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Biomedical subjects

W E Stumpf

Publications and source records attributed to W E Stumpf.

At least 55 records · Page 3Linked to original sources
PubMed1991

Distribution of estrogen target sites in the 2-day-old mouse forebrain and pituitary gland during the 'critical period' of sexual differentiation.

The present study provides a detailed anatomical description of estrogen target cells in the mouse forebrain and pituitary gland during the sexual imprinting stage of the brain. Six 2-day-old mice (3 males and 3 females) were s.c. injected with 16 alpha-[125I]iodo-11 beta-methoxy-17 beta-estradiol ([125I]MIE2) and two additional mice (one male and one female) were s.c. injected with 1000x unlabeled 17 beta-estradiol 1 h before [125I]MIE2 to check the specificity of estradiol binding. Two hours after injection the mice were decapitated, the brains dissected, frozen sectioned, and processed for thaw mount autoradiography. The highest intensity of nuclear labeling was observed in the preoptic-anterior hypothalamic area, amygdala and cortex entorhinalis. Strong labeling was present in the cerebral cortex and moderate to strong labeling in the lateral septum, bed nucleus of stria terminalis and pituitary gland. Weak to moderate labeling was observed in the bulbus olfactorius, circumventricular organs, basal ganglia, ventral striatum, thalamus, hippocampus and pineal gland. No sex differences were observed in the intensity of labeling and distribution of the estrogen target sites. The topographic distributions of estrogen-concentrating cells in the hypothalamus of the 2-day-old mouse forebrain was similar to the adult pattern but differed prominently in the cerebral cortex, entorhinal cortex and thalamus: the cerebral cortex showed an extensive and intensive labeling, the intensity of labeling in the entorhinal cortex greatly exceeded that observed in the adult and the nucleus anterior medialis thalami was distinctly labeled.

Animals↗
PubMed1991

Progestin receptor cells in mouse cerebral cortex during early postnatal development: a comparison with preoptic area and central hypothalamus using autoradiography with [125I]progestin.

The distribution of progestin target cells in the cerebral cortex and the effect of estrogen treatment was assessed during the critical period of brain development and compared with the preoptic/central hypothalamic regions. [125I]progestin was injected into 0, 2, 8, and 12 day postnatal mice pretreated for 3 days with oil, 5 micrograms/100 g b, wt., or 100 micrograms/100 g b. wt. of estradiol dissolved in oil. Two hours after injection of radiolabeled ligand, brains were frozen and processed for thaw-mount autoradiography. At birth, labeled cells were detected in the deep (lamina VI) and intermediate (lamina V) layers of the lateral cortical regions, increased in laminae V-VI of the lateral cortex and laminae II-VI of the cingulate/paracingulate cortex at days 2 and 8, and decreased throughout the cortex by day 12. Pretreatment of animals with estradiol had no noticeable effect on the nuclear concentration of [125I]progestin in cortical cells, while estrogen weakly enhanced labeling in preoptic/central hypothalamic regions at day 2 and markedly augmented labeling in the 8 and 12 day brain. The results demonstrated that progestin receptor cells are present in the postnatal dorsal cortex, preoptic area, and hypothalamus and that the topography of cortical progestin target cells differs in part from that of estrogen target cells reported earlier.

Animals↗
PubMed1991

Uptake, distribution and binding of vertebrate and invertebrate steroid hormones and time-dependence of ponasterone A binding in Calliphora vicina. Comparisons among cholesterol, corticosterone, cortisol, dexamethasone, 5 alpha-dihydrotestosterone, 1,25-dihydroxyvitamin D3, ecdysone, estradiol-17 beta, ponasterone A, progesterone, and testosterone.

The presence of specific binding sites for radiolabelled vertebrate-type and arthropod-type steroid hormones was investigated in several organs including salivary gland, and central nervous system of third instar Calliphora vicina larvae by thaw-mount autoradiography. Ponasterone A, a 20-hydroxyecdysone agonist and 20-hydroxyecdysone are the only steroids which bind to nuclear high affinity binding sites. These binding sites are DNA associated while nucleoli show no tracer binding. Ecdysone, an endogenous 20-hydroxyecdysone precursor, is taken up by target cells but no significant nuclear binding occurs. 1,25-Dihydroxyvitamin D3 concentrates in cytoplasm only and its uptake is highest compared to all other steroids. Progesterone and testosterone show weak accumulation in the cytoplasm, while for cholesterol, corticosterone, cortisol, dexamethasone, dihydrotestosterone and estradiol-17 beta, no noticeable uptake occurs. For ponasterone A, a clear time dependence of uptake and intracellular distribution is visible, suggesting the existence and involvement of specific ecdysteroid uptake and transport mechanisms. These results suggest the presence of binding sites for various mammalian steroids in insects. Whether vertebrate steroid hormones or metabolites of them play a role in insects or whether the uptake and binding is based on chemical similarities alone without biological significance remains to be further investigated.

