Studies on the reproductive cycle on the female cobra, Naja naja. II. In vitro ovarian steroid synthesis.

The synthesis of steroids in vitro by minced ovarian tissue from the cobra, Naja naja, using [³H]pregnenolone and [³H]dehydroepiandrosterone([³H]DHA) as precursors was studied. From [³H]pregnenolone the major products were progesterone, pregnanolone (3α-hydroxy-5β-pregnan-20-one), 17α-hydroxyprogesterone, androstenedione, and testosterone. DHA and 17α-hydroxypregnenolone were tentatively identified, but insufficient material was available for positive characterization. From incubations using [³H]DHA as precursor, the only products identified were testosterone, androstenedione, and estradiol-17β. Significant amounts of radioactivity were associated with an estriol fraction from both the pregnenolone and the DHA incubations but were not further characterized. Time-lapse studies revealed an extremely rapid conversion of [³H] pregnenolone to progesterone, with a maximum occurring after 15 min in tissue taken from a cobra in April at the height of the reproductive period. Addition of cofactors to the medium markedly stimulated the synthesis of progesterone and pregnanolone from [³H]pregnenolone, but appeared to inhibit the production of other ovarian steroids. Mammalian LH, when added to the incubation medium, was found to stimulate the biosynthesis of 17α-hydroxyprogesterone, androstenedione, and testosterone from [³H]pregnenolone. Addition of fresh, homogenized snake pituitary or mammalian FSH appeared to increase the yield of testosterone but none of the precursors in the pathway, and there was a suggestion that FSH alone increased the rate of aromatization.     

In vitro steroidogenesis by the nonzoned adrenocortical tissue of the skink, Tiliqua rugosa.

Steroid formation by adrenocortical tissue from the shink, Tiliqua rugosa, has been studied using established in vitro techniques. Both in conventional incubations, with timed sampling, and in incubations with dialysis, aldosterone, and corticosterone were major products. From endogenous precursors, and from [¹⁴C]acetate, yields of the two products were of the same order, whereas from [³H]pregnenolone maximal yields of corticosterone were at least tenfold greater than aldosterone. Maximal rates of steroid formation from the radioactive precursors occurred within the first few minutes of incubation, but maximal rates of steroid formation from endogenous precursors occurred significantly later, between 1–2 hr.In incubations with dialysis [¹⁴C]aldosterone was significantly less dialysable than [³H]aldosterone under all conditions, whereas [¹⁴C] and [³H]corticosterone were homogeneous. In contrast, neither aldosterone nor corticosterone formed from endogenous precursors were bound under control conditions, although binding was increased following dexamethasone pretreatment, and decreased following stimulation with Tiliqua pituitary extract (but not Synacthen), with concomitant changes in yields and specific activities.Inter alia the results suggest that products formed from [¹⁴C]acetate and from [³H]pregnenolone may be maintained in separate pools within the tissue, and this accounts for their different metabolic fates. The bound pool, penetrated only by [³H]acetate, yields more aldosterone than the free, and may be termed a “biosynthetic pool.” In addition there exists a “secretory reserve pool.” This is suggested by the difference between rates of steroid secretion from endogenous and added precursors, and also from the changes in dialysibility seen in steroids formed from endogenous precursors under different conditions of stimulation.In both the compartmental arrangement of steroids, and the production of large yields of aldosterone the adrenocortical tissue of Tiliqua shows similarities to the zona glomerulosa, but not the inner zones of the rat adrenal cortex.     

In vivo and in vitro binding of [3H]estradiol in brain and pituitary of Mongolian gerbils, Meriones unguiculatus.

