Distribution and Structure of the Adrenocortical Homolog in Polypterus palmas Ayres

The identity, distribution and structure of the adrenocortical homolog (AH) was made in Polypterus palmas Ayres using routine light and electron microscopy and histochemistry for the enzyme δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). The AH is confined to yellow corpuscles which are positioned near the posterior cardinal and renal veins in the anterior two-thirds of each kidney. The 46–57 spherial to cigar-shaped corpuscles are placed end to end and are equally distributed in the kidneys. The tortuous cords of epithelial cells of each corpuscle are surrounded by sinusoids and are completely delimited from the haemopoietic and renal tissue of the kidneys. The steroidogenic nature of the cells is demonstrated by their 3β-HSD activity and their ultrastructure, namely lipid droplets, tubular smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae. Giant mitochondria and gap junctions are notable features of AH cells in this fish. The yellow corpuscles of the kidneys in P. palmas represent the AH and this tissue has a distribution which is in accordance with the taxonomic position of Polypteriformes.     

The kidney,adrenocortical homolog,and corpuscles of Stannius in the cockscomb pricklebackAnoplarchus purpurescens

The morphology of the kidney, adrenocortical homolog, and the corpuscles of Stannius was examined in the cockscomb prickleback,Anoplarchus purpurescens, a marine teleost which inhabits the intertidal zone. The paired kidneys of this fish are fused throughout most of their length, there is essentially a single posterior cardinal vein on the right side, they possess renal corpuscles, and there is no distal segment of the tubule. The tubule is specialized, in descending order, into ciliated neck and two proximal segments before entering the system of collecting tubules and ducts. The cells of the latter system are specialized for mucous secretion, as are cells of the main excretory ducts, the paired archinephric ducts. Tubulogenesis occurs in the kidneys in close apposition to the archinephric ducts. The presumptive adrenocortical homolog is located around the posterior cardinal veins in the head kidney while paired corpuscles of Stannius are confined to the posterior end of the kidney. All of the above features are consistent with those found in the kidneys of many other marine teleosts.     

Immunolocalization of steroidogenic enzymes in gonads of European eel, Anguilla anguilla (L.)

The localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase (P450arom) was investigated using polyclonal antibodies during gonad development in wild European eels, Anguilla anguilla (L.), from the River Po Delta (Ferrara, Italy). The first steroidogenic cells, observed in undifferentiated gonads of 14–16 cm yellow eels, showed no P450scc, 3β-HSD or P450arom activity, but positive regions appeared in head kidney insulae from this stage until the silver eel stage. In undifferentiated gonads of 16–20 cm yellow eels the steroidogenic cells were positive to all enzymes. Pre-Leydig steroidogenic cells, identified in Syrski organs of yellow eels of 22–26 cm evolving into testes, were positive to 3β-HSD and P450scc, but negative to P450arom. However, steroidogenic cells in Syrski organs evolving towards ovaries and in small but fully differentiated ovaries were positive to all enzymes. Immature testes of yellow and silver eels had Leydig cells positive to P450scc and 3β-HSD; the same reactions were also observed in some Sertoli cells of silver eel testes containing meiotic cells. Sex differentiation in A. anguilla apparently occurs through an initial female stage controlled by P450arom activity. Leydig and Sertoli cells appear involved in different steps of hormonal control of spermatogenesis: Leydig cells begin their steroidogenic activity before meiosis, while Sertoli cells begin their activity during meiosis.     

Fine Structure of the Adrenocortical Homolog and the Corpuscles of Stannius of Amia calva L.

