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The finding that thyrotropin-releasing hormone does not always act as a thyrotropin (TSH)-releasing factor in non-mammalian vertebrates has led researchers to believe that another hypothalamic factor may exhibit this function. In representatives of all non-mammalian vertebrate classes, corticotropin-releasing hormone (CRH) appears to be a potent stimulator of hypophyseal TSH secretion, and might therefore function as a common regulator of both the thyroidal and adrenal/interrenal axes. CRH exerts its dual hypophysiotropic action through two different types of CRH receptors. Thyrotropes express type 2 CRH receptors, while CRH-induced corticotropin (ACTH) secretion is mediated by type 1 CRH receptors on the corticotropic pituitary cells. The stimulating effect of CRH on both TSH and ACTH release has profound consequences for the peripheral action of both hormonal axes. The simultaneous stimulation of the thyroidal and adrenal/interrenal axes by CRH, possibly fine-tuned by differential regulation of the expression of the different CRH receptor isoforms, provides a potential mechanism for developmental plasticity.

The adrenal homolog of teleosts is not a compact organ as the adrenal glands of most vertebrates but is composed by aminergic chromaffin and interrenal steroidogenic cells located mostly inside the head kidney that, in this taxon, generally has a hematopoietic function. The two tissues can be mixed, adjacent, or completely separated and line the endothelium of the venous vessels or are located in close proximity. The chromaffin cells in some species are also present in the posterior kidney. Histological and ultrastructural work revealed cytological peculiarities of both types of cells as compared to those of other vertebrate species. In particular, the interrenal ones can show some variations in ultrastructure depending on sex, time of the year, and relation to stress events. A periodic renewal of the whole gland tissue is also sustained by some studies. Research regarding development is scanty as compared to mammals and most studies go back to the early years of the past century. The adrenal homolog of teleosts is under hormonal and neuronal control. Moreover, local paracrine interactions may play an important role in modulating a system involved in stress response and osmoregulation. Most previous studies involved a few species with the object of intensive rearing for commercial purposes; in fact cortisol, the main hormone secreted by the interrenal cells, can also influence reproduction and growth. This review summarizes data from morphocytological work and refers to other excellent reviews regarding physiology. Some of the results are compared to data available from other fishes and vertebrate classes with the aim of including them in an evolutionary and environmental framework.

Different conditions in the arrangement of the adrenal gland are observed in urodeles. The gland consists of islets scattered on the ventral surface of the kidneys, the amount, size and position of the islets varying consistently within different families and even within genera. The infraordinal variation also extends to the fine structure of the gland, as observed in 14 species belonging to 6 different families. The ultrastructural characteristics of chromaffin cells and their relationships with interrenal cells appear to be related to the phyletic position. In primitive urodeles (Sirenidae, Proteidae) the chromaffin cells are isolated or in small groups, mostly separated from interrenal cells and often in contact with renal cells. In neourodeles (Amphiumidae, Ambystomidae, Salamandridae, Plethodontidae) the chromaffin cells appear generally grouped and intermingled with steroidogenic cells. Some cytological characteristics of chromaffin cells, such as nerve supply and the shape and electron density of chromaffin granules exhibit a variability related to phyletic position.

An in vitro system for the incubation of interrenal tissue (head kidney fragments) from coho salmon, Oncorhynchus kisutch, was developed in order to examine changes in interrenal sensitivity to ACTH1-24 during smoltification, using cortisol secretion as the endpoint. Time-course studies indicated that maximal cortisol accumulation in incubation media was achieved after 3 hr exposure to ACTH. There was no correlation between head kidney weight, body weight, or sex and the response of the interrenal to ACTH1-24 in vitro. Approximately monthly or bi-weekly experiments were performed during the smoltification period (February-July): tissue was preincubated in hormone-free media for 3 hr, washed twice, and then challenged with 5 X 10(-10)-5 X 10(-7) M (1.5-1500 ng/ml) ACTH1-24 for 3 hr. The pattern of cortisol secretion was similar in February, early March, and late March in the dose range of 5 X 10(-10)-5 X 10(-8) M ACTH1-24. A marked, significant increase in sensitivity to ACTH and in the steroidogenic capacity of the tissue occurred in April, but by May the response was similar to that in the pre-April period. Enhanced sensitivity and steroidogenic capacity were found in interrenal tissue taken from coho salmon in June and July. Maximal in vitro responsiveness of interrenal tissue to ACTH in April was correlated with peak plasma thyroxine titers and enhanced hypoosmoregulatory ability, but not with peak plasma cortisol titers, which occurred in May.

Vargas and Concha [Vargas F., F., and Concha B., J. (1957), Invest. Zool. Chil. 3, 81–87; 88–95] described some encapsulated glands in the posterior part of the kidneys of the Chilean clingfish, Sicyases sanguineus , and called them the interrenals. Present investigations showed that the surgical removal of these glands consistently produced hypercalcemia. No significant effects were seen in plasma sodium, potassium, and magnesium levels and in percentage hematocrit and body weight change, as was once reported previously in killifish [Pang, P. K. T., Pang, R. K., and Griffith, R. W. (1975). Gen. Comp. Endocrinol. 26, 179–185]. We conclude that these glands are the Stannius corpuscles and not the interrenals of this fish. Histological studies showed the high vascularization and the follicular arrangement of cells.