Following injection of [³H]estradiol in ovariectomized gerbils, the highest concentration of radioactivity was found in cell nuclei from the pituitary, followed by preoptic area, hypothalamus, amygdala, and midbrain, with very low levels in the olfactory bulbs and cerebral cortex. Cell nuclear binding was significantly reduced by pretreatment with unlabeled estradiol or nafoxidine but not by progesterone, 5α-dihydrotestosterone, or cortisol. The time course of estradiol binding in whole homogenate and cell nuclear fractions of brain and pituitary was determined by sacrificing animals 0.25, 1, 3, 6, or 12 hr after injection of [³H]estradiol and measuring hormone uptake into the respective cell components. While whole homogenate radioactivity was highest at 0.25 hr, cell nuclear uptake peaked at 1 hr. Radioactivity was still present in cell nuclei from pituitary, hypothalamus-preoptic area, and amygdala 12 hr after [³H]estradiol injection, although at a level only 2% of 1-hr peak values.Macromolecular binding of [³H]estradiol was found in the cytoplasmic fraction of pooled hypothalamus-preoptic area-amygdala. Scatchard analysis of this in vitro binding indicated a K_d of 5.0 × 10⁻¹⁰M. This binding was inhibited more by unlabeled estradiol or the synthetic estrogen, R2858, than by 5α-dihydrotestosterone, progesterone, or cortisol. Pronase almost totally inhibited binding, while DNase and RNase had little effect. The data indicate that the estradiol binding in female gerbil brain and pituitary is similar to that of other species previously studied, although some species differences were found.     

Uptake and distribution of glucocorticoids in cell nuclei of rat heart in cell-free system: dependence on concentrations and forms of added hormones.

Accumulation by rat heart cell nuclei in vitro of free and protein-bound [³H]corticosterone and [³H]dexamethasone was investigated. [³H]Glucocorticoid-protein complexes were prepared by incubation of labelled hormones with diluted blood serum and cytosol containing glucocorticoid cytoreceptor, transcortin-like protein and various amounts of transcortin. At concentrations of 2 × 10⁻⁹ to 25 × 10⁻⁹m binding to cytoreceptor enhances nuclear accumulations of hormones. Uptake of [³H]corticosterone depends on the ratio of binding proteins present in cytosol. The higher the part of glucocorticoid bound to cytoreceptor the larger the amounts accumulated in nuclear fractions. At relatively high concentrations (5 × 10⁻⁸m and higher) nuclei readily accumulate transcortin-bound and free forms of [³H]corticosterone and binding to transcortin evidently facilitates nuclear uptake. Studies with Triton X-100 and KCl extractions of incorporated [³H]glucocorticoids reveal heterogenity of the accumulation process. This process includes uptake of glucocorticoids by outer nuclear membrane as well as intranuclear accumulation. Binding to proteins facilitates in certain conditions uptake of hormones by nuclear membrane. 0.4 m KCl treatment of nuclei incubated with [³H]glucocorticoid-cytosol complex leads to extraction of essential amounts of intranuclear [³H]glucocorticoid fraction a part of which represents macromolecular complexes of hormones. KCl extracts from nuclei incubated with free of transcortin-bound steroid contain a comparatively low percentage of hormone, the greater part of which is present in free form.The data obtained clearly show that at physiological and pharmacological concentrations of hormones the cell nucleus is the target for glucocorticoid action. At the same time the mechanisms of hormonal action on the nucleus at different concentrations is different. At physiological levels of hormones cytoplasmic glucocorticoid receptor plays a key role in the formation of nuclear response to glucocorticoids whereas at pharmacological concentrations some other additional factors connected with the uptake by nucleus of free steroids from cytoplasm as well as extranuclear events can be of value.     

In vivo specific uptake of labeled insulin by liver,adipose tissue,pituitary, and adrenals in the turtle Chrysemys dorbigni

