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G Callard

Publications and source records attributed to G Callard.

11 recordsLinked to original sources
PubMed2001

Different catalytic properties and inhibitor responses of the goldfish brain and ovary aromatase isozymes.

The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p450 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using androstenedione and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [4-hydroxyandrostenedione, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

Androstenedione↗
PubMed1992

Sulfoconjugation of steroids and the vascular pathway of communication in dogfish testis.

The zonal testis of the dogfish (Squalus acanthias) has proven advantageous to study biochemical changes in relation to stage of spermatogenesis, including information on steroidogenic enzymes and steroid receptors. To investigate whether sulfotransferase is part of a mechanism regulating the availability of biologically active hormone in close proximity to receptors, we measured in vitro conversion of [3H]estrone (E1) to sulfoconjugated metabolites in cytosolic subfractions of testes grossly dissected according to germ cell composition (premeiotic-PrM, meiotic-M, and postmeiotic-PoM stages). Assays were carried out in the presence of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) at 22 degrees C and optimized for time (60 min) and protein (500 micrograms/ml). Michaelis-Menten kinetics and saturation analysis gave the following reaction constants for [3H]E1: Km = 0.33 microM, Vmax = 2.5 pmol/min/mg; and for PAPS: Km = 33 microM, Vmax = 1.1 pmol/min/mg; competition studies carried out in the absence or presence of 1- or 5-fold excess radioinert steroids indicated that estrogen (E2 > E1) as well as androgens (T = DHEA > 5 alpha dihydrotestosterone, DHT) were effective inhibitors. Sulfotransferase activity was found to be stage-related, being highest in PoM regions (2.31 +/- 0.24 pmol/min/mg protein) when compared to M and PrM regions (1.22 +/- 0.22 and 1.28 +/- 0.21 pmol/min/mg protein, respectively). Sulfoconjugation and the intratesticular distribution of steroid sulfates were also measured in vivo by perfusion of the intact testis with [3H]androgen or -estrogen. The pathway of blood flow via the genital artery was epigonal organ-->PoM-->M-->PrM (mature-->immature). Perfused [3H]E2, T, and DHT were all extensively metabolized in a one-pass, 1 hr perfusion, less than 10% of perfused [3H] steroid being recovered from testicular tissues as unchanged steroid. In general, recovery of polar metabolites was greater than non-polar metabolites from all three substrates. Sequential hydrolysis with glucuronidase and glusulase indicated that sulfoconjugation is a minor component (< 20%) of several "inactivating" pathways, which include glucuronide conjugation, 17-ketosteroid synthesis, and pathways leading to unidentified polar metabolites. No consistent stage-related distribution patterns were observed for any of the metabolite subfractions; however, total recovered radioactive steroid (polar plus non-polar) formed a decreasing concentration gradient from point of entry of perfusate (PoM region) to point of exit (PrM region). These data support the conclusion that access to receptors by steroid ligands may be controlled by a balance between activating and inactivating pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Identification of an androgen receptor in the zonal testis of the salamander (Necturus maculosus).

The testis of the salamander, Necturus maculosus, is advantageous for studying biochemical changes during spermatogenesis because germ cells and associated Sertoli and Leydig cells are topographically separated by stage of development. Using extracts of staged tissue samples and [3H]testosterone (T) in a standard binding assay, followed by Sephadex LH-20 or DNA-cellulose chromatography to separate free and bound steroid, we have identified a T-binding protein having physicochemical characteristics of a classical androgen receptor (AR): high affinity (Kd = 10(-9) M), limited capacity (Bmax) = 10(-10) M or 350 fmol/g tissue) and androgen specificity (T = 5 alpha - dihydrotestosterone greater than progesterone = corticosterone greater than estradiol). AR was present in nuclear extracts, where greater than 80% of binding sites were occupied by endogenous ligand, but was not detectable in cytosol. On linear sucrose gradients, nuclear AR sedimented at 3-4 S in both low and high ionic-strength buffers and, by this and other criteria, was distinguishable from the nonreceptor androgen binding protein (ABP) of the same species. The diffuse distribution of AR in germinal and nongerminal (glandular) tissues at all developmental stages is consistent with a dual localization in Sertoli cells and Leydig cells, as previously reported in mammals, and further suggests a regulatory role of androgen throughout spermatogenesis.

Androgen-Binding Protein↗
PubMed1992

In vitro steroid secretion by staged spermatocysts (Sertoli/germ cell units) of dogfish (Squalus acanthias) testis.

