Prostaglandin-dependent in vitro stimulation of adrenocortical steroidogenesis by interleukins.

The effects of interleukins on adrenal steroidogenesis and their mode of action were studied using cultured rat adrenal cells. The addition of rat interleukin-1 alpha (IL-1 alpha) or rat IL-2 increased corticosterone levels in the medium in a concentration-dependent manner during 24 h of incubation. The minimum, half-maximum, and maximum effective concentrations of both rat IL-1 alpha and rat IL-2 were almost same (approximately 3, 10, and 100 U/ml, respectively). After a latent period, the effect became apparent after 12 h of incubation. Human IL-1 beta and human IL-6 also showed a stimulatory effect on corticosterone production, whereas human IL-2 was inactive in this system. To clarify the cellular mechanism of these stimulatory effects, we measured the levels of prostaglandin E2 (PGE2) and cAMP in the cells and media as well as the corticosterone levels. Corticosterone production stimulated by IL-1 alpha or IL-2 was accompanied by intracellular and extracellular cAMP and PGE2 accumulation. Although the stimulation of both cAMP and corticosterone was observed only after 12 h of incubation, PGE2 levels increased during the first 4 h of incubation. Corticosterone, cAMP, and PGE2 production stimulated by ILs was almost completely blocked by the addition of 0.1 mM aspirin, a cyclooxygenase inhibitor. Lipoxygenase inhibitors, i.e. AA-861, nordihydroguaiaretic acid, and 5,8,11,14-eicosatetrynoic acid, did not abolish corticosterone production stimulated by ILs. Submaximal doses of IL-1 alpha and IL-2 synergistically stimulated PGE2 production, but did not have even additional effects on cAMP and corticosterone levels. On the other hand, submaximal doses of ACTH, which did not significantly affect PGE2 levels, acted synergistically with IL to increase cAMP and corticosterone levels in these cells. These results indicate that 1) IL-1 alpha and IL-2 directly stimulate glucocorticoid synthesis in a dose- and time-dependent manner; 2) a half-maximum effective concentration of ACTH acts synergistically with IL in stimulating glucocorticoidogenesis; 3) the stimulatory process initially requires PGs, followed by the activation of the adenylate cyclase system; 4) although the profiles of steroidogenic action of IL-1 alpha and IL-2 are quite similar, they may exert their effects through different mechanisms in their early steps of PGE2 production; and 5) the low effective concentrations of both cytokines suggest possible physiological or pathophysiological roles of circulating cytokines in the glucocorticoidogenesis under certain conditions.     

In vitro uterine contractions in the viviparous lizard Tiliqua rugosa: effects of gestation and steroid pretreatment in vivo.

Uterine contractility was investigated in the viviparous lizard Tiliqua rugosa. Arginine vasotocin (AVT) induces rhythmic contractions in vitro in strips of uterine tissue from ovariectomized female T. rugosa. The strength of these contractions was related to the dosage of AVT and reduced by pretreatment in vivo with both progesterone and estradiol-17 beta. The frequency of spontaneous and AVT-induced contractions was enhanced by estradiol-17 beta pretreatment. The strength of AVT-induced contractions in pregnant females was not significantly different from that recorded in nonpregnant females. Spontaneous rhythmic contractions were present only in pregnant females. Ovariectomy did not affect either spontaneous or AVT-induced contractions in pregnant females. The data indicate that ovarian steroids modulate uterine contractility in T. rugosa. It is suggested that, following a decline in plasma progesterone levels, estrogen may be involved in the onset of parturition.     