Insulin labeled with ¹²⁵I was injected into turtles (Chrysemys dorbigni) to study its specific uptake by tissues. The maximum specific uptake of radioactivity by turtle tissues was obtained 1 hr after administration of [¹²⁵I]iodoinsulin. Besides liver and adipose tissue, specific uptake of labeled insulin was detected in some endocrine glands, such as pituitary and adrenals. Both glands were as active in concentrating labeled insulin as liver and adipose tissue. A significant reduction of the uptake was observed when unlabeled insulin was injected together with the labeled hormone. This reduction was dose dependent, and the concentration of unlabeled insulin that prevented 50% of the tissue uptake of [¹²⁵I]iodoinsulin was of 1 to 10 μg/kg body weight. These doses were able to induce blood glucose decrease in the turtle. Prolactin, growth hormone, or glucagon were unable to displace labeled insulin uptake. The major proportion of the radioactive material extracted from liver and pituitary 1 hr after [¹²⁵I]iodoinsulin injection into turtle coeluted with [¹²⁵I]iodoinsulin in Sephadex G-50 column. The presence of radioactive degradation products are consistent with the intracellular receptor mediated degradation hypothesis. These findings suggest the presence of specific insulin binding sites in liver, adipose tissue, pituitary, and adrenal glands from turtles.     

Testicular steroid biosynthesis by the green lizard Lacerta viridis

Testes from the green lizard Lacerta viridis were incubated with [³H]pregnenolone or [³H]testosterone and the products were identified by chromatography, microchemical reaction, and crystallisation to constant specific activity or isotope ratio. The major metabolites of pregnenolone were testosterone (40.8%), androstenedione (5.5%), 5α-androstane-3β,17β-diol (4.4%), and 5α-pregnane-3β,17α,20ξ-triol (15.2%). Androstenedione was the only identifiable metabolite (4.8%) of testosterone.     

Formation of corticosteroids in vitro by interrenal tissue from the teleost fish, Coregonus clupeoides.

Adrenocortical tissue from the freshwater teleost, Coregonus clupeoides, was incubated with [¹⁴C] acetate and [³H] pregnenolone. Evidence was obtained for the formation of doubly labeled cortisol, cortisone, and deoxycortisol, and the 17-deoxycorticoids, corticosterone, deoxycorticosterone, and aldosterone. This corresponds very closely to the range of steroids isolated from circulating plasma in this species.Under conditions of incubation with dialysis, it was found that the tritiated products tended to be freely dialysable, whereas the [¹⁴C] products were significantly less so. The addition of Coregonus pituitary extract to the incubation medium caused both a release of [¹⁴C] steroid from the bound condition and a significant decrease in the yield of [¹⁴C] aldosterone; neither effect was seen with Synacthen. In all cases tritiated products were largely unaffected both in yield and dialysability.The results were consistent with the view that aldosterone is preferentially formed through a pathway involving bound intermediates. The native ACTH-sensitive bound pool of steroids and intermediates is penetrable by exogenous precursors occurring early in the biosynthetic pathway, such as [¹⁴C] acetate, but not by late precursors such as [³H] pregnenolone. The results are comparable with those obtained with other species, including the skink, Tiliqua rugosa and the zona glomerulosa (but not the inner zones) of the rat adrenal cortex.     

Nuclear uptake of radioactivity by cells of pituitary,brain, uterus,and vagina of the asian musk shrew (Suncus murinus) following [3H]estradiol administration

Many reproductive traits of the Asian musk shrew (Suncus murinus) appear to be independent of ovarian influence, even though [³H]estradiol is taken up by uterus and vagina in vitro. This study seeks to examine in situ nuclear uptake of radioactivity by brain, pituitary, uterus, and vagina following [³H]estradiol administration. Ninety minutes after injection of [³H]estradiol, tissues were processed for either dry autoradiography or thaw-mount autoradiography. In the brain major accumulations of labeled neurons occurred in the medial preoptic nucleus, medial amygdaloid nucleus, lateral septal nucleus, arcuate nucleus, and ventrolateral part of the ventromedial nucleus. The anterior lobe of pituitary contained more than 70% labeled cells, with immunocytochemically stained gonadotropes among the most heavily labeled. The posterior lobe contained scattered labeled cells. In the uterus and vagina stromal and myometrial cells were heavily labeled while epithelial cells were moderately to lightly labeled. The data are discussed in relation to other eutherian species and with respect to current physiological data on the musk shrew.     