1. 1. Secretion of cortisol by the interrenal tissue of the trout Salmo gairdneri was studied in vitro by a perifusion method in relation to the effects of electrolyte concentrations in the medium. 2. 2. An increase in osmotic pressure (produced by adding mannitol or NaCl) induced an immediate, but brief augmentation in cortisol release. 3. 3. Suppression of Na+ had no effect while its reintroduction in the medium led to stimulation of hormone release. By contrast, a sharp peak was obtained whenever Cl − concentration was dropped (by 50 mM fractions). These opposite effects of Na+ and Cl− when they vary independently of each other is interpreted with regard to osmoregulation and acid-base regulation. 4. 4. Raising K+ even to high levels (up to 20 mM) produced no change. 5. 5. The absence of Ca2+ had no obvious effect while its addition induced an immediate peak of cortisol release. In addition, external Ca2+ proved necessary for the action of ACTH to occur. 6. 6. These results establish that cortisol release in trout may be directly affected by changes in electrolyte concentrations in the extracellular space.

A perifusion system technique has been used to compare the effects of metyrapone (SU-4885; Ciba) and aminoglutethimide, two compounds which are known to inhibit corticosteroid biosynthesis in mammal adrenal glands, upon aldosterone output by frog interrenal tissue. Rana ridibunda adrenal fragments were continuously perifused for 13 hr with amphibian culture medium. Fractions were set apart every 5 min and aldosterone levels were measured by means of a specific and sensitive radioimmunoassay method. For doses ranging from 1 × 10−6 to 1 × 10−3 M, metyrapone induced a decrease in aldosterone secretion rate. The infusion of three equimolar doses (1 × 10−5 M)_of metyrapone during three consecutive periods of 1 hr or during various times (30, 60, 150 min) within the same experiment made it possible to study the kinetics of the response of the glands. The lag period (10 min), the amplitude of the inhibition (75%), and the duration of the inhibitory effect after withdrawal of metyrapone (50 min) were almost uniform, whatever the duration of the infusion. Similar patterns were obtained using aminoglutethimide, although much higher doses (up to 100-fold) were required to inhibit aldosterone production. These results demonstrate that both metyrapone and aminoglutethimide are potent inhibitors of mineralocorticoids output in amphibia. Conversely, they suggest that a single injection of each inhibitor, in vivo, would only induce a transient and light diminution in corticosteroid secretion.

The anterior pituitary gland, testes and interrenal glands of a series of young males of the teiid Cnemidophorus l. lemniscatus (L.) have been studied by light microscopy in order to correlate the changes occurring during sexual maturation. In the testes of the smallest animals, spermatogenesis does not progress beyond primary spermatocytes and there is no differentiated interstitial tissue. In medium-sized animals, spermatids and some interstitial cells appear, and in the largest lizards, spermatogenesis is completely established and Leydig cells abound. Simultaneously with the development of the testes, interrenal glands undergo great hypertrophy and hyperplasia, especially in the peripheral reactive zone. Starting in animals of intermediate size, the anterior hypophysis exhibits a considerable hypertrophy of two rostral cell types: the chromophobic corticotrophs and the acidophilic PAS-positive cells considered to be interstitiotrophs. These cells show large, vesicular nuclei and prominent nucleoli, signs of enhanced cellular activity. The hypertrophy begins in the dorso-rostral region of the gland close to the median eminence, at the site of entry of the portal vessels. This suggests a hypothalamic influence on the function of these pars distalis cells. The scattered basophilic gonadotrophs or folliculotrophs are scarce, small, and do not vary appreciably among the animals studied. The hyperactivity of corticotrophs may account for enlargement of the interrenal glands. Testicular development is apparently related to an increased activity of interstitiotrophs but to a stable level of activity in folliculotrophs.

The presence and the distribution of corticotropin producing cells in the adenohypophysis of Rana esculenta has been demonstrated by immunohistological means. In untreated and control animals alike, fluorescent cells were localized (a) in the rostroventral part of the pars distalis around portal capillaries; (b) in almost all of the pars intermedia. Daily Metopirone injections brought about a decrease in the number of pars distalis fluorescent cells (Day 3) and eventually disappearance (Day 7). Metopirone had no marked influence on intermediate lobe fluorescence. Twenty-four hours after adrenalectomy, the number of anti-ACTH binding cells decreased about 30 or 40%. These cells totally disappeared 4 days after adrenalectomy. In the intermediate lobe, the number of anti-ACTH binding cells was almost identical to that in the control animals. These results suggest that a polypeptide, immunologically similar to mammalian ACTH, is simultaneously secreted in both pars distalis and pars intermedia cells in the frog pituitary. They also point out the existence of a pars distalis-adrenal feedback mechanism.

Adrenocortical tissue from the freshwater teleost, Coregonus clupeoides , was incubated with [ 14 C] acetate and [ 3 H] 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 [ 14 C] products were significantly less so. The addition of Coregonus pituitary extract to the incubation medium caused both a release of [ 14 C] steroid from the bound condition and a significant decrease in the yield of [ 14 C] 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 [ 14 C] acetate, but not by late precursors such as [ 3 H] 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.

