Patterns of steroid metabolism in teleost and ganoid fishes

GENERAL

AND

COMPARATIVE

Patterns

ENDOCRINOLOGY

of Steroid

3, 245-253

SUPPLEMENT

Metabolism

in Teleost

LORENZO Institute

of Animal

Biology,

SANDRO Second

Obstetrical

of

and Padua,

(1972)

and

Ganoid

Fisk

COLOMBO

Unive&y

of Padun;

PadlLa,

Italy

36150

PESAVENTO Gynaecological Verona: Italy

Clinic, S7lOO

University

AND

DONALD Department and

W. JOHNSON

of Zoology and its Cancer Research Bodega Marine Laboratory, liniversity Berkeley, California 9&7LO

Genetics LabolntoTy of California,

Steroid hormone biosynthesis and catabolism in teleost fishes present numerous distinctive features when compared with the biochemical systems operating in tetrapods. The fish testis, unlike that of mammals, synthesizes androgens with a hydroxyl or keto group at the 11 position; the routes of synthesis and interconversion still need to be completely elucidated. In two species, the testis has been found to be equipped with efficient enzymatic systems for steroid conjugation of unknown physiological significance. The ripe teleost ovary shows a conspicuous production from progesterone of ll-deoxycorticosterone and ll-deoxycortisol, whose function is probably to induce ovulation. However, when ovulation is temporarily impaired, as in species which must migrat.e to spawn, 11-deoxycorticosteroids are absent, and ll-oxygenated androgens of the testicular type are formed, which may be responsible for an antiovulatory action. With the interrenal, steroid profiles obtainable in vitro from radioactive progesterone incubated with minced tissue preparations, have different patterns in different species with good reproducibility. In the posterior (mesonephric) kidney, 21-hydroxylase activity has been noted only in SaEm,o gairdnerii but not in other fishes. Teleost liver is an effective catabolizing site for corticosteroids but contains only a minimum of I@-hydroxysteroid dehydrogenase activity when compared with mammalian liver. In the anterior kidney of the ganoids Amia calva and Lepisosteua osseus, the metabolic pathways leading to the formation of both corticosterone and cortisol have been determined. They seem to be equally important in these fishes. Aldosterone was not detected in any of the fishes examined.

The evolution of rayfinned fish has proceeded along a line completely separated since early Paleozoic times from that leading to tetrapods. In their history, actinopterygian fish have repeatedly expanded and retaining only few surviving regressed, members from vast groups after the exhaustion of their adaptive radiation. Thus present-day Holostei (ganoids) , and Chondrostei may be considered as remnants of

piscine faunas that flourished, respectively, during Mesozoic and Paleozoic eras. Conversely, the superorder of Teleostei with its actual, impressive diversification (about 15,000 species, more than in any other vertebrate group) represents the ultimate expression of this long evolutionary process. Comparative investigators of vertebrate steroid secretions should find greater differences at the top of the two major 245