Bioconversion of steroids by the testes of the American lobster, Homarus americanus,in vitro

Lobster testes have been demonstrated to contain steroid 20-ketone reductase by their capacity to convert [¹⁴C]progesterone to 20α-dihydroprogesterone (20α-DHP). The major product was isopolar and identical with 20α-DHP during thin-layer chromatography, high-pressure liquid chromatography, mass spectrometry, and acetate derivative formation. The vas deferens from the lobster was also capable of the same conversions but to a lesser extent. Lobster testes converted [¹⁴C]pregnenolone to a major product identified as 20-dihydropregnenolone (20-DHPe) by mass spectrometry after purification by thin-layer chromatography and high-pressure liquid chromatography. The presence of Δ⁵,3β-ol dehydrogenase and Δ⁵,Δ⁴-isomerase in the lobster were also indicated. [¹⁴C]Cholesterol was not transformed to steroid hormones by lobster testes under the same experimental conditions.     

Protein synthesis and steroidogenesis in amphibian (Rana pipiens) ovarian follicles: studies on the conversion of pregnenolone to progesterone

Previous experiments demonstrated that protein synthesis was involved in frog pituitary homogenate (FPH)-induced follicular progesterone production. In this study the metabolic conversion of pregnenolone to progesterone, and involvement of protein synthesis in this specific step of the progesterone synthetic pathway, was investigated in vitro cultured ovarian follicles of Rana pipiens. Fully grown follicles were incubated with frog pituitary homogenates or exogenous pregnenolone and progesterone content of follicle extracts and medium were measured by radioimmunoassay. In the absence of FPH, fully grown follicles converted exogenously added pregnenolone into progesterone in a dose-dependent fashion. Follicular progesterone concentrations were consistently higher than medium levels of steroid throughout the culture period. The conversion of pregnenolone to progesterone at different stages of follicle development was also investigated. The amount of follicular progesterone accumulated after culture with exogenous pregnenolone increased proportionally with follicle size. When fully grown follicles were cultured in the presence of cycloheximide, the inhibitor of protein synthesis blocked FPH-induced progesterone production, but conversion of exogenously added pregnenolone to progesterone was not affected. However, progesterone production was inhibited when cyanoketone (CK), an irreversible inhibitor of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), was added in combination with FPH or exogenous pregnenolone. FPH addition after CK pretreatment did not restore the capacity of follicles to convert pregnenolone to progesterone. These results suggest that conversion of pregnenolone to progesterone occurs efficiently even in the absence of FPH over the course of follicle and oocyte growth (vitellogenesis). Furthermore, in fully grown follicles the 3 beta-HSD activity is independent of protein synthesis. The dependence on protein synthesis in the acute action of FPH appears to be prior to conversion of pregnenolone to progesterone and does not involve de novo synthesis of 3 beta-HSD.     

Localization and role of transcortin-like molecules in the anterior pituitary

The uptake and binding of [³H]dexamethasone and [³H] corticosterone in the anterior pituitary has been studied with special reference to the transcortin-like protein and compared with the uptake and binding in the hippocampal region of the rat brain, which lacks this protein.Isolated pituitary cells of adrenalectomized rats contained both glucocorticoid receptors and transcortin-like molecules.A collagenase treatment was used for isolation of the intact pituitary cells. Exposure of cytosol from broken cells of the anterior pituitary to this enzyme destroyed nearly all binding activity for the corticosteroids. The results indicate an intracellular localization of the transcortin-like molecules in the anterior pituitary, although the possibility that plasma transcortin adheres to the cell surface cannot be excluded.Estradiol treatment increased the level of transcortin in the plasma and of transcortin-like molecules in the anterior pituitary. The concentration of the glucocorticoid receptors in the anterior pituitary and the hippocampus remained unaffected.Subcutaneous injection of doses up to 50 μg corticosterone per 100 g body weight did not compete for the binding in vitro of [³H]dexamethasone to glucocorticoid receptors in anterior pituitary cytosol. The same doses of corticosterone reduced binding of the synthetic steroid in cytosol of the hippocampus by 40%.Endogenous corticosterone competed poorly for the uptake of [³H]dexamethasone in cell nuclei of the anterior pituitary. In cytosol 71% of the receptor sites were still available for [³H] dexamethasone binding in vitro.It is proposed that transcortin-like molecules in the anterior pituitary, whether intra- or extracellularly, modulate the effect of corticosterone on pituitary-adrenal activity by mediating the extent of interaction of the steroid with the glucocorticoid receptor.     