Using the dogfish shark (Squalus acanthias) testis model in which different germ cell stages are topographically separated, we previously observed that steroidogenic enzyme activities vary qualitatively and quantitatively during spermatogenesis. To determine whether these data, obtained by radiolabeled tracer analysis of testicular microsomes, accurately predict steroid secretion by intact cultured spermatocysts (germ cell/Sertoli cell units), steroids were radioimmunoassayed directly in spent medium. Steroid output in basal medium was low, but after addition of 25-hydroxycholesterol (25-OH chol, 60 microM), large amounts of progestins (P) and testosterone (T) accumulated. Dehydroepiandrosterone (DHEA), androstenedione (A), and estradiol (E2) were low or nondetectable in the presence or absence of 25-OH chol; however, addition of T (1 microM) as substrate elevated E2 above assay limits. T and P contents of media increased progressively over a 24-hr culture period (Day 1). Replacement and analysis of spent media at 24-hr intervals indicated that secretion was continuous up to 6 days after seeding, although secretory rates (T plus P) and T/P ratios changed with time in culture. Spermatocysts in premeiotic (PrM), meiotic (M), and post-meiotic (PoM) stages all secreted T and P; however, absolute values, T/P ratios, and stage-dependent patterns varied from one experiment to another with no obvious seasonal pattern. Also steroid secretion by staged cysts did not consistently agree with predictions based on steroidogenic enzyme activities measured in earlier cell-free assays, whether or not 25-OH chol was present. This discrepancy was not explained by stage-related patterns of steroid secretion vs retention or by addition of putative in vivo regulators. Agents that elevated P and/or T were dibutyryl cyclic AMP, 1-isobutyl-3-methylxanthine (IBMX), and forskolin, whereas ventral lobe extract and Ca2+ ionophore (A23817) were inhibitory. An autoregulatory feedback loop was indicated by a negative relationship between cyst concentration per well and steroid secretion rate. In contrast to T and P, E2 secretion in the presence of substrate (T, 1 microM) was as predicted from previously determined patterns of aromatase activity (M > PrM > PoM) and increased in response to IBMX. Added [3H]P, [3H]T and [3H]E2 all were extensively metabolized to polar products and P metabolism was stage related (PoM > M > PrM). These data indicate that analysis of free P, T, and E2 in static cyst cultures may underestimate true steroidogenic potential. Assuming further characterization of the system, we conclude that cultured spermatocysts from dogfish testis have potential for studying the multifactorial regulation of steroid synthesis and secretion stage-by-stage during spermatogenesis.

Animals↗
PubMed1992

Androgen and progesterone receptors in shark (Squalus) testis: characteristics and stage-related distribution.

Although testosterone (T) is essential for the normal completion of spermatogenesis, the exact T-sensitive control points are still unknown. Using staged tissues (premeiotic, PrM; meiotic, M; and postmeiotic, PoM) from zonal testes of the spiny dogfish Squalus acanthias, and standard [3H] steroid binding analysis, we characterized a T-binding component with physiochemical characteristics resembling classical androgen receptors (AR). [3H]T binding was of high affinity (dissociation constant = 4.4 x 10(-9) M), limited capacity (maximum binding, 94 fmol/g tissue) and relatively stable (t1/2 = 4 h at 4 C). The T-binding component was present in both cytosolic and nuclear extracts, adhered to DNA-cellulose, and displayed predicted sedimentation properties of an activated receptor in vivo or in vitro (5.06S). T, 5 alpha-dihydrotestosterone (DHT), and mibolerone (Mib), but not methyltrienelone (R1881), competed well for [3H]T binding; however, progesterone (P) was equivalent to T in its ability to displace tracer. Subsequent analysis of [3H] P binding revealed a P-binding component that was present in nuclear and cytosolic extracts, adhered to DNA, but differed from AR in its inability to bind Mib. Competition studies in which excess radioinert Mib was used to block AR revealed ligand specificity characteristics of progesterone receptors (PR): promegestone (R5020) greater than P greater than deoxycorticosterone, but T, DHT, dexamethasone, and corticosterone were ineffective competitors. Also, a nonlinear Scatchard plot was obtained, suggesting two P-binding activities, which differed in their binding affinities (dissociation constant = 0.88 vs. 5.9 x 10(-9) M) and capacities (66 vs. 210 fmol/g tissue). Conversely, using [3H]Mib to avoid interference from PR, we confirmed that T, DHT, and P were equivalent in their ability to displace ligand from AR. Comparison of tissues by stage of spermatogenesis revealed different distribution patterns for AR (PrM greater than M much greater than PoM) vs. PR (PoM much greater than M = PrM). These data provide definitive evidence for separate testicular T- and P-binding mechanisms and indicate the presence of temporally distinct sets of steroid-regulated genes.

