Stress-induced facilitation of the cortisol response in 17alpha-hydroxylase deficient XX mas-1/mas-1 carp (Cyprinus carpio).

Facilitation of the stress response results from a reduction of the inhibitory effects of circulating corticosteroids, allowing an animal to respond to a novel stressor. In this study, the existence of a facilitated cortisol stress response in normal (STD) and 17alpha-hydroxylase deficient XX mas-1/mas-1 (E5) carp was investigated. E5 carp had previously been characterized as having a low cortisol response to stress. Fish were subjected to either cortisol feeding or daily-acute stress, from 45 until 140 days post-hatch (dph) and were then subjected to a novel net-confinement stressor at 141 dph. Growth of E5 fish was reduced in both the daily-acute stress and cortisol-fed groups, but STD fish were only affected by daily-acute stress. Cortisol feeding had no effect on the stress response of STD fish but daily-acute stress significantly inhibited the response to a subsequent novel stressor. In contrast, daily-acute stress facilitated the cortisol stress response of E5 fish to a novel stressor, while cortisol feeding inhibited the cortisol response. Facilitation was accompanied by significant enlargement of the head-kidney tissue (which contains the steroidogenic interrenal tissue) in E5 fish. To our knowledge this is the first report of stress-induced facilitation in a lower vertebrate.

Characterization of cDNAs encoding cholesterol side chain cleavage and 3beta-hydroxysteroid dehydrogenase in the freshwater stingray Potamotrygon motoro.

The interrenal gland (adrenocortical homolog) of elasmobranchs produces a unique steroid, 1alpha-hydroxycorticosterone (1alpha-B). The synthesis of this and most other steroids requires both cholesterol side chain cleavage (CYP11A) and 3beta-hydroxysteroid dehydrogenase (HSD3). To facilitate the study of elasmobranch steroidogenesis, we isolated complementary DNAs encoding CYP11A and HSD3 from the freshwater stingray Potamotrygon motoro. The P. motoro CYP11A (2182 bp total length) and HSD3 (2248 bp total length) cDNAs harbor open reading frames that encode proteins of 542 and 376 amino acids (respectively) that are similar (CYP11A: 39-61% identical; HSD3: 36-53% identical) to their homologs from other vertebrates. In molecular phylogenetic analysis, P. motoro CYP11A segregates with CYP11A proteins (and not with related CYP11B proteins) and P. motoro HSD3 segregates with steroidogenic HSD3 proteins from other fishes. CYP11A and HSD3 mRNA is found only in interrenal and gonadal tissues, indicating de novo steroidogenesis is restricted to these tissues. Because 1alpha-B is thought to act in the elasmobranch response to hydromineral disturbances, we examined the effect of adapting P. motoro to 10 ppt seawater on mRNAs encoding steroidogenic genes. The P. motoro response to this salinity challenge does not include interrenal hypertrophy or an increase in the levels of interrenal CYP11A, HSD3 or steroidogenic acute regulatory protein (StAR) mRNA. This study is the first to isolate full length cDNAs encoding elasmobranch CYP11A and HSD3 and the first to examine the regulation of steroidogenic genes in elasmobranch interrenal cells.

Interrenal steroid 21-hydroxylase in eels: primary structure, progesterone-specific activity and enhanced expression by ACTH.

Cytochrome P450 21-hydroxylase (P450c21) is a key enzyme for corticosteroidogenesis. To understand the regulatory mechanisms of cortisol production in fish, we have cloned a cDNA encoding P450c21, for the first time in non-mammalian vertebrates, from the head kidney of the eel (Anguilla japonica). The overall similarity of the deduced P450c21 sequence was modest (41-44% amino acid identity) between the eel and mammals. However, the functional domains for steroid-binding, heme-binding and proton-transfer sites were well conserved (74-100% identity). The eel P450c21 mRNA was expressed abundantly in the anterior quarter of the head kidney, but was undetectable in the remaining three-quarters or in other tissues including the gill, heart, liver, intestine, kidney, immature gonad and skeletal muscle. Functional expression of the cDNA clone in non-steroidogenic COS-1 cells produced a protein with high 21-hydroxylase activity to convert progesterone to 11-deoxycortisterone but not 17alpha-hydroxyprogesterone to 11-deoxycortisol, although the latter is a preferred substrate for mammalian P450c21. To examine whether 21-hydroxylated progesterone is actually 17alpha-hydroxylated in the eel interrenal, 11-deoxycorticosterone and (3)H-corticosterone were respectively incubated with the interrenal-containing anterior quarter of the head kidney. The separation of the steroids produced by two HPLC systems revealed that cortisol was produced from both substrates, showing the 17alpha-hydroxylation of 11-deoxycorticosterone and corticosterone in the eel interrenal. ACTH infused at 3 pmol/kg per min for 5 h consistently increased plasma cortisol levels and interrenal P450c21 mRNA levels in seawater eels. These results showed that the interrenal-specific eel P450c21 cloned in this study is involved in cortisol production through conversion of progesterone to 11-deoxycorticosterone in the interrenal-containing anterior quarter of eel head kidney. Furthermore, ACTH stimulates cortisol production in part through enhanced P450c21 expression in the eel interrenal.