Mechanism of thyroid hormone inhibition of thyrotropin-releasing hormone action

The addition of thyroid hormone to cultures of GH3 or GH4C1 pituitary tumor cells maintained in medium with hypothyroid serum decreased the concentration of specific receptors for TRH. The relationship between thyroid hormone effects on TRH receptors and TRH responses was examined by testing the concentration dependence, time course, and specificity of these changes. The concentrations of T3 giving half-maximal decreases in [3H]TRH binding and inhibition of the PRL response to TRH were 0.20 and 0.24 nM, respectively. TRH stimulated the rate of [3H]uridine uptake by 50% in cultures incubated without added T3 but did not increase [3H]uridine uptake in cells incubated with thyroid hormone. The PRL response to TRH was substantially inhibited 12 h after the addition of T3, and the uridine uptake response was completely blocked in 8 h. Two other stimuli of PRL secretion, sodium butyrate and isobutylmethylxanthine, were effective in the presence or absence of T3. Thyroid hormone did not reduce the specific binding of either [125I-Tyr1]somatostatin or [125I]iodoepidermal growth factor. Somatostatin decreased the secretion of GH and PRL by pituitary tumor cells grown with or without T3. The data show that the effects of thyroid hormones on TRH receptors are specific and suggest that regulation of receptor concentrations may be the direct cause of thyroid hormone regulation of pituitary responsiveness to TRH.     

Effects of hypophysectomy on vitellogenesis in the elasmobranch Scyliorhinus canicula L

The effects of total hypophysectomy on vitellogenesis in adult female Scyliorhinus canicula were investigated by injection of ³²P-phosphate 5 days after operation. The rate of plasma vitellogenin formation was not significantly altered by the operation, but removal of vitellogenin from the plasma was delayed relative to controls. Similar investigation 3 days after partial hypophysectomy showed that this effect was caused by removal of the intracranial lobes of the pituitary (the neuro-intermediate/median/rostral lobe complex) but not by removal of the ventral lobe. None of these operative procedures was found to produce a significant effect on the rate of yolk granule synthesis. Consideration is given to the possibility that the intracranial pars distalis of the pituitary secretes a hormone which stimulates the uptake of vitellogenin by growing ovarian follicles.     

The effect of temperature on the testicular steroidogenic enzymes of the rainbow trout, Salmo gairdneri.

Testes of the rainbow trout, Salmo gairdneri, have been incubated with [³H]testosterone, [³H]androstenetrione, and [³H]pregnenolone at a range of temperatures from 1 to 37°. With testosterone and androstenetrione as substrates, yields of free 11-oxygenated androgens were maximal between 6 and 21°. Yields of steroid glucuronides however were highest between 21 and 31° with both substrates. It is suggested that one function of the high steroid glucuronide production by the teleost testis is to limit free 11-oxygenated androgen production to the environmentally favorable temperature for reproduction for the species. This may be achieved by removal of substrate and/or product as its glucuronide ester at higher temperatures. With pregnenolone as substrate, the major product was 17α,20β-dihydroxy-4-pregnen-3-one which was formed in maximal yield between 16 and 26°. With this substrate, only small amounts of glucuronides were isolated and it is suggested that this temperature/yield curve, which is very similar to that of total (free + glucuronide) 11-ketotestosterone obtained from androstenetrione, may be the characteristic curve for steroid synthesis in the absence of glucuronidation.     

The influence of mammalian and avian gonadotropins on in vitro ovarian steroid synthesis in the turtle (Chrysemys picta).

