Dry-mount Autoradiography Research Articles

Preparation of fresh-frozen dried ultrathin sections by drying without vacuum.

Fresh-frozen dried ultrathin sections were prepared without fixation, embedding or staining. Small blocks of tissues were chilled by making contact with copper blocks precooled with liquid nitrogen according to a procedure modified from that of Christensen. Sections were cut at -95 degrees C with a Reichert Um 2 ultramicrotome fitted with cryoaccessories, using dry glass knives. The sections were dried for 15 min or more on formvar-coated grids by a flow of dry nitrogen gas at -95 degrees C in a newly devised glass chamber placed in the cryokit compartment. The sections showed only a few holes attributable to ice crystal formation. Holes were found in the intercellular spaces as well as within cells. The sections were observed without staining and gave enough contrast to permit recognition of organelles such as nucleus, perinuclear cisternae, nucleolus, interchromatin granules, perichromatinic granules and secretory granules in the pancreatic exocrine cells. In the basal part of the proximal convoluted tubules of mouse kidney, abundant mitochondria were seen. Mitochondrial cristae and lines corresponding to the basal infoldings of the renal convoluted tubules were less dense than their surroundings. This method can yield ultrathin sections lending themselves to investigations of the distribution of elements with electron-probe X-ray microanalyzer and of diffusible substances by dry-mount autoradiography at the electron-microscopic level.

Estradiol receptor in uterine tissue from neonatal mice. Influence by cyclic AMP

The estradiol receptor in uterine tissue of neonatal mice has been studied. It is present neonatally and fulfills normal criteria as regards sedimentation behaviour, specificity etc. The influence of dibutyryl-cyclic AMP (db-cAMP) on receptor characteristics has been studied using sucrose gradient centrifugation and dry mount autoradiography. A consistent change in the cytosol receptor sedimentation behaviour is found when db-cAMP is included in the incubation medium of whole uteri preparations. A depression in the 8s receptor region is found simultaneously with an increase in the 4s region. There is no uptake of tritiated estradiol as seen in autoradiograms from organ cultures of neonatal uterine cervix preparations, when these have been cultured for 48 h. When db-cAMP is included in the incubation medium, a pronounced uptake is seen in the sections. Different possible points of action of cyclic AMP related to the findings described, are discussed.

Autoradiographic localization of 3H-estradiol or its metabolites in the central nervous system of the developing rat.

The cellular and subcellular localization of radioactivity in the brain of 2-day-old female rats was investigated by dry-mount and thaw-mount autoradiography 2 hr after SC injection of 1.0 µg of 2,4,6,7 3H estradiol-17β per 10 g body wt. A specific topographic pattern of nuclear concentration of estrogen was obtained similar to that of the adult rat in specific neurons of the basal hypothalamus, preoptic region and amygdala, thus providing the first in vivo evidence for estrogen receptors in the central nervous system (CNS) ofthe developing rat during the “critical period” of brain organization. In competition experiments, nuclear concentration of radioactivity was inhibited in all areas studied with estradiol, while no inhibition wasobtained with 5α-dihydrotestosterone. Testosterone clearlyinhibited the nuclear concentrationof radioactivity in all areas studied except the nucleus preopticus medialis in which either noinhibition or only a partial inhibition occurred. The implications of this selective com...

Cellular and Subcellular Localization of3H-Progesterone or Its Metabolites in the Oviduct, Uterus, Vagina and Liver of the Guinea Pig

The cellular and subcellular distribution of 3H-progesterone or its metabolites was studied in the oviduct, uterus, vagina, liver and diaphragm in 30-day-old estrogen-primed and non-primed ovariectomized guinea pigs using drymount autoradiography. In autoradiograms from estrogen-primed and untreated ovariectomized guinea pigs, prepared 15 min or 30 min after the intravenous injection of l,2,6,7-3H-progesterone, a nuclear concentration of radioactivity was observed in the oviduct in the luminal epithelium, connective tissue and muscularis; in the uterus: in the luminal and glandular epithelium, stroma and muscularis; and in the vagina: in the basal layers of the stratified squamous epithelium, connective tissue and interlacing bundles of smooth muscle. In the nonprimed guinea pigs, the nuclear concentration of radioactivity was less pronounced with exception of the glandular cells. In the diaphragm, no selective cellular and subcellular concentration of radioactivity existed. In the liver, similar to diaph...

