Preparation of tissues for localization of sex hormones by autoradiography

Summary The intracellular localization of ³H-oestradiol in various tissues was examined. For this purpose standard autoradiograms were prepared after freeze drying, osmium vapour fixation and Epon embedding. The radioactive material showed a preferential localization in all the tissues studied, viz. uterus, vagina and anterior pituitary gland. In all tissues a major proportion of the radioactivity was associated with the cell nuclei. Radioactive labelling outside the cell boundaries and in the surrounding connective tissue was almost lacking. Likewise, a very low number of silver grains were found over the Epon in the clefts and at the margin of the tissue sections. The observations appear to indicate that the diffusion and probably also the intracellular dislocation of the radioactive material are very limited.The validity of the results was examined. Pre- and postmortem changes, rapid freezing and osmium vapour fixation were not observed to influence the localization of the radioactivity. Furthermore, the localization was the same whether embedding was carried out at –10°C or at 50°C.It is concluded that freeze drying, osmium vapour fixation and Epon embedding provide a valid method for accurate intracellular localization of steroid sex hormones.This work was supported by grants from The Norwegian Cancer Society and by Nordisk Insulinfond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

Neutral endopeptidase 24.11 in rat peripheral tissues: comparative localization by 'ex vivo' and 'in vitro' autoradiography

The neutral endopeptidase 24.11 (NEP) also called 'enkephalinase' thanks to its inactivation of enkephalins in the brain, was also recently shown to be involved in the degradation of the circulating atrial natriuretic peptide (ANP). Inhibitors of NEP are therefore under clinical trials as new analgesics or antidiarrheal agents, protecting centrally or peripherally released opioid peptides and as novel antidiuretics and anti-hypertensives in prolonging the renal and vascular actions of NEP. It was therefore important from a clinical point of view to investigate the distribution in peripheral tissue of a systemically administered NEP blocker. Different concentrations of the radiolabelled inhibitor [3H]HACBO-Gly have been intravenously injected in rat and the distribution studied using whole-body sections at different times by 'ex vivo' and 'in vitro' autoradiography to investigate differences in tissue accessibility of NEP to a circulating inhibitor. In vivo [3H]HACBO-Gly binding was fully prevented by an excess of unlabelled inhibitor and disappeared rapidly mainly through renal elimination. NEP labelling was prominent in kidney, liver, lung, fat deposits in the neck region, the flat bones of the skull, the mandibula, the vertebrae, the long bones of the limbs, articular cartilages and synoviae. A lower labelling was found in the intestine, the glomeruli and the submaxillary glands. [3H]HACBO-Gly binds also to a limited number of peripheral tissues in which the presence of NEP was yet unknown (bones, parts of adipose tissues. Some tissues, not labelled in vivo, exhibited various degrees of labelling under in vitro conditions (the brain, some portions of the gut, the testes, the prostate). Interestingly, few lobules of the submaxillary glands were much more densely labelled suggesting the possible occurrence of NEP heterogeneity. Except for the brain, the physiological function of NEP in various tissues remains largely unknown, but this ectoenzyme is likely involved in inactivation of regulatory peptides such as: ANP (partially in the kidney), SP in the lung and possibly somatostatin and ANP in bone, ANP in adipose tissue, enkephalin in testes, immune peptidic factors in bone marrow. A part of NEP in bone marrow corresponds probably to the common acute lymphoblastic antigen, CALLA, densely expressed on pre-B cells. Finally, it is important to notice that several tissues containing important concentrations of NEP (brain, testes, prostate, eye, gut, brush border) are inaccessible to the i.v. injected inhibitor thanks to the presence of functional barriers.     

Autoradiographic localization of sex steroid hormones in the lymphatic organs of baboons

Summary The localization of radiolabeled estradiol and dihydrotestosterone was examined in the lymphatic organs of both male and female baboons. A total of 12 baboons were divided into two groups, each containing three males and three females. Each animal in one group, both males and females, was injected intravenously with 1 g/kg body weight of ³H-estradiol while those in the second group were each injected with 1 g/kg body weight of ³H-dihydrotestosterone. As controls, one male and one female from each group also received a dose of 100 g/kg body weight of the corresponding unlabeled steroid. One and a half hours after the injections, the animals were sacrificed and the spleen, thymus, and inguinal lymph nodes removed and processed for autoradiography. The localization of ³H-estradiol was similar in both males and females. In the thymus fibroblasts and epithelio-reticular cells, but not thymocytes, localized ³H-estradiol. In lymph node and spleen, nonlymphoid tissue concentrated the labeled estrogen. Additionally, in the paracortical region of the lymph node, an unknown cell type was labeled with estrogen. Only one male baboon demonstrated nuclear localization of ³H-dihydrotestosterone. This was observed in the reticular cells in the spleen and lymph nodes. The same cell type in the organs of the remaining animals was unlabeled.     