The synthesis of steroids from 7α[³H]cholesterol and 7α[³H]pregnenolone by turtle ovarian tissues in vitro was studied. Pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, estrone, estradiol 17β, estriol, and 16-epiestriol were identified as products. All estrogens were detectable in incubates of preovulatory follicular tissue, but only small quantities of estrone were found in incubates of follicular tissue from postovulatory animals and luteal tissue. The effects of mammalian and avian gonadotropins on the metabolism of tritiated precursors were studied. Both mammalian and avian LH were stimulatory when conversion of cholesterol or pregnenolone to major steroid products was examined. In particular, enhancement of estrogen biosynthesis predominated in preovulatory follicular tissue, whereas increased progestin yield was the major effect in follicular and luteal tissue from postovulatory animals. The effects of FSH were minimal compared to the same dose of LH. Thus, a slight increase in estrogen yield was only noted when preovulatory follicular tissue was incubated with cholesterol and mammalian FSH, and neither mammalian nor avian FSH had an effect on pregnenolone conversion by follicles from postovulatory animals. Prolactin had no effect on luteal progesterone synthesis when used alone, but reduced the stimulatory effect of mammalian LH on progesterone synthesis. 11-Desoxycorticosterone was not found to be a product of the turtle ovary under normal conditions or after in vitro ACTH stimulation.     

Steroidogenesis in dispersed cells of human fetal adrenal

Steroidogenesis in dispersed fetal zone cells of midtrimester human fetal adrenal was stimulated acutely by ACTH. Polypeptide hormones such as hCG, alpha MSH, ovine PRL, and LH did not produce a similar stimulation of steroidogenesis. The principal steroid products of ACTH-stimulated fetal adrenal cells were dehydroisoandrosterone sulfate, pregnenolone, pregnenolone sulfate, and 17 alpha-hydroxypregnenolone. Only minimal production of the delta 4-3-ketosteroids, cortisol, corticosterone, and progesterone, was observed. Cyanoketone (2 alpha-cyano-4,4,17 alpha-trimethyl-17 beta-hydroxyandrost-5-en-3-one; an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity) treatment of the cells caused only a minor increase in 3 beta-hydroxysteroid formation, confirming that 3 beta-hydroxysteroid formation is the principal steroidogenic fate of cholesterol in these cells. SU-10603 [7-chloro-3,4-dihydro-2-(3-pyridyl)naphthalen-1-(2H)one; a steroid 17 alpha-hydroxylase inhibitor] treatment of the cells caused a marked accumulation of pregnenolone sulfate, indicating that the C-19 steroids are produced from C-21 steroids in this tissue and possibly that dehydroisoandrosterone sulfate is synthesized directly from pregnenolone sulfate. ACTH-stimulated pregnenolone synthesis was inhibited by AY-9944 [trans-1,4-bis-(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride; an inhibitor of cholesterol biosynthesis]. Thus, cholesterol synthesized de novo was the likely steroidogenic precursor in the acute hormonally stimulated fetal adrenal cells.     

A vitellogenic hormone with a large and a small form from salmon pituitaries.

Two subfractions, with molecular weights of 25,000 and 45,000, respectively, were discovered in a fraction of chum salmon (Oncorhynchus keta) pituitary extract, which was not absorbed on concanavalin A (Con A)-Sepharose. This fraction, designated as Con AI, had been rechromatographed on Con A-Sepharose to assure exclusion of the glycoprotein (Con AII) gonadotropin. The two forms of the Con AI hormone displayed vitellogenic activity in the hypophysectomized winter flounder (Pseudopleuronectes americanus) as evidenced by their ability to stimulate the uptake of H₃³³PO₄ and [³H]leucine into the whole fish ovary and the protein fraction of the ovary, as well as the incorporation of the radioisotopes into the lipophosphoprotein fraction in vivo. The absence of any significant interference from the Con AI vitellogenic hormones in the radioimmunoassay for the salmon Con AII hormone, reinforced by an earlier finding of specificity of action of the latter hormone in the chick testicular radiophosphate uptake and immature trout gonadal cAMP production assays (D. R. Idler, L. S. Bazar, and S. J. Hwang, 1975a, Endocrine Res. Commun., 2, 215–235), provides strong grounds for the contention that two different types of gonadotropic hormones are elaborated by the chum salmon pituitary.     