@ 1972 by

Academic

Press,

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phyletic branches, that is between teleosts and mammals, than among members of the same branch. This, indeed, seems to be the case. In fact, when compared with the rather similar steroid profiles of tetrapods, teleost fish exhibit many distinctive metabolic features. These include: (a) presence of an oxygen function at the 11 position in testicular (Table l), and in some cases, even ovarian (Eckstein and Eylath, 1969, 1970; Eckstein, 1970) androgens; (b) marked steroid conjugation by mature testes of at least two species (Grajcer and Idler, 1963; Colombo et al., 1970) ; (cj very high conversions in vitro of progesteronel to 11-deoxycorticosteroids, namely 11-deoxycorticosterone and ll-deoxycortisol, by mature ovarian tissue (cf. Colombo and Bern, in press) ; (d) absence or extremely low production of aldosterone by interrenal tissue (cf. Chester Jones et al., 1969; Colombo and Bern, 1971) ; and (e) location of 21-hydroxylase activity in the posterior (mesonephric) kidney of at least one species (Colombo et al., 1971). However, along with these differences, a fundamental correspondence of steroid metabolic pathways and secretions can 1 Trivial and systematic names of the steroids mentioned in the text: pregnenolone, pregnb-enpregn-53,&01-20-one ; lZa-hydroxypregnenolone, dehydroepiandrosterone, ene3/3,17a-dial-20-one ; androst-5-en-3,8-ol-17-one ; 5-androstenediol, androstA-ene-3P,17/3-diol; progesterone, pregn-4ene-3,20-dione ; ll-deoxycorticosterone, pregn-4pregn-4-enecorticosterone, en-21-o&3,20-dione; llp,21diol-3,20-dione; ll-dehydrocorticosterone, aldosterone, pregn-4-en-21-ol-3,11,20-trione; 17a-hypregn-4-en-18-al-11,8,2l-diol-3,2O-dione; droxyprogesterone, pregn-4-en-17ar-ol-3,2Odione; pregn-4-ene-llP,17a-diol-3,202l-deoxycortiso1, pregn-4-ene-17@,21-dioldione ; 11-deoxycortisol, 3,2Odione ; cortisol, pregn-4-ene-ll/3,17ar,21-trioicortisone, pregn-4-ene-17a,21-diol3,2Odione ; 3,11,20-trione ; tetrahydrocortisol, S/3-pregnanetetrahydrocortisone, 3~,11~,17cr,21-tetrol-2O-one; 5/%pregnane-3cr,l7a,21-triol-11,2O-dione; androstenedione, androst-4-ene-3,17-dione; llp-hydroxyandrostenedione, androskkn-11/3-ol-3,17dione; adrenosterone, androst-4-ene-3,11,17-trione; testosterone, androstAen-17p-ol-3-one ; Ilp-hydroxytestosterone, androst-4-ene-llp,l7~-diol-3-one; llketotestost.erone, androst&en-17,b’-ol-3,ll-dione.

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JOHNSON

easily be recognized between teleosts and land-living vertebrates. At present, little is known about the more primitive Actinopterygii, but the search for ‘(ancestral” steroid patterns has disclosed teleost-like characteristics (Idler et al., 1970). The purpose of this report is to discuss a few selected topics on steroid biosynthesis and catabolism in teleostean and holostean fishes, adding some new information. TELEOSTEI

Testis Though scanty, the literature on testicular ste.roidogenesis in teleost fish has already indicated the existence of some peculiar phenomena. Production of steroids in a conjugated form is of special interest. In this respect, it is somewhat surprising to note that, since the pioneering report by Grajcer and Idler (1963) describing the extraction and isolation of conspicuous amounts of testosterone p-glucuronoside both from the testes and the plasma of spawning males of Oncorhynchus nerka, additional contributions failed to appear. It, has only recently been found that the mature testicular tissue of Gob&s paganelZus is also endowed with enzymes both for steroid ,&glucuronylation, and probably, for steroid sulfurylation (Colombo et al., 1970). It seems that in the testicular tissue of both teleosts, the tendency towards steroid conjugation is more marked than that reported for mammals. Thus, fish testis may be a convenient system to study the significance of steroid conjugation by endocrine organs, a problem at present wit,h no clear answer. Another well-documented fact is that. teleost androgens differ substantially from those normally produced by mammalian testes, inasmuch as they have a hydroxyl or keto group at the 11 position (Table 1). Discrepancies do exist, nevertheless, in the routes of biosynthesis and interconversion of these compounds as discussed in detail by Idler and MacNab (1967)) and Idler et al. (1968). Our results are also at variance with those of other authors. In fact,