A comparison is made between the fine structure of yellow corpuscles and white corpuscles located within the kidneys of the holostean fish, Amia calva L. The yellow corpuscles are composed of epithelial cells possessing all the features of steroid-producing tissues, namely an abundance of vacuoles, tubular smooth endoplasmic reticulum, and mitochondria with tubular cristae. The Golgi apparatus is also a conspicuous component of their cytoplasm. These cells are homologous to adrenocortical cells of higher vertebrates and they have cytoplasmic projections which extend into the lumina of surrounding sinusoids. The white corpuscles possess epithelial cells of variable appearance but all cells contain secretory granules and an extensive rough endoplasmic reticulum. The secretory granules appear to originate at the Golgi apparatus and occasionally are observed intact in the intercellular space. However the method of release of these granules was not clearly defined. These corpuscles are similar to the corpuscles of Stannius which have been described in modern bony fish. The presence of multivesicular bodies and smooth endoplasmic reticulum in some cells may reflect the origin of the corpuscles of Stannius from the tubular nephron. A. calva appears to be a suitable organism for comparative studies into the function of the adrenocortical homolog and corpuscles of Stannius in “primitive” fish.     

The andrenocortical homolog in the lake sturgeon, acipenser fulvescens rafinesque.

The adrenocortical homolog in the chondrostean fish, Acipenser fulvescens, is confined within yellow corpuscles located at the dorsal junction of the two kidneys near the cardinal veins. The concentration and distribution of this tissue in chondrosteans appear to be a condition intermediate between that of cyclostomes and teleosts. The homology of this tissue to adrenocortical tissue of higher vertebrates is demonstrated in its delta5-3beta-hydroxysteroid dehydrogenase activity and the ultrastructure of the cells. The cells contain large lipid droplets and an abundance of smooth endoplasmic reticulum and mitochondria. The abundance of smooth endoplasmic reticulum, a large Golgi apparatus and an extensive elaboration of the plasma membrane suggest the high degree of steroidogenic activity of these cells. Apposed plasma membranes make contacts through the unique cell contacts described in the adrenocortical cells of mammals (Friend and Gilula, '72). The Golgi apparatus is described as a most conspicuous component of the cytoplasm and is presented as further evidence for the important involvement of this organelle in corticosteroidogenesis in vertebrates. These morphological observations are discussed in relation to the known secretory products of this tissue in the sturgeon, and it is suggested that there is a need for further examination of the corticosteroids produced by lower vertebrates.     

A study on the 3β- and 17β-hydroxysteroid dehydrogenase activities in the gonads of Monopterus albus (Pisces: Teleostei) at various sexual phases during natural sex reversal

Activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in Monopterus gonads were studied at different sexual phases during natural sex reversal. Before sexual transformation, positive reactions for 3β-HSD in the follicular epithelium were found in the granulosa cells of some large, maturing follicles in some females during the breeding season. Weak reaction for this enzyme was also detected in some scattered interstitial cells found occasionally in some ovaries. At the intersexual and the male phases, intense 3β-HSD activities were demonstrated exclusively in the interstitial Leydig cells. No 17β-HSD activities were observable in the gonads at any stage of development. The reaction intensity of 3β-HSD in the interstitial cells exhibited a marked increase during the process of sex change from female to the intersexual and the male phases and there is a definite correlation with the density and nuclear size of these cells. It is concluded that in Monopterus , the granulosa cells in the ovary and the interstitial cells of the intersexual and male gonads are the major sites for the biosynthesis of oestrogens and androgens, respectively, and that the intensive development of interstitial tissue with increasing steroidogenic enzyme activities at the intersexual and male phases was directly related to the increase in androgen production in vitro reported previously. The occasional presence of some 3β-HSD positive interstitial cells in the ovary suggests that interstitial cell development might precede testicular lobule formation during natural sex reversal.     

Vascularization,innervation, and ultrastructure of the endocrine cell types of stannius corpuscles in the teleost Gasterosteus aculeatus

The blood circulation of the Stannius corpuscles, like that of the kidneys to which the corpuscles are attached, represents a portal system. The corpuscles receive blood from the dorsal caudal vein and from a vein coming from the hypaxial musculature. They are drained by veins which enter the caudal parts of the kidneys and therefore endocrine substances released by the corpuscles pass through the kidneys before they enter the general body circulation. The corpuscles are penetrated by sympathetic nerves coming from a small subvertebral ganglion. It is likely that these nerves innervate the muscular coat around the blood vessels. The muscular coat surrounding the renal blood vessels, the collecting tubules and part of the ureters, is innervated by nerves from the same ganglion. The secretory activity of the gland cells appears to be controlled by blood borne factors, because neither synaptic contacts with these cells, nor gap junctions among the cells, have been found in thin sections and freeze-etch replicas of the corpuscles. The corpuscles contain two cell types, both presumed to have endocrine function. Histochemical and ultrastructural data indicate that the gland cells produce glycoproteins. It is likely that the contents of the secretory granules are released by exocytosis. One cell type is structurally similar to the cells described in many other teleosts and thought to be engaged in the synthesis of a hypocalcemic hormone. The ultrastructure of the second cell type resembles cells described only in other migratory species: salmonids and eels. It may be involved in the control of monovalent ions.     