Impact of architectural disruption on adrenocortical steroidogenesis in vitro

The adrenal cortex is an architecturally complex tissue, with cellular zonation thought to determine steroidogenesis. The impact that disruption of this tissue's architecture has on steroidogenesis in vitro, particularly adrenal androgen (AA) production, is unclear. We hypothesized that the extent of architectural disruption during tissue preparation would impact the study results. To test this hypothesis, we compared adrenocortical steroidogenesis in freshly prepared tissue slices, minces, and cell suspensions. Normal human adrenals (n = 5, three males and two females, age range 17-43 yr) were obtained at the time of organ donation. The three adrenal tissue preparations were incubated in serum-free medium with 10 microM pregnenolone substrate +/- 1 microM ACTH. The production of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol in the media were measured by radioimmunoassay. Initial time course intubations using adrenals from a single donor generally demonstrated that minces and suspensions had a greater steroid production compared with slices. In another series of 6-hr incubations using adrenals from four donors, production of dehydroepiandrosterone sulfate was found to be quite sensitive to architectural disruption, i.e. slices less than minces less than suspensions (0.88 vs. 2.1 vs. 3.0 microg/gm tissue, respectively, P < 0.0001). Alternatively, cortisol and androstenedione production was higher in minces compared with slices or suspensions (25.6 vs. 37.7 vs. 18.7 ng/gm tissue, P < 0.0028, and 254 vs. 709 vs. 456 ng/gm tissue, P < 0.0042, respectively). Production of dehydroepiandrosterone was apparently not significantly affected by the type of tissue preparation (28.2 vs. 22.2 vs. 31.2 ng/gm tissue, P < 0.297, respectively). It is unlikely that generalized tissue disruption alone accounted for the observed differences, as the trends among tissue preparations were not consistent among steroids. We conclude that the type of tissue preparation of fresh adrenal tissue impacts significantly on steroidogenesis in vitro.     

Transmembrane potentials and steroidogenesis in normal and neoplastic human adrenocortical tissue.

Trans-membrane potentials and steroidogenesis were measured in superfused slices of non-tumor and neoplastic human adrenocortical tissue. Non-tumor tissue was obtained at the time for renal transplant or from tissue removed along with tumors. Non-tumor human adrenocortical tissue had electrophysiological and steroidogenic properties similar to those of the rat and rabbit. In normal medium ACTH stimulated steroidogenesis but had no effect on the membrane potential. In K+-free medium, the cells hyperpolarized, and subsequent addition of ACTH caused depolarization. Trans-membrane potentials of adrenocortical tumors were lower than those of non-tumor cells. Ommission of K+ from the medium caused hyperpolairzation of the tumor cells, but the trans-membrane potentials did not reach the values of hyperpolarized non-tumor cells. ACTH, added to the K+-free medium, caused little or no change in membrane potential of tumor cells except in one case of a virilizing adenoma, which responded very much like non-tumor tissue. Except for the virilizing adenoma, tumor tissue slices produced little or no detectable fluorogenic steroid, even in the presence of large amounts of ACTH or cyclic AMP. The virilizing adenoma responded with increased steroidogensis to ACTH and cyclic AMP.     

Prolactin stimulates steroidogenesis by human luteal tissue in vitro

Human luteal tissue recovered from varying stages of the luteal phase was minced and incubated for 3 h and the effect of human chorionic gonadotrophin (hCG), prolactin and hCG + prolactin on progesterone and oestradiol production measured. While hCG generally enhanced both progesterone and oestradiol synthesis, prolactin alone at either 20 or 200 micrograms/l had no significant effect on steroidogenesis. When prolactin was added along with hCG in four of six corpora lutea, however, progesterone production significantly increased and in three of six corpora lutea oestradiol production was increased above that induced by hCG alone. It is concluded that prolactin may play some role in the control of steroidogenesis by the human corpus luteum.     

Identification of testosterone sulfate in the plasma of the male lizard Tiliqua rugosa

Plasma (solvolyzed and unsolvolyzed) from the male lizard Tiliqua rugosa was analyzed using gas chromatography, high-performance liquid chromatography (HPLC), and mass spectroscopy. HPLC with electrochemical detection was also used to characterize ketosteroid conjugates. Testosterone was identified in the solvolyzed plasma extract, and a compound corresponding to testosterone sulfate was detected in unsolvolyzed extracts. Concentrations were approximately 500 nmol/liter in intact male plasma, less than 100 nmol/liter in castrates, and undetectable in female plasma. Steroid glucuronides appeared to be absent from plasma. Epitestosterone conjugates were not detected, although the free steroid is found in high concentrations in T. rugosa. The presence of testosterone sulfate in the blood of T. rugosa may indicate a possible role for sulfoconjugates in controlling the availability of biologically active androgens.     

In vitro effect of hCG on steroidogenesis in the testicular tissue from a patient with complete androgen resistance.