Neurons of the hypothalamus concentrate (3H)progesterone or its metabolites.

Selective concentration of [(1)H]progesterone or its metabolites is observed in nuclei of neurons in certain hypothalamic regions of the guinea pig 15 minutes after injection of [1,2,6,7-(3)H]progesterone, by use of dry-mount autoradiography. Highest concentrations of progestin target neurons exist in the nucleus arcuatus, the nucleus preopticus periventricularis, and the nucleus preopticus suprachiasmaticus. Previous administration of unlabeled progesterone inhibits the nuclear concentration of radioactivity, but cortisol has no effect. Estradiol priming enhances the nuclear uptake of radioactivity. The results demonstrate the existence of progestin target sites in the hypothalamus and suggest a direct action of progestin on certain hypothalamic structures.

Autoradiographic localization of radioactivity in the rat brain after the injection of 1,2-3H-testosterone.

The localization of radioactivity in the brain of immature intact and mature castrated Sprague—Dawley rats was investigated by drymount autoradiography at 1 hr after the injection of l,2–3H-testosterone. Radioactivity is found to be selectively concentrated and retained in specific neurons in the nucleus (n.) arcuatus and the n. ventromedialis hypothalami, the n. preopticus medialis, the n. interstitialis striae terminalis, the n. septi lateralis, the hippocampus and the amygdala. The topographic distribution of androgenconcentrating neurons agrees well with areas in the brain that have been associated with the regulation of gonadotropin secretion and male sex behavior. The autoradiographic data are consistent with the hypothesis that androgen feedback occurs at hypothalamic and extrahypothalamic sites in the central nervous system. (Endocrinology 92: 251, 1973)

Modification by progesterone of oestradiol-induced cell proliferation, RNA synthesis and oestradiol distribution in the rat uterus.

Summary. Progesterone pretreatment (5 mg/day) of ovariectomized— adrenalectomized rats for at least 12 hr abolished the response of the uterine luminal epithelium to oestradiol (0·2 μg) with respect to (i) mitotic activity, (ii) nucleolar enlargement, and (iii) [3H]uridine uptake, and suppressed the uptake of [3H]oestradiol by the epithelial cells, as judged by dry-mount autoradiography. Progesterone pretreatment for periods exceeding 36 hr redirected the mitogenic action of oestradiol and its stimulatory action on [3H]uridine uptake from the epithelium to subepithelial stromal cells. The latter are not responsive to the action of oestradiol on its own. High resolution (EM) autoradiography revealed preferential accumulation of [3H]uridine over the stromal nucleoli, supporting the view that the bulk of the RNA synthesized as an early response to oestradiol is ribosomal precursor RNA. In the glandular epithelium, progesterone prevented the induction of mitotic activity, but not the stimulation of [3H]uridine uptake, by oestradiol; the ability to accumulate [3H]oestradiol remained unaffected. Rats injected with oestradiol during delayed implantation showed a mitotic response in the stroma, but not in the luminal or glandular epithelium, and failed to accumulate [3H]oestradiol in the luminal epithelium. In pregnant and pseudopregnant rats, mitotic activity declined sharply in epithelial and glandular cells after Day 2 post coitum. This was followed by a sharp rise in the rate of thymidine incorporation and cell division of the subepithelial stromal cells. The reciprocal changes in these tissues, previously described in the pregnant mouse, are ascribed to an interaction between progesterone and oestradiol. It is inferred that significant oestrogen secretion commences late on Day 2 post coitum, i.e. well before the time postulated for the preimplantation `oestrogen surge.' The rate of DNA synthesis in pseudopregnant rats reached a peak on Day 5 post coitum, coinciding with maximal uterine sensitivity to deciduoma-inducing stimuli, but mitotic rate in both pregnant and pseudopregnant rats declined sharply before the morning of Day 5, suggesting a prolongation of the G2 phase of the mitotic cycle at that time.

