Biomedical subjects

G V Callard

Publications and source records attributed to G V Callard.

At least 55 records · Page 3Linked to original sources

PubMed1988

A high abundance androgen receptor in goldfish brain: characteristics and seasonal changes.

Testosterone (T) exerts its actions in brain directly via androgen receptors or, after aromatization to estradiol, via estrogen receptors. Brain aromatase activity in teleost fish is 100-1000 times greater than in mammals and would be expected to significantly reduce the quantity of androgen available for receptor binding. Experiments were carried out on the goldfish Carassius auratus to determine if androgen receptors are present in teleost brain and whether their physicochemical properties reflect elevated aromatase. Cytosolic and nuclear extracts were assayed with the use of [3H]T and charcoal, Sephadex LH-20, or DNA-cellulose chromatography to separate bound and free steroids. Binding activity was saturable and had an equally high affinity for T and 5 alpha-dihydrotestosterone (Kd, approximately 2.4 X 10(-9) M). Although mibolerone was a relatively weak competitor, the putative teleost androgen 11-ketotestosterone, methyltrienolone (R1881), estradiol, progesterone, and cortisol were poor ligands. Characteristics that distinguish this receptor from a steroid-binding protein in goldfish serum are the presence of binding activity in both nuclear and cytosolic extracts, a low rate of ligand-receptor dissociation, electrophoretic mobility, sedimentation properties in low vs. high salt, and tissue distribution (forebrain greater than or equal to pituitary greater than mid-/hindbrain). DNA cellulose-adhering and nonadhering forms were detected, but these did not differ in other variables measured. Although goldfish androgen receptors resembled those of mammals in all important physicochemical characteristics, they were unusually abundant (5-68 pmol/g tissue) compared to levels in rat brain, but comparable to levels in prostate and other male sex hormone target organs. Moreover, there were seasonal variations in total receptors, with a peak at spawning (April) 4- to 5-fold higher than values in reproductively inactive fish (July/August). This temporal pattern and magnitude of change corresponded to previously reported changes in brain aromatase. Thus, both phylogenetic and physiological correlates point to a functional interdependence between androgen receptors and aromatase in the brain. These studies in goldfish indicate that brain androgen receptors have a long evolutionary history and have been highly conserved through the vertebrate series.

Animals↗

PubMed1987

A comparison of aromatase, 5 alpha-, and 5 beta- reductase activities in the brain and pituitary of male and female quail (C. c. japonica).

In numerous vertebrate species including Japanese quail (Coturnix coturnix japonica), actions of testosterone (T) on neuroendocrine target tissues are mediated in part by conversion to estrogenic and androgenic metabolites. In order to assess which pathways were favored in each identified androgen target area in quail brain and whether there were discernible sex differences, we developed an assay for simultaneously quantifying aromatase, 5 alpha-, and 5 beta-reductase. In addition, we made the first definitive identification of aromatase in quail pituitary and compared all three enzyme activities in the pituitary of males and females. Enzymes were measured in tissue homogenates by the conversion of [3H]androstenedione to [3H]estrone, [3H]5 alpha-androstanedione, and 5 beta-androstanedione. Aromatase activity was restricted to limbic tissues (anterior hypothalamus greater than posterior hypothalamus greater than septum greater than archistriatum containing nucleus taenia) while hyperstriatum, cerebellum, and midbrain containing nucleus intercollicularis were aromatase-negative. Quail pituitary aromatized androgen at rates equivalent to anterior hypothalamus/pre-optic area (aHPOA). 5 alpha- and 5 beta-reductase were present in all tissues tested. Aromatase was significantly higher in aHPOA and pituitary of males, whereas 5 alpha-reductase was significantly higher in female pituitary. These data suggest that a complex of androgen-metabolizing enzymes controls the neuroanatomic (spatial) distribution of active hormone in neuroendocrine tissues and that quantitative differences between males and females may account for sex differences in behavior.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1987

A novel steroid-binding protein in the testis of the dogfish Squalus acanthias.