The present study was performed to determine the in vitro steroidogenic capacity of a gonadal sample from a patient suffering from a complete androgen resistance syndrome. Testosterone and estradiol production by the testicular tissue from this patient as well as gonadotropin binding to a membrane fraction prepared from this tissue were measured. hCG bound with high affinity but with a very low capacity and the gonadotropin induced a clear dose response for both testosterone and estradiol production. The ED50 of hCG on testosterone and estradiol production were 2.5 and 5.0 nM, respectively. We conclude that estradiol originates from Leydig cell activity, since estradiol synthesis does not depend on testosterone availability and it shows a clear hCG dose response.     

Corticosteroids and control of nasal salt gland function in the lizard Tiliqua rugosa

The scincid lizard Tiliqua rugosa possesses a well-developed nasal gland composed of both mucoserous and salt-secreting cells. Confusion over its secretory capacities (see H. Saint- Girons , M. Lemire , and S. D. Bradshaw, 1977, Zoomorphologie 88, 277-288) has been resolved and NaCl- and KCl-injected animals can secrete a hyperosmotic fluid with an F/P ratio of about 3.6. The concentration of Na+ in the secretion varied from a mean of 434 mmol/liter when sodium loaded to 167 mmol/liter when potassium loaded. Potassium concentrations varied from 226 to 433 mmol/liter, respectively. Na:K ratios thus vary from 1.98 with NaCl loading to 0.42 with KCl loading, demonstrating the gland's capacity to vary the nature of the secretion. Rates of fluid production did not differ significantly between NaCl- and KCl-loaded individuals and varied from 13.3 to 19.6 microliter (100 x g hr)-1. Adjacent studies on the north African agamid lizard Uromastix acanthinurus suggested that aldosterone may influence the rate and composition of the nasal gland secretions and this possibility was investigated in Tiliqua by hormone binding studies. High affinity binding for both corticosterone and aldosterone was demonstrated during the breeding season, with a Kd of 5.2 x 10(-9) and 12.9 x 10(-9) M, respectively. Binding of aldosterone to nasal gland receptors was not evident in nonbreeding animals and the binding of corticosterone was primarily nonspecific in these individuals. These data suggest that hormone receptor concentrations and affinity vary on a seasonal basis and in concert with reproductive activity.     

The in vitro biosynthesis of epitestosterone and testosterone from C19 steroid precursors in the testis of the lizard Tiliqua rugosa

The metabolism of androgens in the testis of the lizard Tiliqua rugosa has been studied in vitro by incubating cellular homogenates with radiolabeled C19-steroid substrates. The identification 17 beta-oxidoreductase and 3 beta-hydroxysteroid dehydrogenase/isomerase activities. Aromatase, 5 alpha-reductase, and 17 alpha/beta-epimerase activities were not detected. The 17 alpha-oxidoreductase activity was temperature dependent (maximal at 32 degrees), while the 17 beta-oxidoreductase activity was temperature independent. Time yield and dual-label studies indicated that testosterone biosynthesis mainly involves the 4-ene pathway (via androstenedione), whereas the formation of epitestosterone uses both the 4-ene and 5-ene (via 5-androstene-3 beta, 17 alpha-diol) pathways. The function of alternative pathways in androgen biosynthesis is discussed, as is the role of temperature in the intratesticular regulation of androgen production.     

Plasma arginine vasotocin, progesterone, and luteal development during pregnancy in the viviparous lizard Tiliqua rugosa

The relationship between plasma levels of arginine vasotocin (AVT), progesterone, and corpus luteum formation and degeneration was studied in the viviparous lizard Tiliqua rugosa. Hormone levels were monitored in free-ranging, pregnant females which were located for sampling by means of attached radio transmitters. There was an increase in plasma AVT levels in the 30 days immediately prior to parturition. Concurrent with this event was a decline in plasma progesterone levels from relatively high levels in mid-term to basal levels prior to parturition. This is associated with degenerative changes in the corpus luteum which include pyknosis of the nuclei of the cells of the cell mass and increasing prevalence of intercellular spaces, while the thecal layer became increasingly compacted. Ovariectomy experiments indicate that the major source of progesterone during pregnancy in T. rugosa is ovarian.     

In vitro study of the effect of adenosine on frog adrenocortical cells.