Animals↗
PubMed1991

Ecdysteroid binding sites localized by autoradiography in the central nervous system of precommitment fifth-stadium Manduca sexta larvae.

Brains and subesophageal ganglia from day 3.5 fifth stadium larvae of Manduca sexta were incubated in vitro with 4 nM tritiated ponasterone A, a 20-hydroxyecdysone analog, to determine whether uptake and specific binding of ecdysteroids occur at a cellular level. These tissues, which were taken just prior to the commitment peak in the hemolymph-ecdysteroid titer, showed saturable uptake of 3H-ponasterone A after 40-60 min of incubation. Uptake was blocked by the addition of 400 nM unlabelled ponasterone A, or of 500 nM or 1000 nM 20-hydroxyecdysone. RH 5849, a synthetic 20-hydroxyecdysone agonist with a long half-life, for which ecdysteroid receptors have low affinity, also reduced ponasterone A uptake at a concentration of 10 microM. Autoradiographs of 4 microns sections of brains revealed distinct nuclear concentrations of silver grains over cell populations in the pars intercerebralis, pars lateralis, and ventral tritocerebrum. Nuclear labelling was also found in many small cells around the mushroom bodies and the neuropil, and between the inner and outer larval optic lobes. Nuclear labelling of cells in the subesophageal ganglion was observed in the fronto-medial and lateral regions, in small cells around the neuropil, and caudally in a few large neurons. In addition to cells with nuclear labelling, both brains and ganglia at this development stage contained cells with exclusively cytoplasmic or both nuclear and cytoplasmic labelling. None of these apparent binding sites were observed in the competition experiments, suggesting that the binding is specific.

Animals↗
PubMed1991

Autoradiographic analysis of the in vivo distribution of 3H-imipramine and 3H-desipramine in brain: comparison to in vitro binding patterns.

Using high resolution autoradiographic techniques, the distribution of radioactivity in forebrain and brainstem was assessed after IV injection of 3H-impramine or 3H-desipramine. Results were compared with regional binding of the drugs to brain sections in vitro. Similar topographic binding of 3H-imipramine and 3H-desipramine was observed in vitro among brain regions, except in the paraventricular nucleus of the hypothalamus and locus coeruleus, where binding was greater for 3H-desipramine. For both 3H-desipramine and 3H-imipramine, some brain regions that exhibited high binding in vitro also showed high accumulation after in vivo injection. However, certain regions that contained high densities of binding sites for the antidepressant drugs as measured by in vitro binding showed very low accumulation of radioactivity after in vivo treatment. Such regions included the dentate gyrus of the hippocampus, layer 1 of piriform cortex, caudate-putamen, pontine and midbrain central gray, and cerebellar granular layer. Compared to in vitro binding of the drugs, the distribution of imipramine and desipramine in vivo appears more anatomically selective. For imipramine, primary sites of action in vivo, as indicated by the topographic distribution in brain, appear to be the locus coeruleus, hippocampus, lateral septal nucleus, and amygdala. For desipramine, the greatest accumulation in vivo was found in the locus coeruleus, paraventricular nucleus of the hypothalamus, and anterior thalamic nuclei.

Animals↗
PubMed1991

The steroid hormone of sunlight soltriol (vitamin D) as a seasonal regulator of biological activities and photoperiodic rhythms.