1. 14 C-labelled isoxazole was utilized to locate the pronephroi as probable sites of 3 β -hydroxy-steroid dehydrogenase activity in Myxine . 2. Histological investigation revealed presumptive interrenal cells in the pronephroi. 3. The numbers of presumptive interrenal cells increased significantly after injection with ACTH. 4. A fluorescent anti-body technique for localizing ACTH and injection of radioactive-iodine labelled ACTH tended to confirm the pronephric tissue as a target organ for ACTH.

Adaptation of brown trout to a black or white background had no statistically significant influence on the activity of the pituitary-interrenal axis in unstressed fish, in fish under conditions of mild chronic stress, or in fish under conditions of severe, acute stress. This finding is in contrast to several other studies on salmonid fish in which background color adaptation has been shown to modulate the pituitary-interrenal response to environmental stress. It is argued that these differences in results may be related to the prior history of interrenal activation in the experimental fish and that in previously unstressed brown trout the activity of the pituitary-interrenal axis is independent of melanotroph activity in the pars intermedia when fish are exposed to subsequent stress.

We determined the in vitro effects of changes in extracellular monovalent ion levels and osmotic pressure on the spontaneous and adrenocorticotropin (ACTH)-stimulated interrenal activity of coho salmon (Oncorhynchus kisutch). We used a perifusion system of incubation and monitored interrenal activity by measuring the effluent cortisol content with a radioimmunoassay. An increase in the medium osmolality with mannitol, from 206 to 290 or 353 mosmol, caused an increase in the spontaneous release of cortisol only slightly (compared with the much greater increase induced by porcine-ACTH). A similar minor increase was observed when NaCl was elevated from 130 to 180 mM. On the other hand, the spontaneous release of cortisol was not affected by increasing the KCl level from 3.2 to 9.6 mM, but was clearly increased when KCl was raised from 3.2 mM to a supraphysiological level of 27.2 mM. Ionic or osmolality changes, within the physiological range observed in coho salmon plasma, did not affect the characteristics of interrenal secretion of cortisol in response to porcine-ACTH. If our results with interrenal cells in vitro are representative of the basic functioning of the cells in vivo, then one would have to conclude that changes in concentrations of plasma monovalent ions or in osmotic pressure may not play a significant physiological role in the regulation of interrenal steroidogenesis or corticosteroid release in coho salmon.

To investigate the possible direct effect of two aldosterone antagonists (Canrenoate-K and Prorenoate-K) upon mineralocorticoid biosynthesis a perifusion system technique has been developed. Frog interrenal tissue was selected for its ability to secrete huge amounts of aldosterone (twice as much as corticosterone in resting conditions). Throughout the experiment, secretion of aldosterone was measured every ten minutes by means of a sensitive and highly specific radioimmunoassay method. Increasing concentrations of both Canrenoate-K and Prorenoate-K (ranging from 10(-4)M to 10(-3)M) caused a dose-related inhibition of aldosterone output. At a dose of 3.16 x 10(-4)M, Prorenoate-K appeared to be somewhat more potent (57.8% inhibition) than Canrenoate-K (47.8% inhibition). Infusion of both Canrenoate-K and Prorenoate-K at a dose of 5 x 10(-4)M during 1 or 2 hours induced a similar sharp decrease in mineralocorticoid secretion. Thus, it appears that Canrenoate-K and Prorenoate-K beside their well known effects at renal tubular receptor sites do also inhibit aldosterone biosynthesis. These results indicate that in vivo administration of aldosterone antagonists may first involve a transient decrease in aldosterone secretion. Furthermore, they suggest that mineralocorticoid biosynthesis might be regulated by a short loop feedback mechanism.

Previous studies have demonstrated the presence of glucocorticoid receptors in the adrenal gland and suggested a direct inhibitory effect of glucocorticoids on the adrenal cortex. The present study was designed to investigate the possibility that dexamethasone could inhibit adrenal steroidogenesis in amphibia. For this study, we have applied a well-defined perifusion model, using interrenal fragments from frogs ( Rana ridibunda ). The amounts of corticosterone and aldosterone released in the effluent perifusate were radioimmunoassayed by means of specific antisera which did not cross-react with dexamethasone. Administration of dexamethasone (10 −6 and 10 −6 ) did not significantly alter the basal secretion of corticosterone and aldosterone. In addition, dexamethasone (10 −5 ) did not modify the response of the interrenal gland to graded doses of ACTH. Finally dexamethasone did not inhibit the stimulation of corticosterone and aldosterone production induced by a single infusion of the angiotensin II analogue [Sar 1 -Val 5 ] All. From these results, it is concluded that in amphibia, chronic dexamethasone administration does not exert a direct suppressive effect on adrenal secretion.

Interrenal function was studied in the freshwater turtle Chrysemys picta by competitive protein-binding analysis of plasma corticosterone in vivo and corticosterone synthesis by interrenal cell suspensions in vitro. Although circulating levels of corticosterone in intact, unanesthetized turtles (0.22 μg/100 ml) are lower than in other vertebrates, hypophysectomy reduced and ACTH increased these levels. Synthetic ACTH1–24 was more potent in vivo than pACTH, and in vitro 10–1000 pg/ml gave a log dose increase in corticosterone production. Interrenal cell suspensions were also responsive to crude homogenates of turtle pituitary. Bleeding was a potent stimulus to interrenal secretion in vivo mediated in part via endogenous corticotropin release.