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METABOLISM

Arai and Tamaoki (1967a,b) were unable to isolate from testicular homogenates of Salmo gairdnerii incubated with androstenedione-4-14C either 11/3-hydroxyandrostenedione or adrenosterone; only testosterone and its 11-oxygenated derivatives were found. However, Idler et al. (1968), through incubations in vitro with androstenedione-l*C and dehydroepiandrosterone-3N concluded that in Salmo salar, 11/3hydroxyandrostenedione was only a minor metabolite of both precursors in the testis, being instead the major product in the interrenal. On the other hand, in our laboratory, minced testis of Anguilla anguilla at the silver stage converted androstenedione4-14C mostly to ll,&hydroxyandrostenedione and, t,o a lesser extent, to adrenosterone (L. Colombo and S. Pesavento, unpublished) I Similar results were obtained after the incubation of progesterone-4-14C with testicular tissue of Anguilla anguilla (L. Colombo and S. Pesavento, unpublished), Roccus saxatilis, and Gillichthys mirabilis (L. Colombo, J. Pieprzyk and D. W. Johnson, unpublished). Further in vitro work is needed to clarify this situation and integration of in wivo and in vitro data seems highly desirable. Yields of products in vitro do not necessarily reflect either their relative secretion rates in viva or blood level re,tios in the living animal (Vinson and Whitehouse, 1970). This is particularly evident in the case of 11,8-hydroxytestosterone which is easily formed in vitro by Snlmo salar testis (Idler et al., 1968), but cannot be detected in the plasma of this species (Schmidt and Idler, 1962). OZYLTY Recent research on the biochemistry of ovarian steroids in teleost fishes has revealed characteristic biosynthetic activities which may have an important bearing on the female reproductive physiology. We are referring both to the production of lldeoxycorticosteroids by fish ovaries near ovulation, and to the elaboration and eventual accumulation of II-oxygenated androgens by ovaries whose ovulation seems inhibited. We have already discussed some aspects

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of our finding that progesterone-4-14C can be efficiently converted in vit?*o Co II-deoxycortisol and/or, to a minor extent, to 11-deoxycorticosterone, in addition to l7~yhydroxyprogesterone, androstenedione and testosterone, by mature ovarian tissue from Gillichthys mirabilis, Microgadus proximus, and Leptocottus armatw (Colombo and Bern, 1971). In our opinion, this result rather conveniently integrates the study of Sundararaj and Goswami (2969: 1970) who have shown that in Heteropneustes fossilis, both ovine luteinizing hormone (LB) and II-deoxycorticosterone are potent ovulating agents. LII appears to have a latent period longer than that of ll-deoxycorticosterone, whereas the latter can be synergistically potentiated by cortisoi. These autho.rs postulated a tropic action of LII on the interrenal to stimulate the production of corticosteroids which, in turn, wouid bring about ovulation. Ou’ever, since Il-deoxycorticosteroida can be produced also in the ovary, L simply promote their synthesis in t without changing into a “corticotropin.” This hypothesis is now being tested in our laboratory. Another point. we wish to emphasize is t.hat at least ll-deoxycorticosterone may be formed also in mature ovarian tissue of other vertebrates. In fact, we have isolated this steroid after incubation of pregnenolone-4-14C with ovaries from two rept,iles, namely, Xantusia. vi&is and Storerin dekayi (L. Colombo, %. Yaron, and E. Daniels, unpublished) ~ as well as after incubation of progesterone-4-r4C with human ovarian tissue (L. Colombo and S. Pesavento, unpublished). Lately, Eckstein and Eylath (196 1970) have put. forward the hypothesis that ll-oxvgenated androgens may exert a renressive action on fish ovulation. They have, indeed, shown that in the female of Jfuqil capita, the inability to spzw-n in freshwater is accompanied in the ovary by higher 11,8-hydroxylase activit,y and greater accumulat~ion of 11-ketotestosterone than occurs in specimens living in sea water, where spawning can OCCUK. Tbe presence of a higher concentration of II-