Partial characterization of frog (Rana catesbeiana) hepatic glucocorticoid receptor

The binding of the synthetic glucocorticoid dexamethasone to Rana catesbeiana frog hepatic cytosol has been characterized partially. Specific binding of [³H]dexamethasone at 0° reached a maximum after 2 hr of incubation. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 5.51 × 10^?9M and a binding capacity of 1.99 × 10^?13 mol dexamethasone/mg protein. The binding was a reversible process ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5.10}\text{hr}$). Binding of [³H]dexamethasone was not affected by pretreatment of cytosol with ribonuclease A or deoxyribonuclease but was reduced drastically when cytosol was pretreated with trypsin. The receptor was specific for glucocorticoids as [³H]dexamethasone was displaced by dexamethasone, corticosterone, and cortisol to a greater extent than by aldosterone, progesterone, 17β-estradiol, and testosterone. On sucrose gradients in low salt, two areas of [³H]dexamethasone were found (3 and 6.8 S), while in 0.3 M KCl only a single peak with a sedimentation coefficient of 3 S was noted.     

Steroid biosynthesis by the ovary of the European eel, Anguilla anguilla L., at the silver stage.

The ovary of the European eel, Anguilla anguilla, at the silver stage, was incubated either as an intact tissue preparation or as a homogenate with and without cofactors in the presence of [4-¹⁴C] pregnenolone and [4-¹⁴C]progesterone. Intact tissue incubates displayed a more complex metabolite profile than reinforced homogenates, and deprivation of exogenous cofactors reduced the profile even further. Among the metabolites derived from pregnenolone, the following steroids were identified by their isopolarity and isomorphicity with standard compounds: 17α-hydroxypregnenolone; dehydroepiandrosterone; progesterone; 17α-hydroxyprogesterone; and androstenedione. The last three steroids plus testosterone, 17β-hydroxyandrostenedione, and adrenosterone were identified using progesterone as a precursor. Metopirone inhibited the formation of 11-oxygenated androgens. 11-Deoxycorticosteroids were not found, indicating the absence of steroid 21-hydroxylase activity in the eel ovary. Integration of the product yield-time curves demonstrates that in vitro the activities of the enzymes 3β-, 17β-, and 11β-hydroxysteroid dehydrogenases were less apparent than those of steroid 17α,20-C₂₁-desmolase, and 17α-, and to a lesser extent 11β-hydroxylase. Irrespective of the incubation conditions, pregnenolone produced more Δ⁵-3β-hydroxy-thanΔ⁴-3-ketosteroids, suggesting a predominance of the former biosynthetic pathway. Among the unidentified metabolites, water-soluble compounds were formed from both precursors in intact tissue incubates.     

Androgen biosynthesis by testes of the goldfish Carassius auratus in vitro: The effect of temperature on the formation of steroid glucuronides

Testes of the goldfish, Carassius auratus, were incubated with [³H]testosterone, [³H]-androstenetrione, and [³H]pregnenolone at a range of temperatures from 1 to 46°. With testosterone as substrate, high yields of the glucuronides of testosterone and 11-ketotestosterone were obtained between 26 and 31°, but formation of free 11-ketotestosterone was favored by lower temperatures (11–26°). When androstenetrione was used as substrate, the sole metabolites were 11-ketotestosterone and its glucuronide which were obtained in maximal yields of 26–31 and 31–36°, respectively. With pregnenolone as substrate, the optimal temperature for formation of glucuronides of testosterone and 11-ketotestosterone was 31–36°. Pregnenolone glucuronide was formed in significant quantities only above 41° indicating a substrate specificity of the glucuronyl transferase enzyme. We were unable to isolate either testosterone or its 11-oxygenated derivatives from the free steroid fraction, the major components of which were pregnenolone and an unidentified hydroxylated derivative of 4-pregnene-3, 11-dione which showed an optimum temperature for formation of 26–36°.