Estrogen receptors in quail brain: a functional relationship to aromatase and aggressiveness

Estradiol (E2) mediates many of the activational effects of testosterone (T) on masculine reproductive and aggressive behaviors. Using Japanese quail (Coturnix coturnix japonica) as an animal model, together with a newly devised procedure for quantifying aggressiveness, we recently showed that aggression is E2-dependent and that individual differences in behavioral intensity are correlated with aromatase in the hypothalamus/preoptic area (HPOA). In this study we characterized estrogen receptors (ER) in quail brain and tested the hypothesis that aromatase in brain regulates T-induced behavioral responsiveness by regulating the quantity of E2 available for receptor binding. Based on standard binding assays and Sephadex LH-20 chromatography, quail brain ER was shown to be estrogen-specific, of high affinity (Kd = 0.88 nM), and of limited capacity with highest concentrations in limbic brain areas (Bmax 23-27 fmoles/gm HPOA). In addition, this ER adhered to DNA-cellulose under activating conditions. The quantitative relationship between aromatization, ER, and aggressiveness was tested in reproductively inactive (nonaggressive) males by treatment with T +/- the aromatase inhibitor 4-hydroxyandrostenedione (OHA). After 5 days, T markedly stimulated aggressiveness, and elevated aromatase and nuclear (occupied) ER in HPOA. Simultaneous treatment with OHA blocked effects on aggressiveness and aromatase, and lowered nuclear ER, but increased cytosolic (empty) ER. Total ER (nuclear plus cytosolic) was higher after T treatment whether or not OHA was administered, suggesting that androgen per se induces ER in quail HPOA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in pituitary responsiveness during the ovulatory cycle of the Japanese quail, in vitro

Anterior pituitary glands from ovulating Japanese quail (Coturnix coturnix) were used to investigate variation in sensitivity to chicken luteinizing hormone-releasing hormone (cLHRH I; Gln8-LHRH). Grouping the pituitaries by ovulatory stage provided preliminary evidence of changes in sensitivity to LHRH during the ovulatory cycle. Pituitaries taken from quail before the preovulatory LH surge were responsive to cLHRH I, while pituitaries from the other times of the cycle showed minimal response to cLHRH I. Female pituitary glands release less LH than those of males. These data indicate a change in sensitivity to LHRH in the female quail that may be due to changes in gonadal steroids or the pool of releaseable LH from the pituitary.     

A specific androgen-binding protein (ABP) in Necturus testis and its zonal distribution

The urodele amphibian Necturus maculosus has a zoned testis, which is advantageous for separating Leydig cells from germinal elements and for studying stage-dependent biochemical changes. Using [3H]testosterone (T) in a standard binding assay and dextran-coated charcoal (DCC) or Sephadex LH-20 to separate free and bound steroids, we identified an androgen-binding protein (ABP) in Necturus testis cytosols. This protein was of high affinity (Kd = 10(-9) M) and was saturable (Bmax = 10(-9) M) and specific for androgen (T; 5 alpha-dihydrotestosterone, DHT) but could be distinguished from the androgen receptor of Necturus testis by its relative abundance (300-550 fmol/mg protein), short half-time of dissociation (3 min at 22 degrees C), inability to adhere to DNA-cellulose, and absence from nuclear extracts. Additionally, when analyzed on sucrose gradients, the ABP of Necturus testis sedimented at 6-7 S in both low or high ionic strength buffers. In that estradiol (E2) is a poor competitor for T-binding, this protein resembles a sex steroid-binding protein previously identified in urodele serum but differs from the ABP and testosterone-estradiol-binding globulin (TEBG) of rodents, humans, goldfish, and sharks. It is differentially distributed within the testis, with the highest levels in immature lobular regions composed of Sertoli cells and germ cells in premeiotic stages and lower levels in regions composed primarily of Leydig cells. The cellular source and function of this protein in Necturus testis remain to be determined.     

Ovarian steroid synthesis and the hormonal control of the elasmobranch reproductive tract

Synopsis The reproductive biology of the chondricthyan fishes is remarkably sophisticated. Using both oviparous and viviparous reproductive modes, the group has generally adapted the style of bringing forth relatively few young at one time, each representing the investment of a great deal of maternal energy. The oviparous species foreshadow the situation common in oviparous reptiles and universal in birds. On the other hand, viviparous species range from simple internal incubators, in which large yolked eggs are retained, to other species in which the complexity of placentation and yolk reduction approach the eutherian condition. Further, in certain viviparous elasmobranchs the phenomenon of histotrophic nutrition attains an importance and complexity not seen in any other vertebrate group including mammals. Internal fertilization and amniote patterns of reproductive tract development also operate in virtually all elasmobranchs. The summary of work presented here suggests that these female reproductive styles are associated with a reproductive endocrinology which is the archetype for amniote vertebrates.     

Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

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)     

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

Expression of SR-BI (Scavenger Receptor Class B Type I) in turtle (Chrysemys picta) tissues and other nonmammalian vertebrates

In this study, the tissue distribution of the expression of an HDL-receptor (SR-BI; Scavenger Receptor Class B Type I) was investigated in the turtle using an antiserum to murine SR-BI. Several turtle tissues including liver, heart, small intestine, kidney, oviduct, ovary, and testis were shown to express an 82 kDa membrane protein. In addition, SR-BI expression in livers of other nonmammalian species such as the chicken, frog, goldfish, shark, and skate is also reported. Ovarian SR-BI expression varies seasonally in the turtle as do plasma levels of apoA-I and cholesterol ester. It is possible that changing levels of SR-BI, the receptor for apoA-I, is physiologically relevant to the demands of the turtle ovarian cycle and cholesterol distribution.