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ketotestosterone, which is a potent fish androgen (Arai, 1967)) would apparently cause a block in the process of ovulation, since oocyte growth is similar in both biotopes. This interesting report induced us to conduct an analogous experiment on Anguilla anguilla, which neither matures sexually nor spawns in inland fresh or brackish waters. Thus, to detect llp-hydroxylase activity, progesterone-4-% and androstenedione-4-% were incubated with minced ovarian tissue from silver eels at the onset of their migration into the sea. As expected, from the first substrate 11/3obtained, hydroxyandrostenedione was along with 17e-hydroxyprogesterone, androstenedione, and testosterone, whereas from the second one llp-hydroxyandrostenedione and adrenosterone were formed, along with testosterone. The question now remains whether production of 11-oxygenated androgens is strictly limited to certain species with blocked ovulation or is simply enhanced in these species being, however, of widespread occurrence. We never observed these steroids among the products of fish ovaries near ovulation (see above), but ll-ketotestosterone was apparently synthesized in vitro from androstenedione7a-3H by the mature ovary of Tilapia aurea (Eckstein, 1970)) a fish which does not migrate to spawn. Anterior or Head Kidney Interrenal Tissue)

(containing

We have recently examined all available information, derived from experiments in vitro, regarding the corticosteroidogenic ability of the teleost interrenal (Colombo and Bern, 1971). This subject has also been critically reviewed by other authors who have prepared up-to-date summaries of data (Chester Jones et al., 1969; Vinson and Whitehouse, 1970). In this paper we would like to add only a few comments. In the application of techniques in vitro with added precursors to problems of steroid metabolism in teleost fishes, considerable improvements have been made both in the refinement of the analytical procedures adopted and in the number of

AND

JOHNSON

parameters measured. Thus, identifications of radioactive metabolites have been lately completed with kinetic studies (Idler et al., 1968; Colombo et al., 1970) and now the time-course of the whole steroid profile of an incubate can be followed by coupling bidimensional thin-layer chromatography with radioautography (Colombo et al., 1971). An interesting finding concerning the teleost interrenal is that steroid profiles obtained in vitro from progesterone-4-14C with minced tissue preparations have different patterns in different species. This specific variability seems more pronounced with interrenal incubates than with gonadal ones, probably because of the more complex composition of the anterior or head kidney. Extensive arrays of products are normally encountered among which many corticosteroids can be recognized. Under carefully checked conditions, the reproducibility of each profile is satisfactory even if quantitative variations may occur. So far we have examined seven species belonging to four different orders. (cf. Colombo and Bern, 1971). It is worth noting that in the last species studied, Anguilla anguilla, at the silver stage, no 17-deoxycorticosteroid pathway appeared operat’ive, and only the metabolic route leading to cortisol was found (L. Colombo and S. Pesavento, unpublished), in agreement with a previous observation by Sandor et al. (1967). To obtain a characteristic steroid profile, it is imperative to use a minced tissue preparation repeatedly washed to remove suspended materials. In fact, the use of homogenates, either with or without cofactors, reduces drastically the number of products in the profile, favoring an accumulation of only few metabolites proximal to the substrate. Thus, homogenized posterior cardinal veins plus anterior kidney from Anguilla nnguilla could not transform pregnenolone-4-14C beyond progesterone and 17a-hydroxyprogesterone (L. Colombo and S. Pesavento, unpublished). Cofactors usually increase yields of products but have modest effects on the total number of metabolite formed so that

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METABOLISM

the profile is still not characteristic. This result seems to indicate that cell disruption probably brings about poor coordination of the various biosynthetic sequences. Posterior

Kidney

After

the demonstration of 21-hydroxactivity in the posterior kidney of k3almo gairdnerii (Colombo et al., 1971)) we have sought this enzyme also in the posterior kidneys of Leptocottus armatus and Anguilla anguilla. In both cases, however, there was no conversion in vitro of progesterone-4-14C to ll-deoxycorticosterone either by minced or homogenized kidney preparations (L. Colombo, S. Pesavento and D. W. Johnson, unpublished). Therefore, at present, the activity of the truak kidney of Salmo gairdnerii remains unique among vertebrates. ylase