Nuclear Concentration of <SUP>3</SUP>H-estradiol in Target Tissues of a Fetal Rat Demonstrated by Dry Mount Autoradiography

Distribution of radioactivity in various tissues has been studied by dry mount autoradiography after the injection of 3H-estradiol in female fetal rats. 3H-estradiol was found to be concentrated in nuclei of cells of the oviduct. Radioactivity is also concentrated in the cell nuclei of the ovary, suggesting some effect of estradiol on these nuclei of cells. In such non-target tissues, as intestine, kidney, lung, liver and muscle, no nuclear concentration of radioactivity was found and average silver grain density per 100μ2 is much lower than that of the oviduct. Although, in the adrenal, radioactivity was found to be randomly distributed in cytoplasm as well as in nuclei, the average silver grain density per 100μ2 in nuclei was relatively high compared to that of the other non-target organs. It seemed that the estrogen exerts some effect on the adrenal of a fetal rat.

Probable sites for estrogen receptors in brain and pituitary.

In the pituitary and areas of the brain the cellular and subcellular distribution of 3H-estradiol-17β has been studied by dry-mount autoradiography. Anterior pituitary cells and certain neurons show nuclear concentration and retention of the hormone similar to peripheral target tissues. This is interpreted, in conjunction with other data, as suggestive evidence of a genomic effect of estrogen in central target tissues, resulting in a stimulatory “feedback” (production of hormonal messengers) which may lead to a negative feedback effect on gonadal estrogen secretion by indirect action.

Effects of estrogens and progesterone on electrolyte balances in normal dogs.