Neural and systemic somatotrophic effects of the ultraviolet component of sunlight through the skin-vitamin D endocrine system are considered as alternate or additional to the neuroendocrine effects of the visual component of light through the retino-diencephalic input. The extensive distribution of soltriol nuclear receptor cells, revealed by autoradiography with tritium-labeled 1,25 dihydroxycholecalciferol (vitamin D, soltriol) and related effects, indicate an involvement of vitamin D-soltriol in the actinic induction of seasonal biorhythms. This is considered to be independent of the traditionally assigned effects of vitamin D on systemic calcium regulation. Skin-soltriol mediated seasonal, and to a degree daily, genomic activation involves many target regions in the brain. These include neurons in the central nucleus of the amygdala, in the linked part of the bed nucleus of the stria terminalis, in periventricular hypothalamic neurons, dorsal raphe nucleus, reticular thalamic nucleus and autonomic, endocrine as well as sensory and motor components of the brainstem and spinal cord. Additional to the eye-regulated "suprachiasmatic clock", existence of a soltriol-vitamin D regulated neural "timing circuit(s)" is proposed. Both, activational and organizational effects of soltriol on mature and developing brain regions, respectively are likely to play a role in the regulation of neuronal functions that include the modulation and entrainment of biorhythms. Soltriol's central effects correlate with peripheral effects on elements in skin, bone, teeth, kidney, intestine, heart and blood vessels, endocrine organs, and tissues of the immune and reproductive system.

Brain↗
PubMed1991

Autoradiography of progesterone and model compound entry and distribution in Xenopus laevis oocytes.

Xenopus laevis oocytes resume meiosis in response to progesterone. The initial interaction involves surface binding to numerous low-affinity receptor proteins. The mechanism of entry and functions of intracellular steroid are unknown. Because the latter are important for understanding progesterone-induced maturation, a dry-mount autoradiographic technique for analyzing entry and intracellular distribution of radiolabeled steroids was developed and tested. The distinguishing feature of this cryo-technique is sample preparation directly in incubation media using uncross-linked polyacrylamide for inert support. The external ligand functions as an internal standard, so quantitation is by simple ratio (bound/free). The entry kinetics and subcellular binding patterns in large oocytes were studied using this method at nM levels of radiolabeled steroids and model compounds. Progesterone, estradiol, corticosterone, and 1,25-dihydroxycholecalciferol all showed rapid entry (P approximately 10(-6) cm/sec). Entry rates were not saturable with unlabeled steroid. Intracellular patterns of these steroids were highly specific and negatively associated with yolk protein and lipid. Intracellular binding in animal hemisphere ooplasm was 10x that of the yolk-rich vegetative ooplasm. In contrast, dexamethasone, ponasterone-A, and ecdysone displayed entry rates 20-60x slower than progesterone with little compartmentalization. Glycerol, glucose, and leucine entered over 10x slower than progesterone. Cholesterol and Ca++ had entry rates below detection. Evidence for mediated entry of progesterone included the rapid saturation of a cortical compartment equivalent in magnitude to reported receptor numbers. The kinetics and specificity of cortical uptake were consistent with low-affinity, high capacity protein binding. Intracellular binding was seen to correlate with rhodamine 123 patterns, suggesting involvement of mitochondrial or other microtubule-associated structures in steroid responses. Mitochondrial binding is consistent with the limited steroid metabolism seen in oocytes. Since several maturation events are consistent with respiratory uncoupling, reported by others for steroids and isolated organelles, and since mitochondria contain nearly all of the oocyte DNA, a role for these organelles in steroid-induced oocyte maturation was proposed.

Animals↗
PubMed1991

Novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods.

Cross-linked and uncross-linked acrylamide polymers were used to alleviate technical difficulties in cryosectioning and autoradiographic processing of marine arthropods (Pycnogonum litorale), larval insect tissues (Sarcophaga bullata), and amphibian (Xenopus laevis) oocytes. Rapidly polymerized cross-linked acrylamide was used to prepare sections from an animal with a hard thick cuticle, P. litorale. Fragmentation and compression artifacts caused by tissue density differences were avoided and internal tissues were well preserved. Our results indicate that closely applied external support eliminates difficulties typically associated with arthropod cryosectioning. Pre-polymerized, uncross-linked acrylamide provided similar protection when used for sectioning larval tissue from S. bullata. Soft tissues under the cuticle were undamaged in tracheal and epidermal preparations. This polymer was also an excellent embedding material for soft tissues such as salivary glands, quickly penetrating tissue cavities and eliminating air bubbles. Uncross-linked acrylamide was used as an embedding and culturing medium for oocytes from X. laevis. The polymers were non-toxic and allowed the preparation of thin frozen sections containing as many as 50 large oocytes per section.