Liver In mammals, a peripheral modulation of the hormonal activity of cortisol is achieved through a reversible oxido-reductive conversion to the probably inactive cortisone, catalyzed by a U/3-hydroxysteroid dehydrogenase localized mainly in the liver (cf. Rosenfeld eC al., 1967). The equilibrium of this reaction is usually markedly shifted in the reductive direction, though, puzzling enough, most of the urinary catabolites of eortisol may have a 5,8-H-11-ketonic configuration as in man (cf. Bush, 1969). By in v&o incubation of minced or even homogenized liver of rat with cortisone4-33 without added pyridine nucleotide cofactors, we could easily observe a rapid transformation into cortisol, whereas cortisol-4-14C was oxidized to cortisone only to a small extent (L. Colombo and S. Pesavento, unpublished). However, when minced livers of t,he teleosts: Gillichthys mirabilis, Leptocottus armatus, Xalmo gairdnerii, Tilapia mossambica (L. Colombo, unpublished), and Anguilla anguilla (L. Colombo and S. Pesavento, unpublished) were used under comparable in vitro condit,ions no detectable conversion (i.e., less than .Ol%) of cortisone-4-l‘% to cortisol was found, whereas only a minute quantity of cortisone was produced

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from cortisol-4-14C, which proved not to be a technical a,rtifact as earlier suspected (Colombo and Bern, 1971). Liver homogenization did not alter this outcome., In the last two species, tetrahydrocortisol was obtained from eortisol-4-l% and tetrshydrocortisone from cortisone-4-14C. Thus, fish liver appears as an effective catabolizing site for corticosteroids but it, probably lacks sufficient, Ilp-hydroxysteroid dehydrogenase activity t,o ai%ect appreciably the balance of circulating eortisol and cortisone. In t.he lat,ter respect, the anterior kidney may be far better equipped; this is the case, for instance, in Fllapia mossambica (Colombo and Bern: 1971) I HOLOSTEI

Kidney

(with Interrenal

Tissue)

In ganoid fishes, the int’errenal tissue consists of clusters of cells distributed around the postcardinal veins. The major portion is coneentrat,ed in the anterior kidney, which is formed by a mass of hemopoiet.ic tissue containing also a chromaffin component and numerous (up to 50) Stannius corpuscles derived from. the pronephric duct. Nephric tubules are absent from this zone, being confined within the median and posterior kidney together with a smaller fra,&ion of interrenal tissue, In Amia corpuscular outgrowths of the dist,al segment of the nephron tubules are also found in this portion of the kidney (De Smet, 1962, 1963). Recently, Idler and coworkers (1970) have found that cortisol is the principal corticosteroid synthesized in z&o by the interrenal of Amia cdva. Pursuing a similar line of investigation, we have examined the pattern of progesterone-4-I46 metabolism by the minced interrenal-rich kidnep as compared with the interrenal-poor segment, both in Amia calva and in Lepisosteus osseus (L. Colombo and D. W. Johnson, unpublished). In Amia, the anterior kidney produced 17-deoxycorticosteroids, namely Il-deoxycorticosterone and corticosterone, as well as 17a-hydroxycorticosteroids, namely 1

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hydroxyprogesterone and cortisol, whereas none of these compounds appeared in the incubate with the caudalmost third of the posterior kidney. In Lepisosteus, the anterior half of the kidney also gave rise to the above-mentioned metabolites but, in addit,ion, cortisone, 11-dehydrocorticosterone and androstenedione were isolated. The posterior half did not show any detectable corticosteroidogenic activity. Other features common to both fish were: (a) A very poor accumulation (or absence) of 11-deoxycorticosteroids, namely ll-deoxycorticosterone and 11-deoxycortisol, in the incubates; (b) a comparable partition of progesterone between the cortisol and corticosterone pathways; (c) no detectable aldosterone; and (d) a vast array of catabolic products. However, the anterior kidney of Amia, unlike that of Lepisosteus, was deficient in 11/3-hydroxysteroid dehydrogenase and 17cu,20-Czl-desmolase activities, despite the fact that it could utilize progesterone-4-l% more completely. These results indicate that corticosteroid profiles in ganoids are not very different from those of teleosts.