ARTICLESEffects of estrogens and progesterone on electrolyte balances in normal dogsJA Johnson, JO Davis, JS Baumber, and EG SchneiderJA Johnson, JO Davis, JS Baumber, and EG SchneiderPublished Online:01 Dec 1970https://doi.org/10.1152/ajplegacy.1970.219.6.1691MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByEffect of glucocorticoid administration on serum aldosterone concentration in clinically normal dogsAmerican Journal of Veterinary Research, Vol. 71, No. 6Estrogen modulates ClC-2 chloride channel gene expression in rat kidney17 June 2003 | Pflügers Archiv - European Journal of Physiology, Vol. 446, No. 5Response of primary rabbit kidney proximal tubule cells to estrogensJournal of Cellular Physiology, Vol. 178, No. 1Effect of testosterone on carbonic anhydrase and mg2+-dependent HCO3stagered−-stimulated ATPase activities in rat kidney: Comparison with estradiol effectComparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, Vol. 114, No. 2ReferencesArterial sodium concentration in rats with hypertension induced by mestranolContraception, Vol. 48, No. 1Receptors, Mechanism of Action and Biological Responses of Hormones in the Fetal, Placental and Maternal CompartmentsDiverse effects of estradiol-17β: Concurrent suppression of appetite, blood pressure and vascular reactivity in conscious, unrestrained animalsPhysiology & Behavior, Vol. 45, No. 3Premenstrual Tension and Idiopathic EdemaIdiopathic Edema in a MaleThe American Journal of the Medical Sciences, Vol. 288, No. 1Role of sex steroid hormones in relative refractoriness to angiotensin II during pregnancyAmerican Journal of Obstetrics and Gynecology, Vol. 149, No. 2Neuroendocrine mechanisms mediating fluid intake during the estrous cycleBrain Research Bulletin, Vol. 12, No. 2The effects of adrenal and gonadal steroids and K+-canrenoate on the metabolism of aldosterone by rat liver microsomesSteroids, Vol. 42, No. 3Premenstrual syndromeAmerican Journal of Obstetrics and Gynecology, Vol. 139, No. 1Simultaneous localization of [3H]estradiol and neurophysin I or arginine vasopressin in hypothalamic neurons demonstrated by a combined technique of dry-mount autoradiography and immunohistochemistryNeuroscience Letters, Vol. 17, No. 1-2Spontaneous NaCl Appetite Induced by Administration of an Oral Contraceptive and Its Components to RatsSex hormones and related compounds, including oral contraceptivesSpecific estradiol-17β binding component in adult rat kidneyJournal of Steroid Biochemistry, Vol. 11, No. 4Östrogenrezeptoren in der menschlichen NiereDistribution of Oestradiol Receptor Molecules in Human KidneyClinical Chemistry and Laboratory Medicine, Vol. 15, No. 1-12Human kidney steroid receptorsJournal of Steroid Biochemistry, Vol. 7, No. 10Oral contraceptive-induced hypertension—Nine years laterAmerican Journal of Obstetrics and Gynecology, Vol. 126, No. 1Mechanism of Action of the Sex Steroid HormonesNew England Journal of Medicine, Vol. 294, No. 24Cellular Receptors and Mechanisms of Action of Steroid HormonesEstrogens and the kidneyKidney International, Vol. 6, No. 5Cyclic changes of human mammary gland epithelium in relation to the menstrual cycle—an ultrastructural study27 June 2006 | Cancer, Vol. 34, No. 3Effects of Aldosterone on Blood Pressure, Water, and ElectrolytesEstradiol renal receptor molecules and estradiol-dependent antinatriuresisKidney International, Vol. 2, No. 2 More from this issue > Volume 219Issue 6December 1970Pages 1691-1697 Copyright & PermissionsCopyright © 1970 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1970.219.6.1691PubMed5485687History Published online 1 December 1970 Published in print 1 December 1970 Metrics

Nuclear concentration of androgens in rat seminal vesicles and prostate demonstrated by dry-mount autoradiography.

The distribution of androgens in the ventral prostate and the seminal vesicle was studied by dry-mount autoradiography at different time intervals after the injection of tritiated testosterone. At hr, 1 hr and 3 hr, a nuclear concentration of radioactivity existed in the epithelial cells of the prostate and the seminal vesicle, whereas at 5 min radioactivity was diffusely distributed at high intensity in all structures. Maximum nuclear labeling appeared at 1 hr. With increased exposure time, at 7 hr nuclear concentration of radioactivity could also be demonstrated in the epithelial cells of both organs. The intensity of the concentration of radioactive androgens in the nuclei appeared similar in the ventral prostate and the seminal vesicle. No such selective labeling occurred in liver, diaphragm, and in the muscle layers of the prostate and the seminal vesicle. Ventral prostate from rats treated with 3H-estradiol did not show any specific nuclear concentration of estradiol. Smear autoradiograms of cell nu...

Estradiol-concentrating neurons: topography in the hypothalamus by dry-mount autoradiography.

Serial section autoradiograms were prepared of different planes of the hypothalamus and diencephalon of immature female, immature male, and ovariectomized mature rats injected with 6,7-(3)H-estradiol-17 beta. Known causes of diffusion and redistribution of the label, such as fixation, embedding, and thawing, were eliminated by the use of an autoradiographic technique based on the dry-mounting of freeze-dried sections. Neurons that concentrate estradiol exist in distinct and definable anatomical areas that are independent of the sex and hormonal state of the animals. Distribution of these neurons follows known terminations of the stria terminalis, which supports the concept of an endocrine amygdaloid-hypothalamic-hypophysial axis.

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

The cellular and subcellular localization of 6.7-3H-estradiol-17β 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 μg 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.