Ultrastructural and histochemical study on the interrenal cells of the male stickleback (Gasterosteus aculeatus, Teleostea), in relation to the reproductive annual cycle.

The steroidogenic interrenal cells in the adrenal homologue of the male stickleback (Gasterosteus aculeatus) were studied in relation to the reproductive cycle by means of histological and ultrastructural observations, and using histochemical methods for the localisation of 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17beta-hydroxysteroid dehydrogenase (17betaHSD). To determine the various stages of the reproductive cycle, the testes were also examined by histological and histochemical methods (3betaHSD). The results indicate that in this teleost the interrenal cells can undergo a cycle in which phases characterised by different cytological aspects are observed. During this cycle there is a renewal of organelles, in particular mitochondria and SER. Periodic degenerative processes are also found. Organelle cytology showed that the cell cycle has at least 3 different aspects during the year. An analogy with some cytological aspects of the adrenal zonation in mammals is possible. It is postulated that the interrenal gland activity could substitute or supplement androgen production by the testes.

Eel (Anguilla japonica) testis 11beta-hydroxylase gene is expressed in interrenal tissue and its product lacks aldosterone synthesizing activity.

A recombinant expression vector containing Japanese eel (Anguilla japonica) testis cytochrome P450(11 beta) (11beta-hydroxylase) cDNA was introduced into COS-1 cells. Enzymatic activity of the expressed P450(11 beta) for corticosteroid synthesis was analysed by incubating transfected cells with 14C-labelled 11-deoxycorticosterone or 3H-labelled deoxycortisol as substrates. Thin layer chromatography of incubation medium revealed that a high percentage of 11-deoxycorticosterone was converted into corticosterone and 11-dehydrocorticosterone but no aldosterone was detected. Similarly, deoxycortisol was converted into cortisol and cortisone. These results show that eel P450(11beta) does not possess significant aldosterone synthesizing activity. Northern blot analysis detected a 1.8 kb transcript of P450(11beta) using RNA extracted from interrenals of untreated Japanese eel but no hybridization signal was apparent using RNA extracted from brain, spleen, heart, muscle or testis. Immunohistochemistry using an antiserum against P450(11beta) also revealed strong immunostaining in interrenal cells.

Mitochondrial localization of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase in interrenals of the toad Bufo arenarum H.

The enzymatic activity of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones including progesterone, mineralocorticoids, glucocorticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal. The present study demonstrates that in the interrenal of Bufo aernarum H., 3 Beta HSD/I activity localizes in mitochondria and micromes. It also shows that the two distinct pathways to aldosterone previously demonstrated for interrenals of B. arenarum H. exhibited differential subcellular localizations, microsomal for the 4-ene route and mitochondrial for the 5-ene route. Kinetic constants of 3 Beta HSD/I were determined for the oxidation of pregnenolone and the recently described 3 Beta-hydroxy analogue of aldosterone (3 Beta AA). The preferred substrate of the mitochondrial 3 Beta HSD/I enzyme was 3 Beta AA (Km = 0.7 microM and 14.0 microM for 3 Beta AA and pregnenolone, respectively). However, the microsomal enzyme has a greater affinity for pregnenolone (Km = 0.8 microM) than for 3 Beta AA (Km = 17.0). Enzymes from both localizations have similar nucleotide (NAD+) requirements, activities being higher in summer. This dual localization opens novel possibilities for the regulation of interrenal functions.

Ontogeny of immunoreactive steroidogenic proteins (adrenodoxin and cytochrome p-450(21)) in the interrenals of the teleost Lates calcarifer.

Antisera against bovine adrenodoxin and cytochrome P-450(21) (steroid 21-hydroxylase) cross-reacted with the interrenal cells of the adult Asian seabass (Lates calcarifer); the cells were arranged as cords, two cells thick, in the headkidney. During larval development, cells immunoreactive for adrenodoxin were first observed 1 day posthatching (dph); immunoreactivity for cytochrome P-450(21) was first detected at 1.5 dph. Initially, the interrenal cells occurred as a mass in each headkidney, which only became identifiable histologically at 5 dph. The number of interrenal cells increased with age, becoming associated with the cardinal veins at 14 dph. The present study thus indicates that the posthatching rise in cortisol may originate from the nascent interrenal tissue.