Acrylamide↗
PubMed1991

Progestin receptor cells in the 8-day-old male and female mouse cerebral cortex: autoradiographic evidence for a sexual dimorphism in target cell number.

The present study examined the number and distribution of progestin receptor cells in the 8-day-old male and female cortex and compared cortical labeling with that in the preoptic area and central hypothalamus. Eight-day postnatal mice (four males and four females), treated with estradiol, were each sc injected with 0.32 micrograms/100 g BW [125I]progestin (SA, 2200 Ci/mM). Brains were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with a nuclear concentration of radioactivity were localized in lamina VI of the lateral cortical regions of the male and female brain, while only a few cortical cells were seen in laminae II, III, and V of the suprarhinal, lateral, and cingulate/paracingulate regions. Comparison of the number of labeled cells revealed that the female cortex contained significantly more labeled cells than the male at three of the four levels investigated. Similarly, the number of target cells was higher in the female medial preoptic nucleus, but not in the arcuate nucleus and ventromedial hypothalamic nucleus, while the distributions of labeled cells in the male and female preoptic/hypothalamic regions were comparable. Injection of unlabeled progesterone or R5020 1 h before [125I]progestin reduced the nuclear concentration of radioactivity in all target regions and verified the specificity of [125I]progestin for the progestin receptor. The results of these studies indicate that mouse 8-day-old cortex and preoptic area in the female animal have more progestin receptor cells than those in the male and demonstrate that progestin receptor cells are localized in a region of the cortex known to contain few estrogen target cells. These results further suggest that a sexual dimorphism in progestin cell number may result in a differential effect of progestin on the cortex and preoptic area of the mouse, perhaps establishing a dimorphism in development and function.

Animals↗
PubMed1991

Immunocytochemical localization of the glucocorticoid receptor in rat brain, pituitary, liver, and thymus with two new polyclonal antipeptide antibodies.

The intracellular localization of the glucocorticoid receptor (GR) was studied in male rat brain, pituitary, liver, and thymus. Two new polyclonal anti-GR antibodies, GR 57 and GR 59, raised against two synthetic peptides (346-357 and 245-259) that correspond to unique regions of the amino-terminus of human GR were used. Vibratome sections (30-50 microns) of perfused brain and frozen sections (6-8 microns) of pituitary, liver, and thymus fixed in paraformaldehyde were incubated in preimmune serum, immunoserum, epitope-purified immunoserum, or peptide-absorbed immunoserum of either GR 57 or GR 59 and immunostained by the avidin-biotin peroxidase method. GR immunoreactivity (GR-ir) was primarily nuclear in brain, pituitary, liver, and thymus sections from intact rats. Adrenalectomy caused nuclear GR-ir to decrease and cytoplasmic GR-ir to increase. When adrenalectomized rats were treated with corticosterone (100 micrograms and 1 mg) or dexamethasone (1 microgram, 100 micrograms, and 1 mg), GR-ir was again predominantly nuclear. One microgram of corticosterone failed to cause nuclear GR-ir when administered to adrenalectomized rats. Immunoreactive neurons and glial cells were extensively distributed, with varied intensity, throughout the rat forebrain. The areas include cortex, septum, hippocampus, amygdala, thalamus, and hypothalamus. Cells with the strongest GR-ir were located in the caudate putamen, paraventricular, arcuate, and central amygdala nuclei, areas CA1-CA2 of the hippocampus, and laminae 4 and 5 of the cortex. In the pituitary, cells of the anterior and posterior lobes were GR immunoreactive, while those in the intermediate lobe were not. Hepatocytes of the liver and thymocytes and reticuloepithelial cells of the thymus were GR immunoreactive. The results show that GR can be localized immunocytochemically in numerous rat tissues using antipeptide polyclonal antibodies and correlated with the results of biochemical and ligand receptor studies.

Adrenalectomy↗
PubMed1990

Steroid hormones and the cardiovascular system: direct actions of estradiol, progesterone, testosterone, gluco- and mineralcorticoids, and soltriol [vitamin D] on central nervous regulatory and peripheral tissues.