stress, etc., and to help in the solution of practical problems such as artificial insemination and rearing, as well as producing useful information for further research in mammals, including man. We have been the first to notice a small in vitro production of 11-deoxycorticosterone from progesterone-4-13C by the human ovary only because of the comparative nature of our study. We looked for a similarity with the teleost ovary, in which 11-deoxycorticosteroid synthesis is very prominent. In general, the larger the number of zoological groups examined, the more comprehensive are the models which can be proposed to explain various phenomena and the less incomplete are the historical reconst.ructions of their evolution. For these reasons an extensive study of steroid biochemistry in primitive fishes and groups even lower in the chordate scale are highly desirable to establish how ancient the steroid metabolic patterns encountered in the more evolved vertebrates are and how they arose.

Testis

Part of the research reported herein was aided by U. S. National Institutes of Health, grant AM97896 to Professor H. A. Bern, and a NIH postdoctoral fellowship to D. W. J. We are indebted to Professor H. A. Bern, Professor F. Ghiretti, Professor A. Onnis, and Professor A. Sabbadin for their support and encouragement. We are grateful to Miss F. Bottecchia, Miss M. Gadotti, and Mr. F. Palazzo for their help.

An attempt was made to determine the steroids obtainable from progesterone-4-14C in the immature testes of the same three Lepisosteus specimens used in the previous experiment. Although most substrate was transformed, sex hormone biosynthesis was definitely absent (L. Colombo and D. W. Johnson. unpublished).

ACKNOWLEDGMENTS

REFERENCES CONCLUSIONS

We believe that the validity of the comparative approach in steroid endocrinology is well demonstrated. Previous knowledge of steroid metabolism in mammals has been a useful guide in extending this research to teleosts. Comparative investigations are yielding their rewards ; differences have been discovered. This knowledge is likely to contribute to our effective understanding of many physiological phenomena in fish such as sexual maturation, spawning response to migration, osmoregulation,

ARAI, R. (1967). Annot. Zool. Jup. 40, 15. ARAI, R., SHIKITA, M., AND TAMAOKI, B. (1964). Gen. Comp. Endocrinol. 4, 68-73. ARAI, R., AND TAMAOKI, B. (1967a). Can. J. Biochem. 45, 1191-1195. ARM, R., AND TAMAOKI, B. (196715). Gen. Comp. Endocrinol. 8, 305313. BUSH, I. E. (1969). In “Advances in the Biosciences 3,” Berlin, December 13 and 14, 1968, pp. 23-40. Pergamon Press-Vieweg, Oxford. CHAN, S. T. H., AND PHILLIPS, J. G. (1969). Gen. Comp. Endocrinol. 12, 619-636. CHESTER, JONES, I., CHAN, D. K. O., HENDERSON, I. W., AND BALL, J. N. (1969). In “Fish Phys-

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iology,” (Hoar, W. S., and Randall, D. J., eds.), Vol. 2, pp. 321376. Academic Press, New York and London. COLOMBO, L., AKD BERN, H. A. (1971). Excerpta Med. Found. Int. Congr. Ser. 219. Proc. Int. Cwgr. Harm. Steroids 3rd Hamburg, September 7-12. 1970, pp. 904-911. COLOMBO, L., BERN, H. A., AND PIEPRZYK, J. (1971). Gen. Comp. Endocrinol. 16, 74-84. COLOMBO, L., DEL CONTE, E., AND CLEMENZE, P. Gsn. Camp. Endocrinol. (in press). COLOMBO, L., Lupo DI PRISCO, C., AND BINDER, G. (1970). Gen. Comp. Endocrinol. 15, 404419. De SMET, W. (1962). Acta Zool. 43, 201-219. DE &WET, W. (1963). Acta Zool. 44, 269-296. ECKSTEIN, B. (1970). Gen. Comp. Endocrinol.