In an earlier study, we identified and characterized a testicular estrogen receptor in the spiny dogfish (Squalus acanthias); however, in this species estrogen receptors were located exclusively in nuclear subfractions. We describe here a nonreceptor, sex hormone-binding protein (SBP) present in Squalus testicular cytosol and distinguishable in its physicochemical characteristics from both the estrogen receptor and serum SBP of the same species. Cytosol (100,000g supernatant) was prepared by differential centrifugation of testicular homogenates and incubated with [3H]estradiol (E2) or [3H]testosterone (T) (10-15 nM +/- 100-fold excess radioinert competitor) to determine total and nonspecific bound radioactivity. The testicular SBP had a broad specificity: E2 = T greater than progesterone greater than 5 alpha-dihydrotestosterone greater than estrone, but diethylstilbestrol was not an effective competitor. It displayed a high affinity for both E2 and T (Kd = 2.2-2.5 X 10(-9) M), sedimented at 8-10 S in both low- and high-salt sucrose gradients, and migrated more slowly than BSA during polyacrylamide disc gel electrophoresis. The testicular SBP-E2 complex was relatively stable (t1/2 = 160 min) compared to the serum SBP-E2 complex (t1/2 = less than 30 min). The testicular SBP was not found in nuclear subfractions nor did it bind to DNA-cellulose affinity columns. Its intratesticular distribution was stage-dependent: Zone III (mature spermatids) greater than Zone II (spermatocytes) greater than Zone I (stem cells and spermatogonia). Moreover, increased binding activity corresponded exactly to the hypertrophy and differentiation of Sertoli cells in the same zones, pointing to this cell as the possible site of SBP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

A method to quantify aggressiveness in Japanese quail (Coturnix c. japonica).

Testing procedures which rely on paired fights are unsuitable for measuring individual aggressiveness because the outcome of initial encounters influences subsequent dominance status. In an effort to circumvent this problem, we developed a test system which quantifies aggressiveness in individual Japanese quail by measuring locomotor and pecking activity of test birds presented with the visual stimulus of a conspecific. Using this procedure, we demonstrate that aggression is specific to sexually mature males; however, the intensity of the behavior varies widely among individuals. Scores for each bird are constant with time, reproducible, independent of the gender or behavior of the stimulus bird and accurately predict the outcome of a subsequent paired fight. In addition, we show that fighting per se produces increases and decreases in aggressiveness of dominant and subordinate birds, respectively, and re-orders the rank of a group of birds. This test procedure, therefore, confirms earlier studies which indicate that dominance hierarchies are influenced by initial encounters. Finally, repeat testing is reliable and gives an absolute, not relative, measure of aggressiveness. We conclude that this behavioral test can be applied to future studies addressing the social, developmental and endocrine basis of aggressiveness and dominance in quail and other vertebrates.

Aggression↗

PubMed1986

Sertoli cell cytoplasts in the semen of the spiny dogfish Squalus acanthias.

The fine structure of Squalus acanthias (Elasmobranchii) semen was investigated to determine the cellular component responsible for the steroidogenic activity previously demonstrated in the seminal plasma of this species. Semen was found to consist of bundles of spermatozoa, many of which were encased in a sleeve of cytoplasm restricted to the tail region; large, dense bodies lacking a limiting membrane and numerous anuclear cytoplasmic remnants containing lipid droplets, mitochondria, areas of agranular reticulum, and possibly unreleased spermatozoa. These remnants, which we have termed cytoplasts, morphologically resemble in appearance Sertoli cells of S. acanthias at the time of spermiation. This structural similarity, plus the fact that many elasmobranch species slough the apical regions of Sertoli cells during the release of spermatozoa, indicates that the cytoplasts present in the semen of S. acanthias originate from Sertoli cells. Furthermore, the occurrence in these cytoplasts of organelles normally associated with steroid synthesis strongly suggests these structures are the source of steroidogenic enzymes in the semen of S. acanthias. The steroidal contribution to the semen by Sertoli cell cytoplasts may be necessary for either maturation or maintenance of spermatozoa in the excurrent reproductive ducts of S. acanthias.

Animals↗

PubMed1986

Effects of short days on aromatization and accumulation of nuclear estrogen receptors in the hamster brain.