Subcellular localization of the cyanogenic glucoside of sorghum by autoradiography

The use of microautoradiography at the electron microscopic level indicates that the vacuole is the site of accumulation of the cyanogenic glucoside of Sorghum bicolor. When a specific biosynthetic precursor of dhurrin, p-hydroxy[3,5-(3)H]phenylacetaldoxime, was used, 90% of the tritium label was recovered in the vacuoles of tissue prepared for microautoradiography. l-[3,5-(3)H]Tyrosine and d-[1-(3)H(N)]glucose, nonspecific precursors of dhurrin, of differing solubilities and biosynthetic capacity, were also fed to the shoots. The data obtained from these controls indicated that the high recovery of label in the vacuole of aldoxime-fed shoots was not indicative simply of the size of the vacuole, nor was it a result of movement of labeled compounds during preparation of the tissue for electron microscopy. The problem of movement of these labeled compounds during dehydration of tissue was dramatically reduced by chemical dehydration in 2,2-dimethoxypropane in less than 30 minutes rather than with routine dehydration in acetone or alcohol series for 24 hours.     

Ultrastructural localization of zinc in rat exocrine pancreas by autoradiography

Summary ⁶⁵Zn was infused at a constant rate for 10 days into a rat. Glutaraldehyde fixed, Epon-araldite embedded ultrathin sections of pancreatic tissue were coated with Ilford L4 emulsion and at 211 days exposure were developed. Silver grains were found over the zymogen granules and over the rough endoplasmic reticulum of exocrine cells. Islet tissue was not observed in these studies. The failure of other zinc localization methods to demonstrate zinc in acinar tissue is discussed as are some of the pitfalls of the autoradiographic method and suggestions for future improvement.Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, Madison. Supported in part by USPHS Research Grant AM-05606 from the Nat. Institute of Arthritis and Metabolic Diseases.Supported by an NIH post-doctoral fellowship.     

The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.     

Validation of bromodeoxyuridine immunohistochemistry for localization of S-phase cells in decalcified tissues. A comparative study with tritiated thymidine autoradiography

Summary Immunohistochemical detection of the thymidine analogue 5-bromo-2-deoxyuridine (BrdUrd), which is incorporated by S-phase cells, offers a convenient way of studying the proliferation kinetics of cells in normal skeletal tissues and in bone containing/derived tumours. To assess the validity of using this approach on decalcified, paraffin embedded tissues, the BrdUrd method was compared with tritiated thymidine (³H-TdR) autoradiography, using rat tibiae labelled with both³H-TdR and BrdUrd, fixed in Carnoy's fluid and decalcified in EDTA, prior to routine paraffin embedding. The distribution of BrdUrd-labelled cells correlated with the sites of cell proliferation in the growing rat tibia.Independent studies with each method on paired serial sections of double-labelled tissue, showed a highly significant correlation (r=0.81, p<0.0003) in the numbers of labelled cells seen in autoradiographs and immunostained sections from the proximal tibial growth plate. Combined BrdUrd immunohistochemistry and³H-TdR autoradiography showed that the majority of labelled cells in cartilage, bone marrow, and fibrous perichondrium and periosteum had incorporated both labels. These results show that BrdUrd immunohistochemistry is a valid technique for the study of dividing cells in mineralized tissues after decalcification.     

