Richard F. Hoyt

ORIGINAL RESEARCH—BASIC SCIENCE: Differential Regulation of the Expression of Estrogen, Progesterone, and Androgen Receptors by Sex Steroid Hormones in the Vagina: Immunohistochemical Studies

OBJECTIVE Significant structural changes occur in the rat vagina in response to sex steroid hormone deprivation and replacement. However, the mechanism by which these changes occur is not clearly understood and our current hypothesis is that these effects are mediated, at least in part, by the expression of sex steroid hormone receptors. The goal of this study was to assess changes in steroid hormone receptor expression and distribution in response to sex steroid hormone deprivation and administration. METHODS Female rats were either kept intact (controls) or ovariectomized. Ovariectomized animals were treated with vehicle, estradiol, testosterone, progesterone, or hormone combinations. Using immunohistochemistry, hormone receptor distribution was assessed in all layers of the vaginal wall. RESULTS After ovariectomy, estrogen receptor alpha (ERalpha) was up-regulated and progesterone receptor (PR) was down-regulated. Estradiol replacement restored these ovariectomy-induced changes, and this effect was dose-dependent. Androgen receptor (AR) expression was unaffected by ovariectomy or estradiol replacement. However, testosterone treatment resulted in increased AR density in the muscularis. Addition of either testosterone or progesterone to estradiol mitigated but did not abolish the effects of estradiol alone. CONCLUSION Estradiol down-regulated ERalpha and up-regulated PR expression in the vagina, suggesting this may be a mechanism to prevent continued proliferation of the epithelium by surges of estradiol during the estrous cycle.

Small-granule (neuro)endocrine cells in the infracardiac lobe of a hamster lung. Number, subtypes, and distribution.

Small-granule APUD (amine precursor uptake and decarboxylation) endocrine cells were surveyed in 600 3 microns glycol methacrylate-embedded, periodic acid-Schiff (PAS)-lead hematoxylin-stained serial sections comprising 95% of the infracardiac lobe of a hamster lung. Results were confirmed by less systematic study of other hamster lungs. Positions of endocrine cells were marked on cardboard profiles of bronchi and bronchioles for assembly into a 70 X enlarged three-dimensional model from which size and branching of the airway were determined. Records were made for computer analysis of the number and staining patterns of endocrine cells, the nature of contiguous epithelial cells, and the presence of underlying smooth muscle and blood and lymph vessels. APUD cells occurred in 95% of all airways, at a mean density of six solitary cells and 10 cell clusters (neuroepithelial bodies) per millimeter of airway length, measured along the bronchial-bronchiolar long axis. Nineteen percent of endocrine cell loci (29% of all cells) were found at bronchioloalveolar portals in all regions of peripheral lung. Twenty percent of loci (28% of all cells) occurred about the origins of lateral airway branches; these included 4% of loci on carinal points of bifurcation. Two groups of APUD cells had distinctive anatomic relationships: 1) 13% of loci (20% of cells) were related to pulmonary capillaries and venules, mainly at bronchioloalveolar portals; and 2) 39% of loci (53% of cells) overlaid peribronchial muscle, mainly in larger airways where changes in diameter might affect ventilation. In this lobe, APUD cells were not related to goblet or mast cells; 74% of loci abutted Clara and/or ciliated cells, 17% great alveolar cells. Few loci were associated with pulmonary arteries and veins. Five APUD cell types were identified by PAS-lead hematoxylin staining. Types I, II, and V, with granules approximately 0.2 micron in diameter, made up 38%, 45%, and 2% of cells, respectively. Types III and IV, 10% and 5% of cells, respectively, had larger granules. Types I, II, III, and V occurred as solitary cells as well as in neuroepithelial bodies. One-third of the neuroepithelial bodies contained a single cell type; the rest were mixed. Type IV cells, with coarse lead hematoxylin-positive granules, usually were found in large neuroepithelial bodies containing two to four cell types and were never seen occurring alone. We conclude that 1) hamster lungs contain different kinds of APUD cells; 2) those likely have a variety of functions; 3) distinctions merely between solitary and clustered cells may not be significant; 4) the histophysiology of many neuroepithelial bodies probably is more complex than previously suspected; and 5) PAS-lead hematoxylin is superior to argyrophilia and amine fluorescence for light microscopic counting and analysis of pulmonary APUD cells in this species.