Exposure of hamsters to short days increases sensitivity to the negative feedback effects of testosterone (T) but decreases responsiveness to the behavioral effects of the hormone. Since T is metabolized in the brain to 5 alpha-dihydrotestosterone (DHT) and estradiol, which differentially affect gonadotropin secretion and sex behavior, it is reasonable to postulate that daylength can modulate neural responses by quantitative or qualitative alterations in T metabolism and subsequent receptor binding of active hormone. Experiments reported here focused on aromatization and the nuclear accumulation of estrogen receptors. Adult male hamsters were maintained for 6-12 wk in long (14:10 LD) or short (8:16 LD) daily photoperiods. Both intact and castrated animals were used to assess direct effects of short days versus changes due to short-day-induced testicular regression. Discretely dissected regions of the brain (preoptic area, POA; hypothalamus, HTH; and corticomedial amygdala, CMA) or limbic blocks (LIM) comprised of all three regions were assayed for estrogen-synthesizing activity (aromatase) and estrogen-binding activity (receptors). Aromatase was estimated in vitro by conversion of [7-(3)H] androstenedione to [3H] estrogen and in vivo by measuring increases in nuclear estrogen receptor levels after injection of aromatizable androgen. Receptor-binding activity was assayed in crude cytosolic and nuclear extracts by incubating samples with [3H] estradiol +/- 100-fold excess inert estradiol, and separating free and bound steroids by Sephadex LH-20 gel filtration. When aromatase was assayed in homogenates prepared from discrete brain regions of individual hamsters, significantly lower activity was found in the HTH of short-day animals than in long-day controls. This effect was seen in both intact and castrated animals, which indicates that it was not mediated by the testis. Decreased enzyme activity in the POA and CMA of short-day hamsters was not significant, nor was there an effect of castration independent of short days. Low levels of nuclear estrogen receptors were present in LIM of intact males, but these were reduced after castration or concomitant with testicular regression after short-day exposure. This suggests that the hamster testis normally secretes estrogen or aromatizable androgen. A single injection of estradiol or aromatizable androgen (T or androstenedione) increased nuclear receptors in LIM of castrated animals. Cytosolic receptors were not different in short-day vs. long-day hamsters, nor were there differences in nuclear receptor levels after a single estradiol injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗

PubMed1986

Tissue and species specificity of unmasked nuclear acceptor sites for the estrogen receptor of Squalus testes.

The testicular estrogen receptor of the shark Squalus acanthias is restricted to nuclear subfractions when tissue is homogenized in low salt buffers and adheres tightly to nuclei and DNA-cellulose even when exposed to high salt conditions. Therefore, we examined the binding characteristics of this receptor to chromatin subfractions from homologous and heterologous tissues. Squalus chromatin linked to cellulose and partially deproteinized by 0-8 M guanidine hydrochloride (GuHCl) gave extraction patterns similar to those obtained with mammalian and avian chromatin. Chromatin as prepared in our laboratory contained no bound estrogen receptor. The binding pattern of the [3H]estradiol-labeled nuclear estrogen receptor to chromatin fractions from Squalus testicular zones I/II (containing spermatogonia, spermatocytes, and high receptor levels) revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 2-4 M GuHCl (350% increase over unextracted chromatin), with a 40% decrease from maximal binding at 5-8 M GuHCl. By contrast, binding to chromatin from zone III (containing spermatozoa and little or no detectable receptor) showed no major peak and 4 times less binding at 3 M GuHCl-extracted chromatin. We have previously shown that zones I and II contain the majority of testicular receptors and, presumably, are the primary sites of estrogen action, whereas receptor activity in zone III is minimal (less than 5%), indicating a secondary or nontarget tissue. Squalus testicular [3H]estradiol-receptor complexes bound minimally to rabbit uterine chromatin. Likewise, [3H]estradiol-receptor complexes from rabbit uterus, Squalus oviduct, or mouse testis bound minimally to Squalus testicular chromatin. Thus, maximal binding occurred only with Squalus zones I/II chromatin and Squalus testicular receptor. The binding of [3H]estradiol-receptor complexes to testicular chromatin (zones I/II) was of high affinity (Kd = 1.9 X 10(-10) M) and low capacity and was optimal in the presence of 150 mM KCl, but was unaffected by the addition of urea in an amount similar to that of Squalus body fluids (300 mM). [3H]Estradiol binding to chromatin required undenatured receptor and was competitively inhibited by radioinert estradiol-receptor complexes, confirming saturability of acceptor sites. The affinity of the Squalus estrogen receptor for homologous chromatin was in the same range as that reported for other systems, despite the unusual nuclear extractability characteristics of Squalus receptor. This study provides new evidence for tissue and species specificity of receptor binding to chromatin acceptor sites.