Contrasting effects of ACTH and cyanoketone on delta 5-3 beta-hydroxysteroid dehydrogenase activity in interrenal glands of tadpoles of Rana catesbeiana in vitro.

The present communication describes an investigation of stimulation and inhibition of delta 5-3 beta-hydroxysteroid dehydrogenase in interrenal glands of tadpoles of Rana catesbeiana. Frozen sections of interrenal glands, together with kidneys, were prepared histochemically for assay of delta 5-3 beta-HSD activity. Concentrations of 0.01, 0.1, 1, and 10 IU/ml of ACTH or of 0.01, 0.1, 1, and 10 micrograms/ml of cyanoketone were added to the incubation media. The reaction products of the histochemically prepared slides, in terms of absorbance, were scanned at a defined area with a computerized microscope spectrophotometer. The results indicate that ACTH causes a significant dose-response stimulation of delta 5-3 beta-HSD activity in tadpole interrenals; cyanoketone, on the other hand, causes significant dose-dependent inhibition.

Ultrastructural localization of delta 5-3 beta-hydroxysteroid dehydrogenase in the interrenal cells of the goldfisch (Carassius auratus).

The ultrastructural localization of the enzyme delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was studied in the goldfish interrenal cells by using the potassium ferricyanide method. Immersion fixation in a mixture of 1% paraformaldehyde and 0.25% glutaraldehyde results in a consistently good ultrastructural preservation and enzyme localization. A precipitate of copper ferrocyanide indicating the localization of 3 beta-HSD activity was observed in close vicinity to the smooth endoplasmic reticulum or in contact with the outer surface of its membrane. A small number of precipitated grains were also found in the lumen of mitochondrial cristae. Addition of phenazine methosulfate increased the intensity of the reaction without changing the localization of the copper ferrocyanide grains. The findings suggest that the outer surface of the smooth endoplasmic reticulum is the major site of 3 beta-HSD activity in goldfish interrenal cells.

Electron microscopic localization of 3beta-hydroxysteroid dehydrogenase and NADH-ferricyanide reductase activities in amphibian interrenal cells.

3beta-hydroxysteroid dehydrogenase and NADH-ferricyanide reductase activities were localized at the ultrastructural level in amphibian interrenal (adrenocortical) cells previously fixed in a mixture of formaldehyde and glutaraldehyde. Potassium ferricyanide was used as an electron acceptor. Copper ferrocyanide deposits resulting from 3beta-HSD activity were seen in close association with the external faces of the membranes of the smooth endoplasmic reticulum. Very rare grains of precipitate appeared in mitochondrial cristae. The addition of phenazine methosulfate to the incubation medium had no effect on these localizations. The interrenal cells showed also a strong NADH-ferricyanide reductase activity. The copper ferrocyanide grains were abundant in the mitochondrial cristae and in the hyaloplasm, where they were not preferentially associated with the smooth endoplasmic reticulum.

Hydroxysteroid dehydrogenases in the interrenal gland and the ovary of stork-billed kingfisher, Pelargopsis capensis (Linn.): a histochemical study.

The distribution of delta5 3beta-hydroxüsteroid dehydrogenase (delta5 3beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), Glucose-6-phosphate dehydrogenase (G-6-PDH) and NADH-diaphorase enzymes has been histochemically studied in the interrenal gland and the ovary of the stork-billed kingfisher, Pelargopsis capensis (Linn.). In the interrenal gland, the activity for all the enzymes studied, occurred in the interrenal cells. All these enzyme activities occurred in the theca interna of normal growing follicles, atretic follicles and interstitial gland cells of the ovary. A weak activity for all the enzymes occurred in the hypertrophied granulosa cells of the atretic follicles. The significance of these findings is discussed.

Functional morphology of the interrenal and chromaffin cells in the teleosts, Rasbora daniconius (Hamilton), Barbus stigma (Cuv. et Val.) and Channa gachua (Hamilton).

The interrenal cells in Rasbora daniconius, Barbus stigma and Channa gachua are mainly found around the postcardinal vein and its major branches in the haemopoietic head-kidney. The chromaffin cells which are identified by the positive chromaffin reaction are found in the walls of the postcardinal vein or dispersed among the interrenal cells. delta5-3beta-HSDH and G-6-PDH activity was observed in the interrenal cells of all three teleosts. The present work indicates that the interrenal cells are capable of steroid biosynthesis and the chromaffin cells contain biologically active catecholamines.