Previous reports have shown that adenosine in rat inhibits both spontaneous and ACTH-induced release of corticosteroids through activation of adenosine A1 receptors. In the present study, we have investigated the possible effect of adenosine in the secretion of corticosteroids in amphibians using a perfusion technique for frog adrenocortical slices. Infusion of adenosine, at concentrations ranging from 10(-7) to 10(-4) M, had no effect on the basal output of corticosterone and aldosterone by frog interrenal cells. Similarly, adenosine did not affect the response of frog adrenocortical slices to ACTH, vasoactive intestinal peptide, or angiotensin II. The stable adenosine A1 receptor agonist N6-phenylisopropyl adenosine (PIA) was also totally devoid of effect on the spontaneous or ACTH-induced release of corticosteroids. These results show that in amphibians, adenosine does not modulate adrenal steroidogenesis.     

Control of steroidogenesis by the calcium messenger system in human adrenocortical cells

The involvement of the calcium messenger system in the control of steroidogenesis in the rat and bovine adrenal cortex has been studied extensively. However the role of these second messengers in the control of human adrenocortical function is not established. This was therefore studied by incubating collagenase-dispersed human adrenocortical cells with the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). The effects of the calcium channel blocker verapamil on basal and stimulated steroidogenesis were also studied. Both TPA (1 pmol/l-10 mumol/l) and A23187 (1 nmol/l-10 mumol/l) caused a dose-dependent increase in cortisol, aldosterone and corticosterone production. Verapamil (10 mumol/l) inhibited the increase in aldosterone, corticosterone and cortisol produced in response to ACTH(1-24), potassium, and desacetyl-alpha MSH. Unlike previous results in the rat, these effects were not specific for aldosterone secretion. The results suggest that, as in other species, calcium mobilization and protein kinase C activation have a role in the control of steroidogenesis in the human adrenal cortex. However, in contrast to the rat, these mechanisms appear to be involved in the control of steroidogenesis in both the zona glomerulosa and inner zone cells.     

Human recombinant activin-A modulates the steroidogenesis of cultured bovine adrenocortical cells.

The effect of human recombinant activin-A on adrenal steroidogenesis was studied in cultured bovine adrenocortical cells. Activin-A significantly reduced cortisol output from ACTH (10nmol/l)-stimulated adrenocortical cells incubated for 24 hours in a dose-dependent manner (10, 100 and 500ng activin-A/ml suppressed cortisol secretion by 19, 33 and 40%), although no significant effect was observed in the case of 3 h incubation. Dehydroepiandrosterone (DHEA) secretion from ACTH-stimulated adrenocortical cells incubated for 24 h was also decreased by the addition of activin-A in a dose-dependent manner. (10, 100 and 500ng activin-A/ml suppressed DHEA secretion by 22, 56 and 58%). These inhibitory effects of activin-A (100ng/ml) on cortisol and DHEA secretion were partially blocked by the addition of follistatin/FSH-Suppressing Protein (200ng/ml). In contrast, activin-A treatment resulted in no significant decrease in aldosterone secretion. There were no significant effects of activin-A on basal secretions of cortisol, DHEA or aldosterone from adrenocortical cells. These results suggest that activin-A has a direct inhibitory effect on ACTH-stimulated bovine adrenocortical steroidogenesis.     

Identification of epitestosterone in the plasma and testis of the lizard Tiliqua (Trachydosaurus) rugosa

Blood and testicular extracts of the scincid lizard Tiliqua (Trachydosaurus) rugosa were analyzed using thin-layer, column, and high-performance liquid chromatography. The major androgens isolated were epitestosterone, testosterone, and androstenedione. Epitestosterone was characterized by chromatography in several systems, and by derivative formation. Epitestosterone was further identified in testicular extracts by gas chromatographymass spectrometry. Immunoreactive material was detected in tissue extracts using an antiserum specific to epitestosterone. The maximum concentration of epitestosterone in blood, measured by radioimmunoassay, was four times that of testosterone (approximately 900 and 200 nmol/l, respectively). Epitestosterone could not be detected in the blood of intact females and the concentration of this steroid was low in castrate males. The maximum testicular concentrations (pmol/testis) were 390 (nonincubated) and 2050 (incubated) for epitestosterone, and 2025 (nonincubated) and 1040 (incubated) for testosterone. Both plasma and testicular concentrations showed considerable seasonal variation. The identification of endogenous epitestosterone confirms the results of earlier investigations using radioactive substrates. The occurrence of this steroid as a major product of the testis in T. rugosa is discussed in relation to androgens in reptiles and other vertebrates.