Animals↗

PubMed1985

Aromatase and 5 alpha-reductase in the teleost brain, spinal cord, and pituitary gland.

The distribution of aromatase and 5 alpha-reductase was investigated in the brain, spinal cord, and pituitary of adult goldfish (Carassius auratus) and toadfish (Opsanus tau) of both sexes. Tissue homogenates were incubated with [3H]androstenedione in the presence of an NADPH-generating system and, following validation of assay conditions, radiolabeled products (estradiol, estrone, 5 alpha-androstanedione), were measured as an index of enzyme activity. Neuroendocrine tissue of both species produced exceptionally large amounts of estrogen, thus confirming previously observed differences between teleosts and other vertebrates. By contrast, 5 alpha-reductase levels resembled the vertebrate norm. In general, aromatase was concentrated in the pituitary and various forebrain regions, especially the hypothalamus/preoptic area; however, estrogen yields from the medulla and anterior spinal cord of toadfish were high compared to adjacent midbrain, hindbrain, and cord regions. This same neural region in toadfish, but not in goldfish, is known to control a sex dimorphic behavior, the courtship boatwhistle. In contrast to aromatase, 5 alpha-reductase was more uniformly distributed throughout the brain, although somewhat higher activity was obtained in the pituitary. High levels of aromatase in the neuroendocrine tissues of teleosts recommend them as animal models for further studying the enzyme, its regulation, and its role in governing androgen-dependent responses in central targets.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1985

Characterization of estrogen receptors in the hamster brain.

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.

Animals↗

PubMed1985

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood.

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.

Aging↗

PubMed1985

Exclusive nuclear location of estrogen receptors in Squalus testis.

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing.

Animals↗

PubMed1985

Location of enzymes of androgen and estrogen biosynthesis in the testis of the ground squirrel (Citellus lateralis).

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

Stage-dependent changes in steroidogenic enzymes and estrogen receptors during spermatogenesis in the testis of the dogfish, Squalus acanthias.

In the spiny dogfish (Squalus acanthias), germ cells and adjacent steroidogenic elements are topographically segregated within the testis according to stage of development. In the experiments reported here, we have taken advantage of this favorable anatomical arrangement to demonstrate stage-specific variations in steroidogenic enzymes and estrogen receptor number. The testes of Squalus collected in July-November were sectioned transversely and further subdivided into three zones as follows: I) germinal bed plus immature lobules with spermatogonia; II) lobules with primary or secondary spermatocytes; III) lobules with spermatids or mature spermatozoa. The morphology of these zones was verified by light microscopy and, in a separate study, by electron microscopy. Through the course of spermatogenesis, Sertoli cells increased dramatically in size and in the abundance of steroidogenic organelles. By contrast, interstitial tissue was sparse in all stages of development, and only relatively undifferentiated Leydig-like cells were present. Microsomes prepared from each zone were incubated with [3H] progesterone, [3H]17 alpha-hydroxyprogesterone, or [3H]androstenedione to evaluate androgen and estrogen biosynthetic potentials. Based on product formation, 17 alpha-hydroxylase and C-17,20-lyase activities increased progressively from less mature to more mature regions (zone III greater than II greater than I), whereas aromatase was greatest in regions undergoing meiosis or early spermiogenesis (zone II). These enzymes were not detected in semen, although C21 substrates were converted to unidentified polar metabolites in high yield. Estrogen receptors were concentrated in immature zones (zone I greater than II greater than III), and the percentage of occupied receptors revealed the same distribution. Semen and epigonal tissues were receptor negative. We conclude that Sertoli cells are responsible for steroidogenesis in Squalus testis and that hormone production is keyed to the spermatogenic cycle. The data are consistent with an important role for androgens during spermatid maturation and/or after sperm release, but reveal that estrogen actions are expressed primarily during early spermatogenic stages. This report demonstrates the usefulness of unconventional animal models for obtaining new information of general relevance.

