Specific binding of 3H-estradiol to rat prostate nuclear matrix

Specific estradiol binding activities can be demonstrated in nuclear matrix preparations obtained from intact rat prostate nuclei. Some of the characteristics of these in vitro binding activities to intranuclear components are presented and compared to those exhibited by purified nuclear fractions. Examination of the effects of exposure to castration and testosterone on the number of nuclear matrix binding sites revealed that the quantity and quality (Type) of receptors was modified. Furthermore, these changes are prevented when protein synthesis was inhibited.     

3H-estradiol binding to cytosol receptors of the rat olfactory epithelium

Two types of cytosol receptors of 3H-estradiol with high affinity and limited quantity of binding sites (KDI = 1-2 nM, BmaxI = 8 fmoles/mg protein; KDII = 10 nM, BmaxII = 8 fmoles/mg protein) were determined in the rat olfactory tissue. The amount of high affinity receptors of type I does not change with maturation of the rats, and has no sex difference. The role of estradiol receptors in the olfactory tissue of the rats is discussed.     

Localization of 3H-estradiol in the uterus of pregnant and non-pregnant armadillos

Summary The uptake and retention of radiolabeled estradiol by the uterus was examined in the armadillo. One pregnant and two non-pregnant armadillos were treated with 1.4 g/kg body weight of ³H-estradiol (E₂) by injection into the left ventricle, and one non-pregnant animal was injected with both the labeled hormone and 140 g/kg body weight of unlabeled E₂. One and a half hour after injection, the animals were sacrificed and the uteri were removed and processed for autoradiography. In the non-pregnant animals, nuclear localization was observed in the interstitial cells and glandular epithelium of the endometrium and the connective tissue cells and smooth muscle of the myometrium. Additionally, there was a gradation of uptake in the epithelial cells of the endometrium in that the glandular cells of the basal region were heavily labeled, while those cells in the sinusoidal, and luminal regions contained successively less label. The luminal cells were poorly labeled. In the pregnant female, the smooth muscle and glandular cells hypertrophied and their nuclei contained less label than was observed in the non-pregnant animals. The arteries of the myometrium were more easily distinguished in the pregnant animals and the nuclei of the endothelial cells and smooth muscle were more consistently labeled than those of the non-pregnant armadillos.     

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.     

Quantitative autoradiographic assessment of 3H-estradiol uptake in immunocytochemically characterized pituitary cells

Summary Dry-mount autoradiography was combined with peroxidase immunocytochemistry to examine estrogen uptake in four pituitary cell types. Quantification by silver grain counts was used to compare ³H-estradiol uptake in nuclei of pituitary cells 60 min after i.v. injection into short-term (control) and long-term ovariectomized and in long-term thyroidectomized rats. Under all three hormonal states, the order of labeling intensity was: gonadotropes > somatotropes > lactotropes > thyrotropes. Long-term ovariectomy caused a significant increase in estrogen uptake of gonadotropes, somatotropes and lactotropes, while uptake in thyrotropes decreased. Long-term thyroidectomy decreased uptake in somatotropes, lactotropes and thyrotropes while gonadotropes remained unchanged.Supported by NICHHD grant HD-03007 to D.A.K., NICHHD grant HD-03110 to the Biological Sciences Research Center of Child Development Research Institute and a grant from the Rockefeller Foundation to the Laboratories for Reproductive Biology.     

Radioautographic study of the localization of 3H-estradiol, 3H-histamine, and 3H-cyclic adenosine monophosphate in rat uterus]

The method of radioautography has demonstrated that 3H-estradiol adheres to the nuclei of some myometrium cells, 3H-histamine is accepted by the cytoplasm of the majority of the myometrium cells, 3H-cyclic-AMP is selectively bound by the endothelial cells of capillaries and small vessels in all layers of the uterus. From the data obtained it is possible to conclude that estradiol and its mediators, histamine and cyclic-AMP, specifically interact with different cells and with different cell structures.     