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Thus, brain 3β-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3β-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3β-HSD, we asked whether 17β-estradiol (E₂) regulates DHEA metabolism in adult zebra finch ( Taeniopygia guttata ) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [³H]DHEA and radioinert E₂ in vitro . Within only 10 min, E₂ significantly reduced 3β-HSD activity in both male and female brain. Interestingly, the rapid effects of E₂ were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.     

Electrophoretic analysis of DNA-binding proteins during induction of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase in wild type and mutants of Streptomyces hydrogenans

Abstract Treatment of the wild-type strain HY 0 of Streptomyces hydrogenans with estradiol, a specific inducer of 3β,17β-hydroxysteroid dehydrogenase (17β-HSD) formation, caused several soluble proteins to bind to DNA-cellulose with altered affinity. Hydrocortisone which induces biosynthesis of 3α,20β-hydroxysteroid dehydrogenase (20β-HSD), and progesterone which induces production of both 17β- and 20β-HSD, had no effect on DNA-binding properties of the proteins. In mutants with altered activity/inducibility of 17β- and 20β-HSD only one DNA-binding protein (protein 23) still showed an altered DNA affinity in response to estradiol-treatment and this in only one strain. In other mutants the DNA affinity was not altered during induction with estradiol but the DNA affinity of protein 23 varied between low, low-and-high, and high affinity, depending on the strain. In the mutant where DNA affinity was altered by estradiol treatment the change was opposite to that found in the wild type.     

Production of monoclonal antibodies against 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

Abstract A procedure is described for the production of monoclonal antibodies (mAbs) against 3α,20β-hydroxysteroid dehydrogenase (3α,20β-HSD) from the actinomycete Streptomyces hydrogenans ATCC 19631. Clones which were obtained after fusion of immune cells were screened by solid-phase ELISA and immunoblotting. About 5.2% of the clones secreted immunoglobulins with specificity for 3α,20β-HSD. The purified mAbs were found to belong to subclass IgG₁ and to recognize both the native enzyme as well as its identical subunits which were obtained by SDS denaturation. However, the activity of the tetrameric holoenzyme was only weakly diminished in the presence of these mAbs.     

THE OCCURRENCE OF HISTOCHEMICAL ACTIVITY OF 3β-HYDROXYSTEROID DEHYDROGENASE IN THE DEVELOPING TESTES OF POECILIA RETICULATA

In the mature testes of the guppy, Poecilia reticulata , some groups of cells, distributed sparsely in the interspace between the peripheral germ cell layer and the hilar duct system, show evident histochemical response for Δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). In the testis of newly delivered guppies, somatic cells are present in the testicular hilus as a compact mass without revealing any structural differentiation. In the testis of juvenile fish of the 8mm stage about 7 days after birth, interstitial cells resembling histologically those of adult testes become differentiated from the somatic cell mass and, though only in some specimens, coincidentally begin to display weak but obvious histochemical response for 3β-HSD. Thereafter the occurrence of enzyme activity becomes increasingly regular in the developing testes, and attains the adult pattern of distribution in testes of all specimens after the 11 13mm stage or 17 ∽ 20 days of age.
The appearance and enhancement of 3β-HSD activity in the testis is concurrent with the differentiation and development of the testicular duct system. Treatments of newly delivered fish with methyltestosterone (30 ∽ 50 μg/g diet) distinctly stimulate the development of the duct system, which suggests a possible role of androgen secretion occurring in the early phase of the testicular development in the control of testicular organogenesis in the guppy.     