17-alpha-Hydroxyprogesterone↗

PubMed1984

Development of agranular reticulum in Sertoli cells of the testis of the dogfish Squalus acanthias during spermatogenesis.

Biochemical analyses of Squalus testis indicates that key enzymes involved with androgen production increase progressively from immature regions containing spermatogonia to mature regions in the late spermatid stage of maturation (Canick et al., 1983). In an effort to identify cells possessing the cytological characteristics of steroid production and to determine the structural correlates of the observed functional changes, we have carried out an electron microscopic study of Squalus testis. This report demonstrates that Sertoli cells contain a well-developed agranular reticulum, mitochondria with tubulovesicular cristae, and numerous lipid droplets. Moreover, as germ cells mature, there is an increase in abundance of agranular reticulum in the adjacent Sertoli cells. By the time of spermatid elongation, this has reached dramatic proportions and fills the Sertoli cell as a mass of tubules. These results lead us to conclude that the Sertoli cell is responsible for secretion of the increasing amounts of androgen during the spermatogenetic cycle in Squalus.

Animals↗

PubMed1984

Identification of Leydig-like cells in the testis of the dogfish Squalus acanthias.

The site of hormone synthesis in the testis of elasmobranchs has been the subject of much controversy. This is primarily due to the problem of whether Leydig cells are present or absent in the testes of many species of elasmobranchs. In previous studies we have shown that key enzymes associated with the biosynthesis of androgen increase in activity during the spermatogenetic cycle of Squalus acanthias (Canick et al., 1983). To determine the site of this activity we undertook an electron microscope study of Squalus testes to identify cells that possessed the structural correlates of steroid production. This report describes cells present in the interstitial tissue that are morphologically analogous to Leydig cells occurring in the testes of higher vertebrates. Although these cells possessed an agranular reticulum, tubulovesicular mitochondria, and lipid droplets, they were mesenchymal in appearance. We have, therefore, preferred to describe these cells as Leydig-like.

Animals↗

PubMed1984

Organization of interstitial tissue in the testis of the salamander Necturus maculosus (Caudata:Proteidae).

In Necturus maculosus the organization of the interstitial tissue varies according to the stage of spermatogenesis. Leydig cells at various stages of differentiation and myoid cells are always present in this tissue. The Leydig cells are undifferentiated at all phases of germ cell activity and only hypertrophy following spermiation and degeneration of Sertoli cells. These Leydig cells are structurally analogous to mammalian Leydig cells. They do not form part of the lamina propria of the seminiferous lobules and hence cannot be referred to as lobule-boundary cells previously described in the urodele testis (Lofts, '74). When the Leydig cells hypertrophy, numerous unmyelinated axons appear in the interstitial tissue. These axons, often devoid of Schwann-cell cytoplasm, occur in close proximity to Leydig cells. Because the levels of both Substance P and neurotensin increased in the testis of Necturus maculosus as Leydig cells differentiated, we concluded that these neural elements may regulate Leydig-cell function locally, through the release of neuropeptides.

Animals↗

PubMed1984

In vitro conversion of androgen to estrogen in amphioxus gonadal tissues.

The ability to convert androgen to estrogen (aromatization) is a constant feature of gonadal and neural tissues in all major vertebrate groups. In experiments reported here, the existence of this pathway was investigated in the protochordate amphioxus (Branchiostoma lanceolatum). Following incubation with [3H]19-hydroxyandrostenedione, gonadal homogenates contained authentic estrone and estradiol-17 beta, as determined by derivative formation and recrystallization to constant specific activity. Cephalic ("brain") and other segments were aromatase negative. The results indicate that a potential for estrogen biosynthesis in the gonads predates that in other tissues and arises prior to the evolution of true vertebrates.

Androstenedione↗

PubMed1984

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus.

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.

Androgens↗