An unusual bronchial carcinoid tumor: Light and electron microscopy

An unusual bronchial carcinoid tumor was studied by light and electron microscopy. The tumor cells, which appeared to be monotonously uniform in hematoxylin and eosin stained sections, were found to be morphologically heterogeneous at the ultrastructural level with regard to the size, number, and morphology of the endocrine granules. Presumptive endocrine granules were seen in all tumor cells, but some cells contained only small round granules (2000 A largest diameter), other cells contained large round granules (some with as large a diameter as 1.0 mu), and some cells contained large polymorphic granules. Many of the cells stained positively at the light microscopic level when selective stains for endocrine cells were applied. All types of granules showed argyrophilia at the ultrastructural level. Numerous clusters of endocrine cells were observed in the otherwise normal bronchial and bronchial glandular epithelium. The spectrum of granule morphologies, as seen in the tumor cells, was displayed in cells of the intraepithelial clusters. Some mucous cells and sparsely ciliated cells within these clusters contained argyrophilic granules. Multiple continuities existed between the epithelial endocrine cell clusters and the underlying tumor mass. The intraepithelial clusters represent foci of carcinoma in situ, the genesis of which is discussed.

Estradiol Ameliorates Diabetes-Induced Changes in Vaginal Structure of db/db Mouse Model

INTRODUCTION Women with diabetes experience diminished genital arousal, reduced vaginal lubrication, vaginal atrophy, dyspareunia, and increased infections. Limited studies are available investigating the effects of diabetic complications on the vagina. AIMS The goals of this study were to investigate type 2 diabetes-induced changes in vaginal structure, and to determine if estradiol treatment ameliorates these changes. METHODS Eight-week-old female diabetic (db/db) mice (strain BKS*Cg-m+/+Lepr(db)/J) and age-matched control normoglycemic female littermates were used to investigate the effects of type 2 diabetes on vaginal tissue structural integrity. Diabetic animals were divided into two subgroups: diabetic treated with vehicle only and diabetic treated with pellets containing estradiol. At 16 weeks, the animals were sacrificed, and the vaginal tissues were excised and analyzed by histological and immunohistochemical methods to assess diabetes-induced changes in vaginal tissue and the extent by which these parameters are restored by estradiol treatment. MAIN OUTCOME MEASURES The effects of type 2 diabetes and estradiol supplementation were investigated on vaginal histoarchitecture. RESULTS Diabetic animals exhibited high blood glucose levels (>600 mg/dL), increased body weight (43.0 +/- 6.0 g vs. 24.4 +/- 2.0 g), and reduced plasma estradiol levels (65.5 +/- 6.6 pg/mL vs. 80.77 +/- 13.2 pg/mL) when compared to control animals. Diabetes resulted in significant thinning of the epithelium (P <or= 0.05), marked decrease in the muscularis area (P </= 0.05), distinct truncation of elastic fibers, and significant reduction of the nitrergic immunoreactive nerve fibers (P <or= 0.05). Treatment of diabetic animals with estradiol restored epithelial thickness (P <or= 0.05), muscularis area (P <or= 0.05), and elastic fiber distribution, and partially restored the density of nitrergic nerve fibers. CONCLUSIONS The data in this study demonstrate that type 2 diabetes disrupts vaginal structural integrity and that estradiol supplementation ameliorates the diabetes-induced vaginal pathology.

An unusual bronchial carcinoid tumor analyzed by conjunctive staining

Abstract Two staining methods selective for pulmonary APUD endocrine cells-PAS-lead hematoxylin and a more elaborate procedure termed conjunctive staining-were applied to 2 μm. glycol methacrylate sections of a human bronchial carcinoid tumor, in order to display the advantage they hold over routine pathologic methods in revealing the presence as well as cytochemical characteristics of endocrine cell granules in the tumor cells. PAS-lead hematoxylin alone showed the tumor population to be mixed, with a wide range of staining in the granules, extending between the extremes of PAS only and lead hematoxylin only. The conjunctive staining protocol is described in detail. In tissue previously reacted for argyrophilia, it demonstrates, sequentially, fluorescence for serotonin, PAS alone, and PAS-lead hematoxylin, all in a single section. By comparing photomicrographs of a given field recorded during the process, exact point by point correlations of staining behavior can be made, limited only by the resolving power of the microscope. Conjunctive staining of the tumor revealed 10 distinct cytochemical cell signatures, far exceeding the number of types of endocrine cells known to exist in normal human lung. Preliminary results indicate that three types with PAS and lead hematoxylin positive granules may occur in adult human bronchi, although previous studies have reported but a single type of endocrine cell. Numerous tumorlets in the bronchial epithelium overlying the tumor were an unusual feature of the case. These were composed of cells staining similarly to cells in the tumor mass. Some were connected to it by cords of cells, whereas other tumorlets were interconnected in the epithelium, but a number of smaller ones were clearly isolated from other tumor cells, as shown by serial sections. A few tumorlets contained cells with both mucous and lead hematoxylin positive granules. With a population density that exceeded the expected distribution of normal small granule endocrine cells in the bronchial epithelium and including cells with mixed endocrine and nonendocrine characteristics, the tumorlets provided testimony favorable to the view that carcinoid tumors like this one arise from indifferent cells of the bronchial epithelium that have been driven by oncogenic forces toward expressing an APUD cell phenotype.