The existence of seasonal changes in the secretory activity of the interrenal glands in European Green frogs has been investigated in vivo, in a natural environment, and in vitro, using a perifusion system technique. In vivo, plasma corticosterone levels have been systematically measured at six different moments during a 24-hr period (03, 07, 11, 15, 19, and 23 hr), for almost all the months, during 2 consecutive years. In this part of the study, corticosterone concentrations were assayed in duplicate, in an average of seven animals at each of the six definite moments of the 24-hr cycle. Significant fluctuations in circulating corticosterone have been recorded: a single peak in late spring (May and June) coinciding with the spawning period; a decrease during summer and autumn; the nadir in the depth of winter (February); and a sharp increase at the emergence from hibernation (end of March). The amplitude of the fluctuations ranged from 0.28 ± 0.07 μg/100 ml in late February to 2.68 ± 0.36 μg/100 ml in late May. No significant difference in plasma corticosterone concentrations was observed between males and females. The in vitro studies consisted of comparing the responses of perifused frog interrenal tissue to increasing doses of homologous pituitary extracts, at various times of the year. According to the date of the experiment, important differences in maximum corticosterone snd aldosterone secretions were recorded. As an example, under submaximum ACTH stimulations, 188 and 193% increases in corticosterone and aldosterone outputs, respectively, were recorded on June 4th, whereas the corresponding figures on October 17th were 38 and 54%. The maximum capacity of interrenal tissue to secrete corticosterone and aldosterone after stimulation by homologous pituitary extract was observed in May and June, whereas the minimum capacity was recorded in winter. These results are in complete agreement with our in vivo data. From the present results, we conclude that corticosterone production undergoes seasonal fluctuations in European Green frog. Since the fluctuations cannot simply originate from variations of ambient temperature (F. Leboulenger, C. Delarue, M. C. Tonon, S. Jegou, and H. Vaudry, 1978, Gen. Comp. Endocrinol. 36, 327–338), our in vitro results support the view that plasma corticosterone rhythms are due, at least in part, to seasonal variations of interrenal sensitivity to ACTH.

The presence of cholinergic nerve terminals was demonstrated in the interrenal gland of the lizards Lacerta dugesi and Lacerta pityusensis. Terminals were more numerous in the subcapsular than in the central zone.

The fine structure of interrenal cells in the salamander, Hynobius nebulosus, from prometamorphic larvae to its adult stage, was observed with electron and light microscopes. In all the animals examined the interrenal cell clusters were located at the medial edge of the ventral surface along the total length of each kidney, suggestive of a primitive nature. In larval salamanders, the interrenal cells contained small to moderate numbers of lipid droplets, and their cytoplasm was filled with a tubular network of smooth endoplasmic reticulum (SER) and numerous mitochondria with tubulo-vesicular cristae. In the adult and young adult salamanders, however, most interrenal cells were filled with lipid droplets, so that both tubular SER and mitochondria were decreased in amount in inverse proportion to the increased lipid droplets. These mitochondria frequently contained a crystalloid structure composed of closely packed tubules which were continuous with the cristae. These findings suggest that the interrenal cells in the larvae are more active than those in the adult or young adult salamanders. In addition, the interrenal cells at the end of metamorphosis contained a greater number of enlarged mitochondria with loosely distributed tubular cristae and with a less dense matrix and more numerous membrane-bounded dense bodies, 0.1-0.3 micron in diameter, than those at the prometamorphic stage. These findings suggest that the cells are their most active at this stage. In the prometamorphic larvae, bundles of filaments frequently occurred in the cytoplasm, especially around the nucleus. These filaments gradually decreased in number with the advance of age, and in young adult salamanders they appeared only occasionally.

SUMMARY The effect of ACTH on cortisol concentration in the plasma of the carp has been studied; cortisol was determined using a competitive protein-binding radioassay. Blood was sampled at 12.00 h (the diurnal minimum) by cardiac puncture. The plasma cortisol level of undisturbed carp (blood obtained 1–3 min after netting) was 46 ± 14 (s.e.m.) ng/ml. One hour after injection of ACTH (1·0 i.u./100 g body wt., i.m.) the level of cortisol increased to 415 ± 62 ng/ml and reached a maximum of 656 ± 62 ng/ml after 2 h. The level of cortisol in saline-injected control carp after 2 h was 72 ± 33 ng/ml. A transient rise of cortisol was noted in both groups 30 min after injection (315 ± 51, 315 ± 62 ng/ml). This was attributed to a surge of endogenous ACTH, presumably due to handling. Carp head kidneys were superfused in vitro. They released cortisol spontaneously, but the releasing rate declined exponentially and reached a minimum after 135 min. Addition of ACTH to the medium was followed by a prompt increase in cortisol release. The rate of cortisol release was maximal 30–45 min after addition of ACTH and was identical to the rate at the beginning of the superfusion.