14, 303-312. ECKSTEIN, B., AND EYLATH, U. (1968). Comp. Biochem. Physiol. 25, 207-212. EC&STEIN, B., AND EYLATH, U. (1969). Gen. Comp. EndocrZnoE. 13, 503. ECKSTEIN, B., AND EYLATH, U. (1970). Gen. Comp. Endocrinol. 14, 396403. GRAJCER, D., AND IDLER, D. R. (1963). Can. J. Biochem. Physiol. 41, 23-30. IDLER, D. R., AND MACNAB, H. C. (1967). Can. J. Biochem. 45, 581-589. IDLER, D. R., SANGALANG, G. B., AND WEISBART, M.

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Excerpta Med. Found. lnt. Congr. Sea. Int, Congr. Horm. Steroids 3rd Hamburg, September 7-12, pp. 72-73. IDLER, D. R., TR~SCOTT, B., AKD STEWART; Li. C. (1968). Exeerpta Med. Found. Int. Congr. Sey. 184. Proc. Int. Congr. Endocrinol. M&co D. F. June 30-July 5, pp. 724-729. LUPO DI PRISCO, C., MATERAZZI, G., AND CHIEFPE. G. (1970). Gen. Comp. Endocrinol. 14, 595-598. ROSENFELD, R. S., FUKUSHIMA, D. I<., AND GALLAGHER, T. F. (1967). In “The Adrenal Cmtex” (Eisenstein, A. B., ed.), pp. 103-131. Little. Brown and Company, Boston. SANDOR, T., LANTHIER, A., HENDERSON, I. W., AND CHES’I’ER JONES, I. (1967). Endocrinology 904912. SCHM~T, P. J.> AKD IDLER; D. R. (1962). Gen. Comp. Endocrinol. .2, 204214. SUNDARARAJ, B. I., AND GOSWAMI, S. V. (1969). Gen. Comp. Endocrinol. Suppl. 2, 374384. SUNDARARAJ, B. I., AND GOSWAMI, S. V. (1970). Excerpta Med. Found. Int. Congr. Ser. 210. Proc. Int. Congr. Worm. Steroids 3rd Hamburg, September 7-12, p. 72. VKNSOK, G. P., AND WHITEHOUSE, B. J. (192%). In “Advances in Steroid Biochemistry and Pharmacology” (Briggs, M. H., ed.), pp. 163342. Academic Press, London and New York,

210. Froc.

DISCUSSION CALLARD: Why do you think that 11-deoxycorticosterone is involved in reptilian corpus iuteum function? COLOMBO: Because its formation in vitro from pregnenolone-4-14C occurred only in the corpus luteum and not in the remaining ovarian tissue from pregnant snakes of the species Storeria dekayi. CALLARD: Do you have any evidence for the presence of this hormone in plasma? COLOMBO: No, I have not. CALLARD: At what temperature were your incubations conducted and what was the percent conversion of pregnenolone to progesterone and 11-DOC? COLOMBO: At 30”. The conversions were approximately 20% and 12al,, respectively, if I remember correctly. FONTAINE: Did you look for an action of gonadotropins on steroid metabolism in the gonads? COLQMBO: No, but I shall soon study the effect of FSH, LH and LTH from mammalian sources on 11-deoxycorticosteroid production by the teleost ovary. VAN OORDT: Did you detect estrogens in the ovary of the teleosts you studied? COLOMBO: I have not looked for estrogens in teleost ovaries since my aim at the time was to study 11-deoxycorticosteroid production. SUNDARARAJ: Have you tried the effect of deoxycorticosterone and compound S on in uivo or in vitro ovulation in Gillichthys? COLOMBO: No, but I plan to study the effect of these steroids on the ovulation process in some other teleosts. DONALDSON: Did you incubate ovarian t,issue with DOC to determine whether there is any further metabolism from DOC? COLOMBO: No, I did not.