Knowledge of steroid hormone sites of action and related effects in cardiovascular and neural regulatory tissues is reviewed. Evidence for nuclear receptor sites is derived mainly from autoradiographic studies with relatively intact tissues and some biochemical studies with tissue homogenates. In the heart and in the walls of blood vessels, estradiol, dihydrotestosterone, corticosterone, aldosterone, dexamethasone, and soltriol (vitamin D) show nuclear binding. In the brain and spinal cord, neuronal regions associated with cardiovascular regulation contain nuclear receptors in specific patterns for each steroid hormones, including progesterone and soltriol. These data indicate that all steroid hormones exert direct actions on the cardiovascular system at its different levels of organization, thus enabling adjustment to the changing demands during reproduction (gonadal steroids), stress (adrenal steroids), and solar seasons (vitamin D-soltriol).

Adrenal Cortex Hormones↗
PubMed1990

Colocalization of atrial natriuretic factor (ANF) and estradiol in hypothalamic neurons by combined autoradiography-immunohistochemistry.

The distribution of estrogen target neurons which contain atrial natriuretic factor (ANF) in female rat hypothalamus was investigated by thaw-mount auto-radiography combined with immunocytochemistry using tritium-labeled estradiol and antibodies against ANF. Colocalization of the two hormones was found in the arcuate nucleus, periventricular nucleus, lateral ventromedial nucleus, ventral premammillar nucleus and lateral basal hypothalamus. The percentage of ANF containing cells which concentrate estradiol varies among the different hypothalamic nuclei with the highest number of ANF-positive cells showing nuclear concentration of 3H-estradiol (80-90%) in the nucleus premammillaris ventralis, but less (5-15%) in the other nuclei. These data, together with topographical correspondence in extrahypothalamic brain regions between sites of action of estradiol and production of ANF, suggest extensive interrelationships and modulatory effects of estradiol on ANF production and secretion in the brain, similar to the atrium of the heart.

Animals↗
PubMed1990

Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography.

Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.

Administration, Topical↗
PubMed1990

Sites of action of soltriol (vitamin D) in hamster spleen, thymus, and lymph node, studied by autoradiography.

Siberian hamsters (Photopus sungorus) were injected with 3H dihydroxycholecalciferol (vitamin D, soltriol). Autoradiograms of spleen, thymus, and lymph nodes revealed nuclear concentration of the hormone in a select population of cells in all of these organs. In the spleen, labeled cells were abundant in the red pulp, but sparse in the white pulp. In the periarterial lymphatic sheath (PALS) labeled cells were found predominantly at the outer rim, with a few scattered labeled cells in the inner PALS and in the marginal zone. Lymphocytes, including pyronin-positive plasma cells, did not display nuclear labeling. In the red pulp, some of the labeled cells contained pigmented inclusions in the cytoplasm, while most of the labeled cells did not appear phagocytic under the conditions of the experiment. In the thymus, labeled cells were most numerous in the medulla, but sparse in the cortex. Many of the thymic target cells were larger than the unlabeled lymphocytes, with a large and pale nucleus, sometimes containing a distinct nucleous, and with large and dendritic cytoplasm, having the appearance and distribution of epithelio-reticular cells. In lymph nodes, scattered labeled cells were conspicuous in or near the subcapsular sinus, while other cells did not concentrate radioactivity in their nuclei. The results indicate that nuclear receptors and direct genomic actions for soltriol exist in certain cell populations of lymphatic tissues that probably include reticular cells and a subpopulation of macrophages. These target cells may mediate effects of the steroid on lymphocytes that appear to have no or only very low numbers of nuclear receptors.

Animals↗
PubMed1990

Distribution of progestin-binding cells in estrogen-treated and untreated neonatal mouse uterus and oviduct: autoradiographic study with [125I]progestin.

Progestin-binding sites in uteri and oviducts of estrogen-treated and untreated 8-day-old mice were studied by thaw-mount autoradiography with [125I]progestin. In the untreated uteri, nuclear concentration of radiolabelled progestin was observed in all tissues of the uterus, with strongest nuclear labelling in luminal and glandular epithelia and in stroma. In the estrogen-treated uteri, the degree of labelling was markedly augmented in stroma and muscle, but much reduced in the luminal and glandular epithelia, compared to untreated uteri. In untreated oviducts, nuclear labelling was observed in stroma and muscle in all regions and in epithelium in the isthmic and uterine regions. The epithelium in infundibular and ampullar regions was only scarcely labelled. The estrogen-treatment augmented the labelling in stroma and muscle of the oviduct as in the uterus, but the labelling in epithelium was not affected. These results indicate that estrogen-treatment induces progesterone receptor differentially among tissue compartments both in the uterus and oviduct.

Animals↗