Acute exposure of brook trout to acid water (pH 4.0) caused a significant decrease in interrenal (adrenocortical cell) nuclear diameter, concomitant with an increase in the intensity of nuclear staining. Cytochemical analyses of Feulgen-DNA (F-DNA) of interrenal cell nuclei revealed these cells to be diploid (2C) and nonreplicating. There was no loss in DNA evidenced in interrenal nuclei of acid-stressed trout. In the same fish, there appeared to be a progressive decrease in azure B-RNA levels measured cytophotometrically over a 3- to 24-hr-exposure interval. A marked but transient elevation in circulating cortisol levels occurred at 1–3 hr of exposure with a return to control levels after 24 hr of exposure. No visually discernible changes in Sudan black B staining for lipids were evidenced after acute acid exposure. The findings support the conclusion that there is a marked inhibition of RNA synthesis and presumably of steroidogenesis in interrenal tissue following acute exposure of trout to low pH.

In the adrenal chromaffin tissue of the tropical teiid lizard Cnemidophorus l. lemniscatus, adrenaline-storing cells are present together with noradrenaline-storing cells in the peripheral layer and its expansions, and exclusively constitute the central islets, but in any case they only occur in close contact with the interrenal tissue. Such a distribution gives support to the idea that a high concentration of corticosteroids is needed for the differentiation of adrenaline-storing cells.

Summary In order to elucidate the role of prostaglandins of the E and F series on adrenal steroidogenesis, we have studied corticosterone and aldosterone production by frog interrenal. Rana ridibunda interrenal dice were perifused with amphibian culture medium for ten hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. Perifusion of interrenal fragments with increasing concentrations of PGE 1 and PGE 2 (ranging from 8.8 nM to 2.8 μM) led to a dose-related increase in both corticosterone and aldosterone biosynthesis, the magnitude of the stimulation being 1.3 fold higher for aldosterone than for corticosterone. High concentrations of PGF 2α (2.8 μM) were only responsible for a slight increase in corticosteroid biosynthesis while PGF 1α was almost inactive. Indomethacin an inhibitor of prostaglandin biosynthesis caused a marked decrease of spontaneous production of corticosterone (−84%) and aldosterone (−75%) but did not alter the stimulation of steroidogenesis induced by ACTH. From these data, it was concluded that 1) exogenous prostaglandins control corticosteroid production in amphibia ; 2) endogenous prostaglandins are required for spontaneous biosynthesis of corticosteroids ; 3) endogenous prostaglandins are not involved in ACTH-induced steroidogenesis.

1. Experiments were undertaken to examine the role of various endocrine glands in regulating plasma solute concentrations in the euryhaline elasmobranch, Dasyatis sabina , exposed to various environmental dilutions. 2. Hypophysectomy (with the exception of the ventral lobe of the proximal pars distalis) resulted in a depression of plasma osmolarity; this reduction was due primarily to lowered urea levels. 3. Removal of the rostral lobe of the pars distalis in Dasyatis caused a significant increase in plasma osmolarity, urea and sodium concentrations. Treatment of rostral lobectomized stingrays with mammalian prolactin reversed the effects of this operation, reducing blood urea and sodium levels. Pituitary prolactin activity (measured using the Gillichthys xanthophore assay) increased approximately 100-fold within 24 hr after stingrays were transferred from sea water to 33% sea water, lending further support to the hypothesis that a prolactin-like hormone functions in osmoregulation. 4. Mammalian ACTH had no apparent effect on plasma solute levels in rostral lobectomized animals. ACTH did, however, reverse the effects of prolactin in rostral lobectomized animals when these hormones were given simultaneously. 5. Plasma osmolarity was significantly reduced by interrenalectomy. 6. Thyroidectomy resulted in an elevation of plasma urea levels; replacement therapy with thyroxine reversed the effects of thyroidectomy. 7. These data indicate that pituitary (particularly a prolactin-like hormone), interrenal and thyroid hormones are involved in the osmotic adaptation of this euryhaline elasmobranch to dilute environments.

The role of prostaglandins (PGs) in calcium-induced corticosteroid secretion by frog adrenal (interrenal) gland examined in vitro using a perifusion technique. Increasing concentrations of CaCl 2 (4–10 mM) stimulated in a dose-dependent manner aldosterone, PGE 2 and 6-keto-PGF 1α production, whereas TXB 2 was not affected. The kinetics of the adrenal response to CaCl 2 indicated that the increase in PG output always preceded that of steroid. Administration of cobalt (4 mM), a calcium-channel inhibitor, blocked the calcium-induced stimulation of PGs and corticosteroids. Infusion of indomethacin (5 × 10 −6 M), a specific cyclooxygenase inhibitor, significantly decreased the basal production of PGs and steroids, and prevented the stimulatory effect of CaCl 2 (6 mM). Infusion of the calcium ionophore A 23187 (10 −6 M), for 20 min, induced a marked stimulation of PG and steroid production. Taken together, these data support the notion that biosynthesis of prostaglandins is associated with calcium-induced corticosteroid secretion in frog adrenal cells.