Factors influencing fetal macrophage development: III. Immunocytochemical localization of cytokines and time‐resolved expression of differentiation markers in organ‐cultured rat lungs

Exogenous TNFα, IL‐1β, M‐CSF, and GM‐CSF all stimulate growth of macrophages arising in explanted fetal rat lungs. The present study examines the intrinsic availability of these factors in intact and organ‐cultured lungs and utilizes expression of cytokines and marker proteins to explore the differentiation pathway followed by phagocytes in vitro.

Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model☆

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr(db)/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.

Factors influencing fetal macrophage development: I. Reactions of the tumor necrosis factor-α cascade and their inhibitors

When fetal rat lungs are explanted to organ culture, precursor angular cells soon convert to nascent macrophages that multiply rapidly as they mature into efficient phagocytes. The present study examines the influence of proinflammatory early cytokines of the tumor necrosis factor‐alpha (TNFα) cascade on this initial expression of the macrophage phenotype.

Factors influencing fetal macrophage development: II. Effects of the PDGF subfamily of protein-tyrosine kinase receptor ligands as studied in organ-cultured rat lungs

Macrophage precursors in pseudoglandular rat lungs rapidly differentiate into phagocytes in organ culture, although this occurs only gradually in vivo. Macrophage colony‐stimulating factor is vital for the process, but the possible importance of other ligands in the platelet‐derived growth factor (PDGF) subfamily is scarcely appreciated.

ORIGINAL RESEARCHEstradiol Ameliorates Diabetes-Induced Changes in Vaginal Structure of db/db Mouse Model

INTRODUCTION Women with diabetes experience diminished genital arousal, reduced vaginal lubrication, vaginal atrophy, dyspareunia, and increased infections. Limited studies are available investigating the effects of diabetic complications on the vagina. AIMS The goals of this study were to investigate type 2 diabetes-induced changes in vaginal structure, and to determine if estradiol treatment ameliorates these changes. METHODS Eight-week-old female diabetic (db/db) mice (strain BKS*Cg-m+/+Lepr(db)/J) and age-matched control normoglycemic female littermates were used to investigate the effects of type 2 diabetes on vaginal tissue structural integrity. Diabetic animals were divided into two subgroups: diabetic treated with vehicle only and diabetic treated with pellets containing estradiol. At 16 weeks, the animals were sacrificed, and the vaginal tissues were excised and analyzed by histological and immunohistochemical methods to assess diabetes-induced changes in vaginal tissue and the extent by which these parameters are restored by estradiol treatment. MAIN OUTCOME MEASURES The effects of type 2 diabetes and estradiol supplementation were investigated on vaginal histoarchitecture. RESULTS Diabetic animals exhibited high blood glucose levels (>600 mg/dL), increased body weight (43.0 +/- 6.0 g vs. 24.4 +/- 2.0 g), and reduced plasma estradiol levels (65.5 +/- 6.6 pg/mL vs. 80.77 +/- 13.2 pg/mL) when compared to control animals. Diabetes resulted in significant thinning of the epithelium (P <or= 0.05), marked decrease in the muscularis area (P </= 0.05), distinct truncation of elastic fibers, and significant reduction of the nitrergic immunoreactive nerve fibers (P <or= 0.05). Treatment of diabetic animals with estradiol restored epithelial thickness (P <or= 0.05), muscularis area (P <or= 0.05), and elastic fiber distribution, and partially restored the density of nitrergic nerve fibers. CONCLUSIONS The data in this study demonstrate that type 2 diabetes disrupts vaginal structural integrity and that estradiol supplementation ameliorates the diabetes-induced vaginal pathology.