Relations between 3H-estradiol uptake and receptor content of estrogen responsive tissues of castrated female rat

The time course of 3H-Estradiol-17 beta (3H-E2) uptake, and estrogen receptor content in estrogen responsive tissues were studied between 0 and 12 h after injection of 0.5 microgram/kg of 3H-E2 or cold E2 injection to castrated adult female rats. The plasma concentration of 3H-E2 between 10 min and 2 h after injection was in the range of the plasma E2 level of cyclic rat. The total 3H-E2 uptake was well correlated with the receptor content in all tissues. The rank order of 3H-E2 uptake was: uterus (Ut) greater than anterior pituitary (Ap) greater than hypothalamus (Ht) greater than plasma. The cytosol 3H-E2 uptake showed its maximal level 10 min after injection in all tissues. Parallel time course between plasma 3H-E2 and cytosol uptake was obtained for each separate tissue. The nuclear 3H-E2 uptake showed its maximal values 2 h after injection with a subsequent decline. Cytosolic estrogen receptor (Rc) content showed a depletion-replenishment cycle after cold E2 injection in all tissues. Nuclear estrogen receptor (Rn) content in Ut increased progressively from 0 to 14 h after injection, but in Ap it showed its maximal level 2 h after injection, declining afterwards. In Ap, nuclear 3H-E2 uptake and Rn level showed parallel time courses. The maximal level of both parameters coinciding with the time of maximal Rc depletion. However, the Rn level in Ut increases more slowly at greater length than the nuclear 3H-E2 uptake, both processes being divergent. These findings are interpreted as the expression of tissular differences in the rate of nuclear receptor formation from the Rc-E complex previously translocated into nucleus and attached to chromatin.     

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.     

Uptake and retention of3H-estradiol by gonadotrophs and lactotrophs in the pituitary glands of the guinea pig,hamster and gerbil

Summary Nuclear uptake and retention of³H-estradiol by luteinizing hormone (LH) and prolactin (PRL) cells was examined in three species of rodents (guinea pigs, hamsters and gerbils) using the combined techniques of immunocyto-chemistry and autoradiography. Castrated animals were injected with³H-estradiol and decapitated 1.5 h later. The pituitary glands were processed for thaw-mount autoradiography followed by conventional immunocytochemical staining for LH and PRL.³H-estradiol accumulated in more than 80% of the anterior pituitary cells in the gerbils, while only 33 and 22% of the cells accumulated³H-estradiol in the hamsters and guinea pigs, respectively. A varying percentage of immunoreactive LH and PRL cells in all three species were found also to contain binding sites for estradiol. Some LH and PRL cells in hamsters and guinea pigs and only some in PRL cells of gerbils were found to be devoid of grains. Quantitative analysis revealed that the number of grains per nucleus differed considerably from cell to cell. LH cells of guinea pigs accumulated much larger amounts of³H-estradiol than did the PRL cells, while the LH cells in the hamsters and gerbils accumulated only slightly more³H-estradiol than the PRL cells.These results confirm the previous observations in rats and baboons that demonstrated tremendous species differences in percentage of cells in the anterior pituitary gland that accumulated³H-estradiol. Also, these data suggest that there are functionally heterogeneous cell types among the LH and PRL cells in hamsters, guinea pigs and gerbils as has been previously demonstrated in rats and baboons.     

In vitro binding of 3H-estradiol to eosinophil and neutrophil granulocytes in various tissues (normal and neoplastic) of newborn and adult rats

In vitro uptake and binding of tritiated estradiol to the specific granules of eosinophil granulocytes has been observed by ultrastructural radioautography in various tissues of the rat (liver and spleen of newborn, small intestine and bone marrow of adult animals). Binding of 3H-estradiol to granules present in the cytoplasm of neutrophil granulocytes has also been observed in bone marrow and in a cholangioma produced by chemical carcinogenesis. Neither type of granulocyte bound 3H-testosterone.