Plasma corticosterone concentrations were low in premetamorphic tiger salamander larvae (Norman Stage I; M. F. Norman (1985) Anat. Rec. 211 , 102–109). Corticosterone levels were significantly elevated at midmetamorphosis (Norman Stage IV) but decreased at the end of metamorphosis (Norman Stage VII). Corticosterone levels remained low 2 weeks after metamorphosis. Interrenal 3β-hydroxysteroid dehydrogenase activity was low in premetamorphic larvae (Norman Stage I) but was significantly elevated by midmetamorphosis (Norman Stage IV) and remained elevated at the end of metamorphosis (Norman Stage VII). There were no significant changes in interrenal cell nuclear size during metamorphosis. There was a significant decrease in body weight as well as a significant increase in hematocrit accompanying metamorphosis. The increase in plasma corticosterone concentration seen during metamorphosis of the tiger salamander is accompanied by an increase in interrenal steroidogenesis.

In order to investigate a possible role of atrial natriuretic factor (ANF) in the control of corticosteroid biosynthesis in amphibians, we have examined the effect of synthetic ANF (Arg 101-Tyr 126) on perifused frog interrenal slices. ANF did not affect the spontaneous secretion of corticosterone and aldosterone. In contrast, ANF (10 −6 M ) inhibited ACTH- and angiotensin II-stimulated corticosteroid production. ANF was more potent in suppressing aldosterone than corticosterone secretion. Immunocytochemical studies using a specific ANF antiserum revealed the presence of ANF-like immunoreactive fibers in the vicinity of interrenal cells. It is thus proposed that, in amphibians, both “hormonal” ANF secreted by myocytes and “neurohormonal” ANF delivered by peptidergic nerve terminals coursing among interrenal cells may partake in the regulation of corticosteroidogenesis.

Δ5-3β-hydroxysteroid dehydrogenase activity (Δ5-3β-HSD) has been studied in the interrenal glands and the seminal vesicles of intact and castrate male catfish. The intensity of the enzyme reaction is about equal in the seminal vesicles of both groups, whereas the intensity of enzyme reaction in the cortical cells is more pronounced in the castrate than in the intact male. The responses of the interrenal glands and the regressed seminal vesicles of the castrate catfish in the postspawning period to exogenous luteinizing hormone (LH), Δ4-androstenedione (Δ4-androst.), desoxycorticosterone acetate (DCA) and hydrocortisone acetate (HA) (experiment 2), and the effect of the LH, Δ4-androst., DCA and HA, and prolactin (LtH) and growth hormone (STH), singly or in combination on the regressed seminal vesicles of the castrate catfish (experiment 3) have also been studied. The data indicate that both LH and Δ4-androst. induce secretory activity in the seminal vesicles but they appear to act through different routes. Since LH brings about an increase in the nuclear diameters in cortical cells and Δ4-androst. does not, it is felt that LH acts through the cortical cells of the interrenal glands while Δ4-androst. acts directly on the seminal vesicles. Further, Δ4-androst. can synergize with LtH and STH in inducing secretory activity in the seminal vesicles, whereas DCA and HA do not. These results, coupled with the fact that Δ5-3β-HSD activity is more marked in the cortical cells of the castrate than in the intact catfish, strongly suggest that following castration, the resulting high titers of gonadotrophin act on the interrenal cortical cells to produce androgens which, in turn, induce hyperactivity in the seminal vesicles.

Adult male common frogs (Rana temporaria) were exposed to increased temperatures (10 and 26°C) and various tissues and organs were examined histologically two and seven weeks later. Appropriate control tissue was obtained from hibernating animals. The following tissues and organs were examined: pars distalis of the adenohypophysis (acidophils type 1, basophils types 2 and 3); interrenal gland; interstitial tissue (Leydig cells) and germinal epithelium of the testis.

The cortical cell of the interrenal gland of the American bullfrog, Rana catesbeiana, was examined in the electron microscope. These cells occur in small groups and cords and are quite irregular in shape. The cortical cell is reminiscent of adrenal cortical cells from other vertebrates. Liposomes are variable in size and density. Smooth endoplasmic reticulum is scant in amount and predominantly of the fine tubular type. Mitochondria have vesicular cristae, a dense matrix, and occasionally have blebs, vacuoles, and myelin-like whorls at their surfaces. Intimate morphological relationships are found among the Golgi apparatus, lysosomes, and liposomes, and among Golgi vacuoles, mitochondria, and liposomes. In addition microfibrils are a prominent feature of the cortical cell. The biochemical events of steroidogenesis in amphibia and other vertebrates are discussed in relationship to the organellar interrelations found in the bullfrog interrenal cortical cells. Based on the available chemical and morphological information a scheme is proposed of movement of the steroidal intermediates through the cell that tentatively identifies the localizations of the various enzyme systems involved in corticosteroidogenesis from acetate to corticosterone and aldosterone.

Seasonal variation in the thyroid and interrenal glands of the lizard Leiolopisma rhomboidalis was noted over a period of 12 months. Material was collected in the tropical region of north-eastern Australia. Least seasonal variation was noted in the thyroid and interrenal glands of the nongravid females. Gravid females exhibited greater variation than nongravid females and less than the males in both endocrine organs studied. The cold season (May–August) was the period of least activity of both thyroid and interrenal glands. Greatest thyroid activity in the males occurred during the hot, dry season (September–December). Greatest interrenal activity of the males occurred during the hot, wet season (January–April). Thyroid activity seems related to reproductive activity-yolk maintenance in the females, and copulation and territory defense in the males. Interrenal activity also, to a lesser degree, appears related to reproduction in this lizard. Without experimental work it is impossible at present to establish more positive interrelationships between thyroid and interrenal gland activity.