Cellular and subcellular 3H-estradiol localization in the pituitary by autoradiography

Summary The cellular and subcellular localization of 6.7-³H-estradiol-17 in the pituitaries of five immature and two mature castrated female rats was studied by autoradiography. The autoradiographic technique used minimizes or eliminates translocation. It is based on low temperature tissue preparation of freeze-dried sections which are dry-mounted on dried photographic emulsion, excluding known sources of translocation artifacts such as liquid fixatives, dehydrating and clearing fluids, embedding media and thawing. The animals were given from 0.093 to 0.63 g of ³H-estradiol and were sacrificed at 15 min, 1, 2, or 6 hours after the injection. A large portion of the anterior pituitary cells was found to be labeled; the extent of this labeling varied with dose, time of sacrifice after the injection, and photographic exposure time, but apparently not with the endocrine status of the animal. The portions labeled were 76 and 86 per cent for the immature and mature rats respectively, exceeding single tinctorial light-microscopic groups. Gomori trichrome chromophiles and chromophobes, cells with intense and weak pyronin basophilia, as well as morphologically defined castration cells, were all partially labeled and unlabeled. Acidophiles appeared to be labeled in a somewhat higher proportion. Cells of the intermediate and posterior lobe were generally unlabeled except for occasionally interspersed cell groups or single cells, especially at the border between intermediate and posterior lobe, probably identical with basophilic invaginations in man and other mammals. The subcellular concentration of radioactivity was always nuclear. The findings are interpreted as suggesting a) feedback control on the pituitary level, in addition to the diencephalic level, b) pluripotentiality of anterior pituitary cells, and c) possible positive feedback mechanism of estradiol with secondary negative effect. Dry-mount autoradiography with labeled hormones, as applied in this study, provides a new methodological approach to the elucidation of pituitary physiology and pharmacology.Supported by USPHS Grants 1-ROl-AM-12, 649-01, GRS Grant FR-5367, ACS Grant IN-41-H, and Otho Sprague Memorial Institutional Grant. — The author thanks Dr. N. S. HALMI for consultation.     

Beta adrenergic receptor localization in rat brain by light microscopic autoradiography

The characteristics of ³H-dihydroalprenolol (³H-DHA) binding to mounted tissue sections of rat brain were studied. The binding had all the characteristics of a beta-receptor. It was reversible, saturable (K_D 2.3 nM, B_max 23 fmol/mg tissue, wet wt) and was inhibited only by beta-adrenergic drugs. The forebrain binding had the properties of a beta-1 receptor, while cerebellar binding had beta-2 characteristics. Beta-adrenergic receptors were widely distributed in the rat brain. High concentrations were localized in the superficial layers of the neocortex, in the caudate-putamen, nucleus accumbens, olfactory tubercles, substantia nigra, nucleus interpeduncularis, subiculum and pia mater. Areas containing intermediate concentrations of receptors included the cerebellum, hippocampus and thalamus. Areas containing low concentrations of receptors included the hypothalamus, amygdala, brainstem and medulla. Co-incubation with low concentrations of zinterol to preferentially block beta-2 receptor resulted in an inhibition of most of the binding to the cerebellum and pia matter and produced only a small and generalized decrease in the rest of the brain. Beta receptors were found in many areas known to contain noradrenergic nerve terminals. Paradoxically some areas with high densities of dopaminergic nerve terminals, had high densities of receptors. Results of electrophysiologica and lesion studies are also discussed.     

Modification of postsynaptic dopaminergic sensitivity by female sex hormones

Stereotyped behavior in response to d-amphetamine or apomorphine was significantly reduced in female guinea pigs following cophorectamy. Responsiveness to either dopamine agonist was significantly increased by the subsequent chronic administration of either estradiol valerate or progesterone. Modification of postsynatpic dopaminergic sensitivity by female sex hormones may underly the clinical association of hyperkinetic movement disorders, psychosis or depression seen with alterations in female sex hormones in man.     

Neuroendocrine effect of sex hormones

The paper provides a generalization of data and the results of own experiments on influence ovarian steroids on the hypothalamus and other brain areas related to reproduction. Ovarian hormones have widespread effects throughout the brain: on catecholaminergic neurons and serotonergic pathways and the basal forebrain cholinergic system, as well as the hipocampus, spinal cord, nigrostriatal and mesolimbic system, in addition to glial cells and blood-brain barrier. The widespread influences of these various neuronal systems ovarian steroids have measurable effects on mood and affect as well as on cognition, with implications for dementia. There are developmentally programmed sex differenced in hippocampal structure that may help to explain differences in the strategies which male and female rats use to solve spatial navigation problems. The multiple sites and mechanisms of estrogen action in brain underlie a variety of importants effects on cognitive and other brain functions--coordination of movement, pain, affective state, as well as possible protection in Alzheimer's disease. Estrogen withdrawal after natural or surgical menopause can lead to a host of changes in brain function and behavior.