Analysis of Variance↗
PubMed1990

Nonmammalian vertebrate models in studies of brain-steroid interactions.

Estrogen formation in brain and pituitary mediates certain androgen actions in central targets. Goldfish (Carassius auratus) and quail (Coturnix coturnix japonica) have been advantageous for studying the role of locally formed estrogen in autoregulating aromatization and in controlling estrogen receptor occupancy, androgen receptor levels, and behavioral expression. Data from these two experimental models reveal a molecular basis for androgen-estrogen synergism in neuroendocrine tissues and for alterations in androgen sensitivity/responsiveness. These mechanisms are essential components of seasonal reproduction in the test species and may have wider relevance for cyclicity in other vertebrates, including mammals.

Androgens↗
PubMed1989

Regulation of spermatogenesis: the shark testis model.

Spermatogenesis is a unique developmental sequence dependent on FSH and androgen. Due to the complex organization of the mammalian testis, however, mechanistic details of regulation are largely unknown. Using the dogfish shark (Squalus acanthias) in which there is a cystic mode of spermatogenesis and a topographic separation of different germ cell stages within the testis, we have obtained new information of general relevance on stage-related biochemical and morphological changes and have proposed a model in which steroids serve as parahormonal regulators of the spermatogenic progression. In addition, techniques developed for culturing staged spermatocysts (intact Sertoli/germ cell units) and isolated, staged Sertoli cells demonstrate the usefulness of this model for studying spermatogenic regulation under defined conditions in vitro.

Animals↗
PubMed1986

Characteristics of a testosterone-estradiol binding globulin (TEBG) in goldfish serum.

Neuroendocrine tissues in teleost fish aromatize androgen to estrogen at extraordinarily high rates. As part of a project in which we are studying the dynamics of sex steroid uptake, metabolism, and receptor binding in goldfish (Carassius auratus) brain and pituitary, we have identified and characterized a sex-steroid-binding component of serum. This protein has been designated a testosterone-estradiol-binding globulin (TEBG) since it bound testosterone (T) and estradiol-17 beta (E2) with high affinity (Kd = 1.9 and 2.1 nM, respectively) whereas other steroids were less effective ligands (5 alpha-dihydrotestosterone greater than progesterone = 11-ketotestosterone greater than estrone = estriol = diethylstilbestrol greater than cortisol). Scatchard analysis, disc gel electrophoresis and sucrose gradient centrifugation all indicate that T and E2 are bound by the same protein. The number of available serum binding sites (Bmax = 10(-7) M) greatly exceeded reported maximal levels of T and E2 in the same species and showed no obvious sex or seasonal differences. However, the steroid-TEBG interaction was unstable, exhibiting very short half-times of association (less than 15 min) and dissociation (less than 10 min). On the basis of comparison of the physicochemical characteristics of TEBG with other intracellular androgen-binding proteins in goldfish brain (androgen receptor, aromatase), we predict that the serum-binding protein would not limit but rather enhance exchange of T and E2 in central tissues.

Animals↗
PubMed1985

Distribution of cell types and aromatase activity in the sculpin (Myoxocephalus) pituitary.

Although aromatase activity is exceptionally high in the teleost pituitary, it is not known which of the secretory cell types are responsible. Pituitary glands from the longhorn sculpin (Myoxocephalus octodecimspinosus) were sectioned transversely into "cephalic" and "caudal" fragments and cultured for 24 hr in medium containing [7-3H]androstenedione. Radiolabeled estrone and estradiol-17 beta production were measured as an estimate of aromatization. In order to determine the distribution pattern of different cell types, the in situ pituitary and dissected fragments were analyzed by standard cytological procedures. Further verification of cell function was obtained by somatostatin (SRIF) and corticotropin-releasing factor (CRF) immunocytochemistry. Estrogen yields obtained from caudal fragments in two separate experiments averaged four times higher per milligram protein than yields from matched cephalic fragments. In addition, female glands synthesized significantly more estrogen than those of males. Due to an anteroflexion of the longitudinal axis and a disposition of the gonadotropic (GTH) cells at the periphery of the gland and surrounding the neurointermediate lobe (NIL), the classical subdivisions of the teleost adenohypophysis were not strictly applicable to the sculpin. The predominance of growth hormone (GH) secreting cells in the caudal fragment suggests their participation in aromatization, a finding which is consistent with a previous study of rodent pituitary; however, a role for gonadotropes and other hypophysial cells in this transformation cannot be ruled out.

Adrenocorticotropic Hormone↗