SUMMARY Incubation of the adrenocortical tissue of Xenopus with labelled steroidal precursors gave aldosterone, corticosterone, 11-deoxycorticosterone and 18-hydroxycorticosterone as the major products. Kinetic studies indicated that the major biosynthetic pathway in Xenopus is the same as that in the duck, cobra and frog, and follows the sequence: pregnenolone → progesterone → 11-deoxycorticosterone → corticosterone → aldosterone + 18-hydroxycorticosterone. The effects of adenohypophysectomy and of injection of mammalian corticotrophin (ACTH) on the corticosteroidogenetic capacity of Xenopus were also studied. The production of both aldosterone and corticosterone increased after ACTH administration and decreased markedly after adenohypophysectomy. Some of the evidence suggests that ACTH was effective at biosynthetic stages following the formation of pregnenolone. The present studies on corticosteroid production were done in conjunction with parallel investigations on osmoregulation and electrolyte and water changes in Xenopus (Edwards, 1969).

Histological and histochemical changes have been studied in the interrenal tissue of the male common frog, Rana temporaria, following ACTH administration and ablation of the pars distalis of the pituitary. A combination of the following parameters appeared to be useful for the determination of interrenal activity in histological material: Δ 5-3β-Hydroxysteroid-dehydrogenase and glucose-6-phosphate-dehydrogenase activity, determined histochemically, were less useful in the determination of interrenal activity. ACTH administration and hypophysectomy invariably affected the entire interrenal gland. This seems to indicate that in the common frog the whole interrenal depends on pituitary ACTH and that a zonation, as seen in the mammalian adrenal cortex, is absent in Amphibia.

The effects of metopirone and aldactone on the histology of the pars distalis of the pituitary, the interrenal gland and the interstitial tissue of the testis were studied in male common frogs. Activation of the interrenal tissue by these substances was paralleled by a stimulation of one of the tinctorial cell types in the distal lobe namely the basophils 3 and of caudal chromophobic cells. Simultaneously a regression of the interstitium testis was observed. It is suggested that either the basophils 3 or the caudal chromophobic cells have a corticotropic function.

El estudio histofisiologico de la glandula interrenal de un lagarto teido (Cnemidophorus l. lemniscatus) permitio poner de manifiesto la que se denomino zona reactiva de la glandula. Por su situacion es periferica, y esta constituida por celulas dispuestas en formaciones redondeadas. Estas celulas, que tienen normalmente aspecto de menor actividad, responden mas rapida e intensamente que las centrales a la accion de la corticotrofina, exogena o endogena, y aparecen entonces mas activas que las ultimas.

In order to determine the role of intermediate filaments in adrenal steroidogenesis, we have studied the effect of IDPN (beta-beta'iminodipropionitrile), an intermediate filaments perturbing agent, on corticosteroid secretion by frog interrenal glands in vitro. A 6-h administration of IDPN (10(-3) M) did not affect the spontaneous release of corticosterone and aldosterone. While IDPN did not alter the response of adrenal fragments to ACTH, the drug caused a marked decrease in angiotensin II-induced stimulation of corticosterone and aldosterone production. These results indicate that, in contrast to microfilaments, which play an important role in spontaneous steroidogenesis, intermediate filaments are not required for basal corticosteroid secretion but are involved in the mechanism of action of angiotensin in frog adrenocortical cells.

Partial hepatectomy was carried out on Xenopus laevis to investigate its influence on the endocrine system. In addition to other endocrine effects, a marked hypertrophy and stimulation of the interrenal gland was observed. Activated cells contain mitochondria with extended and irregularly coiled tubules embedded in a low electron dense matrix. Hepatectomy induces two phases of proliferation [3 and 35 days postoperative (p.o.)]. After 106 days p.o. giant mitochondria possessing narrow and closely packed, parallel tubules surrounded by an electron dense matrix indicate a phase of inactivation. The smooth endoplasmic reticulum and the Golgi apparatus proliferate after hepatectomy. During activation the high lipid content seen in controls in decreased significantly.

A superfusion system was developed in which secretion of cortisol from the interrenal tissue of Sarotherodon aureus could be stimulated by ACTH. Head kidneys from four fish were cut into fragments and superfused with Eagle's basal medium containing 4 mm-NaHCO3 and bovine serum albumin (50 mg/100 ml). The superfused medium was collected every 15 min before stimulation and every 5 min thereafter. Cortisol was measured in the medium by radioimmunoassay. The rate of cortisol secretion increased considerably after a 5 min pulse of 0·1 mu. porcine ACTH/ml, reaching a peak of 11·64 ± 2·40 (s.e.m.) ng/min (n = 5) after 20 min. In the subsequent 35 min the secretion rate decreased to a level of 0·49 ± 0·21 ng/min which was within the range of the baseline (0·2–0·7 ng/min). The amount of cortisol secreted by the superfused tissue in response to the corticotrophin, calculated from the area under the peak, was dose-dependent at the range of 0·06–273 mu. ACTH. Cortisol secretion in this system could also be stimulated by a crude pituitary extract from the same fish. Using the dose–response line, the adrenocorticotrophic activity in the pituitary gland of S. aureus was estimated as 0·2 i.u./g or 1·87 mu. per gland, in porcine ACTH equivalents. It was possible to substitute ACTH with dibutyryl cyclic AMP. The response of the interrenal tissue, i.e. the increase in secretion of cortisol, was dose-dependent at the range of 2–20 mmol/l. The response of the superfused interrenal tissue to ACTH could be extended by the addition of the synthetic phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (0·1 mmol/l), to the medium. These results indicated that the stimulation of cortisol secretion from the interrenal tissue of this fish by ACTH is, most probably, mediated by cyclic AMP as a second messenger.