In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β-hydroxysteroid dehydrogenase (17β-HSD)-like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed-phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α-dihydrotestosterone (5α-DHT) in the telencephalon where 17β-HSD-positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α-DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography-mass spectrometry. Using the pulse-chase technique with [³H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α-DHT, and estradiol. These data demonstrate the existence of an active form of 17β-HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.     

Fine structure of the corpuscles of stannius in the toadfish.

The micro-anatomy of the corpuscles of Stannius of the toadfish, Opsanus tau, an aglomerular marine teleost, has been studied by light and electron microscopy. The corpuscles are composed of extensively anastomosed cords of epithelial cells which maintain intimate contact with blood capillaries. Most of the epithelial cells contain acidophilic granules which also show a positive reaction with the periodic acid-Schiff technique and aldehyde fuchsin. On the basis of fine structural criteria, three cell types can be recognized. The granular cells contain abundant quantities of granular endoplasmic reticulum, ribosomes, Golgi apparatus with prosecretory granules, coated vesicles, polymorphic mitochondria with lamellar cristae, filaments, microtubules, a cilium, a variety of lysosome-like dense bodies, glycogen particles, lipid droplets, secretory granules and intranuclear lipid-like inclusions. One variety of agranular cell (type I) is characterized by the total absence of secretory granules, but it contains large amounts of granular endoplasmic reticulum and ribosomes, conspicuous profiles of Golgi apparatus, coated vesicles and sometimes an abundance of glycogen. Another variety of agranular cell (type II) has poorly developed cytoplasmic organelles. The perivascular space between the capillary and parenchyma contains connective tissue cells and abundant nerve fibers. The different types of epithelial cells observed in the corpuscles of Stannius of this fish may represent functional stages of the secretory cycle in a single cell type.     

Histological, fine-structural and histochemical differences in the testicular glands of gobiid and blenniid fishes

The testicular gland (t.g.) is a glandular tissue situated adjacent to the testis of blenniid and several gobiid species. In the present study the t.g. of Blennius pavo Risso and Gobius niger L. were compared by histological and histochemical methods. In B. pavo the spermatozoa have to cross the t.g. to reach the vas deferens and thus they come into contact with the gland cells, whereas in G. niger the vas deferens is situated between the testis and the t.g. The fine structure and histo-chemistry of the t.g. cells reveal that in B.pavo the cells of the t.g. have exocrine as well as endocrine functions. The t.g. cells of B. pavo contain large amounts of lipids, form a secretion containing acid mucopolysaccharides, show positive reaction for acid phosphatase, and some cells stain for 3β-HSD and G6PD. The function of the t.g. of G. niger is exclusively endocrine. Characteristics of the gland cells of this species are well developed smooth ER and tubulovesicular or paracristal-line mitochondria. The stainings for 3β-HSD, G6PD and UDPGD give strong positive results in the whole t.g., indicating the presence of steroids and steroid glucuronides.     

Gonadal morphology, enzyme histochemistry and plasma steroid levels during the annual reproductive cycle of male and female brown bullhead catfish, Ictalurus nebulosus Lesueur

The annual reproductive cycle of the brown bullhead catfish, Ictalurus nebulosus Lesueur, was investigated over a two-year period. In females, GSI increased in the spring as follicles enlarged and the granulosa became hypertrophied, dropped during spawning in August, then rose in the autumn as follicles enlarged slightly. 3β-Hydroxysteroid dehydrogenase (3β-HSD) activity was limited to thecal nests of large, vitellogenic follicles. Plasma testosterone and estradiol-17β levels increased in parallel with GSI. Levels of both steroids dropped prior to the spawning period, although a peak in estradiol-17β was evident during the spawning period. No 11-ketotestosterone was detected in female plasma. In males, GSI increased in the spring as spermatogenesis proceeded, and dropped during spawning. 3β-HSD activity was confined to Leydig cells and was most intense prior to spawning. Plasma testosterone and 11-ketotestosterone peaked during the pre-spawning period, dropped prior to spawning, then rose slowly during the autumn. A peak in estradiol-17β occurred during the spawning period. Significant differences in GSI and plasma steroid levels during the pre-spawning and spawning periods were observed between the two yearly cycles; they may be related to differences in rainfall during these periods.     

