and Peute, J. (1977). Ultrastructural evidence for Endocrinol. 22, 116- 127. the existence of steroid synthesizing cells in the Gaffney, J., and Goad, L. J. (1974). Progesterone ovary of the starfish Asterias rubens (Echinoder- metabolism by the echinoderms Asferias rubens mata). Cell Tissue Res. 182, 275-279. and Marthasierias glacialis. Biochem. .I. 138, Schoenmakers, H. J. N., and Goedhart, M. J. The an- 309-311. nual reproductive cycle of the ovaries of Asterias Gottfried, H., and Dorfman, R. 1. (1970). Steroids of rubens (Echinodermata). Submitted for publica- the invertebrates. V. The in vifro biosynthesis of tion . steroids by the male-phase ovotestis of the slug Schoenmakers, H. J. N., Lambert, J. G. D., and (Ariolimax californicus). Gen. Comp. Endocrinol. Voogt, P. A. (1976). The steroid synthesizing ca- 15, 120- 138. pacity of the gonads of Asterias rubens. Gen. Grossert, J. S. (1972). Natural products from echino- Comp. Endocrinol. 29, 256. derms. Chem. Sot. Rev. 1, l-25. Smith, A. G., Goodfellow, R., and Goad, L. J. (1972). Ikegami, S., Shirai, H., and Kanatani, H. (1971). On The intermediacy of 3-0~0 steroids in the conver- the occurrence of progesterone in ovary of the sion of cholest-5-en-3/3-ol into Sa-cholestan-3P-ol starfish, Asterias amurensis. Dobursugaku Zasshi by the starfish Asterias rubens and Porania pul- 80, 26-28. villus. Biochem. J. 128, 1371-1372. Lupo di Prisco, C., and Dessi’Fulgheri, F. (1975). Al- Tcholakian, R. K., and Eik-Nes, K. B. (1971). ternative pathways of steroid biosynthesis in Steroidogenesis in the blue crab Callinectes gonads and hepatopancreas of Aplysia depilans. sapidus Rathbun. Gen. Comp. Endocrinol. 17, Comp. Biochem. Physiol. SOB, 191-195. 115-124. Lupo di Prisco, C., Dessi’Fulgheri, F., and To- Teshima, S. -I., Fleming, R., Gaffney, J., and Goad, masucci, M. (1973). Identification and biosyn- L. J. (1977). Studies on steroid metabolism in the thesis of steroids in the marine mollusc Aply- echinoderm Asferias rubens. In “Marine Natural sia depilans. Comp. Biochem. Physiol. 45B, Products Chemistry” (D. J. Faulkner and W. H. 303-310. Fenical, eds.), pp. 133- 146. Plenum, New York. Nambara, T., and Iwata, T. (1973). Analytical chemi- Teshima, S. -I., and Kanazawa, A. (1971a). Biocon- cal studies on steroids. LXIII. Steroid numbers of version of progesterone by the ovaries of crab, androstanones and their oxime derivatives on Portunus trituberculatus. Gen. Comp. Endo- gas-chromatography. Chem. Pharm. Bull. 21, crinol. 17, 152- 157. 899-902. Teshima, S. -I., and Kanazawa, A. (197lb). In l,itro Rio, G. J., Stempien, M. F., Nigrelli, R. F., and Rug- bioconversion of progesterone by the sliced testes gieri, G. D. (1965). Echinoderm toxins. I. Some of crab, Portunus trituberculatus. Nippon Suisan biochemical and physiological properties of toxins Gakkaishi 37, 524-528. from several species of Asteroidea. Toxicon 3, Yasumoto, T., Tanaka, M., and Hashimoto, Y. (1966). 147- 155. Distribution of saponins in echinoderms. Nippon Schoenmakers, H. J. N. (1977). Steroid synthesis of Suisan Gakkaishi 32, 673-676.