Although interrenal tissue from the intact duck does not show a clear zonation when examined by light microscopy, the tissue does develop a well defined zonation following exposure to high and low levels of corticotropic stimulation. Under these conditions clear ultrastructural differences are seen between cells of the subcapsular zone (SCZ) and the inner zone (IZ). Based on these observations, the ultrastructure of tissue from intact birds was examined retrospectively and in addition, cell sizes and the relative volumes and areas of intracellular components were measured morphometrically. These analyses reveal morphological and quantitative differences between cells from the IZ and the SCZ. Cells of the IZ have small rounded nuclei, extensive smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae, whereas cells from the SCZ contain pleomorphic nuclei, less SER and mitochondria with shelf-like cristae. The mean cell volume in the IZ is significantly less than that in the SCZ. In the SCZ cells the volume densities (volume per unit volume cytoplasm) of mitochondria and lipid droplets and the surface densities (area per unit volume cytoplasm) of the outer mitochondrial membranes are significantly greater than those in IZ cells. Conversely, in the cells of the IZ the volume densities of the nuclei and dense bodies and the surface density of the SER are greater than the corresponding values estimated for the cells of the SCZ. Although the mitochondria comprise a smaller fraction of the mean volume of IZ cells than SCZ cells, the total surface area of the cristae is approximately the same in the cells of both zones.

As reported for several other avian species there are clearly distinguishable subcapsular (SCZ) and inner (IZ) zones of interrenal tissue in the Japanese quail. The SCZ contains large columnar cells (type I) with rounded nuclei, polymorphic mitochondria with shelf-like cristae, and relatively small numbers of lipid droplets. The IZ contains two and possibly three types of cells. Type II consists of large columnar cells with moderately dense cytoplasm containing large numbers of lipid droplets and many rounded mitochondria with tubular cristae. Smooth endoplasmic reticulum (SER) and Golgi apparatus are well developed; coated vesicles occur in the Golgi area and at the cell surface. Type-III cells occur in IZ and especially in its more peripheral areas. They are columnar cells with strikingly clear cytoplasm (in comparison with type II) containing mitochondria with plate-like cristae and tubular SER. Type-IV cells are sparsely distributed in IZ and occur rarely in SCZ. Type IV may be a degenerating phase of type III. After adenohypophysectomy or section of portal vessels type-I cells atrophy somewhat with a decrease in lipid droplets; type-II cells, also atrophy with conspicuous increase in size and number of lipid droplets, enlargement of mitochondria, and gradual disappearance of SER; type-III cells decrease in number whereas type-IV cells increase. After injection of ACTH, type-I cells enlarge and their mitochondria, SER and Golgi apparatus become more conspicuous; there is a decrease in lipid droplets in type-II cells and a development of SER, polysomes and Golgi apparatus; there is also a decrease in lipid droplets and a development of SER in type-III cells after injection of 2IU ACTH and an almost complete disappearance of lipid droplets after 4IU ACTH; type-IV cells increase in number.

Corticosterone (B), aldosterone (Aldo), 11-deoxycorticosterone (DOC), 11-dehydrocorticosterone (A), 18-hydroxycorticosterone (18 OH B) and cortisol (F) are identified after incubation of interrenal of Bufo bufo formosus with radioactive progesterone. Yields of radioactive B and Aldo are larger than those of radioactive DOC, A and 18 OH B; yield of radioactive F is the smallest one.

The present study was undertaken to determine the structure-activity relationships of ACTH analogs on corticosteroid production by frog adrenal gland. Rana ridibunda interrenal dice were perifused with amphibian culture medium for 10 hr. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. Perifusion of interrenal fragments with increasing concentrations of synthetic human ACTH 1-39 (ranging from 6.25 X 10(-11) to 10(-9) M) led to a linear log-dose increase in both corticosterone and aldosterone secretion. Thus, this model made it possible to compare the steroidogenic potency of several ACTH analogs. Synthetic alpha-MSH and its des-N alpha-acetyl derivative were found to be approximately equipotent, and 5 X 10(3) times less active than authentic ACTH. The short-chain analog ACTH 1-10 was 2 X 10(4) times less potent than ACTH whereas ACTH 4-10 was totally inactive. A fragment of the N-terminal region of the proopiomelanocortin molecule, gamma 3-MSH, caused a dose-related stimulation of steroid secretion. However, in contrast to what has been observed in the rat, gamma 3-MSH did not potentiate the corticotropic action of ACTH on frog interrenal gland. Since processing of proopiomelanocortin in frog intermediate lobe generates high amounts of alpha-MSH and des-N alpha-acetyl alpha-MSH, these results suggest that in amphibians, several peptides other than ACTH may be involved in the control of corticosteroidogenesis.