Estradiol Secretion by Tadpole Ovaries Masculinized by Implanted Capsules of Cyanoketone

Female tadpoles of Rana catesbeiana were laparotomized at metamorphic stages XI-XIII and an empty capsule or one containing cyanoketone (CK), which is an inhibitor of Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD), was implanted intraperitoneally. Ovarian activity of Δ⁵-3β-HSD was examined histochemically 2 months later, estradiol-17β (E₂) secretion by the ovaries was measured by RIA 4 months later and histological changes of the ovaries were examined 6 months later. The Δ⁵-3β-HSD activity of the CK-treated ovaries was much lower than that of controls. E₂ secretion per froglet by CK-treated ovaries was about one third that of controls (p<0.001). Histological examination showed various degrees of masculinization of the ovaries, about 28% of which were totally transformed into testis-like structures.
As a result of suppressed Δ⁵-3β-HSD activity, dehydroepiandrosterone would have accumulated, resulting in deficient E₂ secretion and, therefore, ovarian masculinization. In tadpoles, this effect does not depend on the pituitary, whereas interrenal hyperplasia and hyperactivity do, indicating that interrenal function is not essential for ovarian masculinization. From these findings and our previous results, we suggest that disturbance of steroidogenesis by CK in the ovaries results in their masculinization.     

Glial fibrillary acidic protein and desmin in salivary neoplasms. Expression of four different types of intermediate filament proteins within the same cell type

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

Testis-testicular gland complex of two Tripterygion species (Blennioidei, Teleostei): differences between territorial and non-territorial males

The morphological differences between the testis and testicular gland of territorial and nonterritorial males of Tripterygion tripteronotus and T. delaisi were examined and correlated with differences in reproductive behaviour. In territorial males of both species the testicular gland is much more developed than in non-territorial males. Larger cellular and nuclear sizes in the territorial males indicate that the activity of the gland cells is enhanced. These cells contain SER, numerous lipid droplets and mitochondria with lamellar cristae. Absence of 3β-HSD activity at these sites points to lack of a steroidogenic potency. In both territorial and non-territorial fish, steroid-producing Leydig cells have been demonstrated in the connective tissue betweeen the testis and the testicular gland, and around the collecting sperm duct. In addition, 3β-HSD activity has been found in the scarce interstitial Leydig cells of territorial fish. Morphometric data indicate an enhanced activity of the Leydig cells in territorial fish.     

Studies on the Mechanism of Action of Estradiol in Regulating Follicular Progesterone Levels: Effects on cAMP Mediated Events and 3β-Hydroxysteroid Dehydrogenase

Previous studies demonstrated that estradiol interferes with pituitary-induced progesterone production and oocyte maturation in cultured amphibian ( Rana pipiens ) ovarian follicles. To elucidate the mode of action of estradiol in modulating follicular progesterone accumulation we have examined its effects on cAMP-induced progesterone production and enzymatic conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Follicular cAMP levels were manipulated with forskolin (an adenylate cyclase activator), isobutyl methyl xanthine (IBMX-phosphodiesterase inhibitor) and exogenously added cAMP. Progesterone production induced by forskolin alone or forskolin in combination with frog pituitary homogenate (FPH) was inhibited by estrogen. Addition of estradiol to culture medium markedly inhibited follicular progesterone accumulation following treatment of follicles with cAMP and IBMX. In the presence of exogenous pregnenolone, non-FPH stimulated ovarian follicles effectively converted the 3β-HSD substrate to progesterone. Treatment of follicles with estradiol inhibited conversion of pregnenolone to progesterone. The results indicate that estradiol acts, following FPH stimulation, at one or more steps subsequent to elevation of cAMP levels to regulate intrafollicular progesterone accumulation and oocyte maturation. Estrogen appears to directly influence the enzymatic (3β-HSD) conversion of pregnenolone to progesterone.     

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.