Various unis :;:i::g of tritia 1551s: slices fro 5;;erxazant and ; Luelled cortisol "~15; . ' wied cortisc ..... ‘b‘. . N. ...... "“3”: l~montr1 "L. '3; £ mr‘OUS Con' "I . IO Tnere y in: 1NyAN . o qutnabC'l £ Ti? ABSTRACT CORTICOID BINDING IN BOVINE MAMMARY TISSUE SLICES AND SERA BY Ronald C. Gorewit Various unlabelled corticoids significantly reduced binding of tritiated cortisol and dexamethasone to mammary tissue slices from lactating cows separated into 700 x g supernatant and precipitate fractions. Similarly, un- labelled cortisol and dexamethasone significantly reduced tritiated cortisol and dexamethasone binding to components within 700 x g fractions of mammary tissue slices from virgin heifers, l-month prepartum and dry (nonpregnant, nonlactating) cows. Unlabelled progesterone, testosterone and l7B-estradiol had no effect on tritiated corticoid binding in mammary tissue slices. Mammary tissue slices from lactating cows, virgin heifers, l-month prepartum and dry cows, incubated at 37°C with various concentrations of tritiated cortisol and dexa- methasone bound these hormones with high affinity (Kd.25x lo-loM). There were 1263 and 1855 molecules of cortisol and dexamethasone bound per mammary cell, respectively, in mammary tissue slices from lactating cows; whereas, virgin :afiers bound 413 an; :etascne per mamar; fléxd336nwlecule: Barycell, respeC‘ :—-:*:.:' 542 molecules 1 :atzle bound approxi: Exaethasone as cor Eatery tissue . States examined cont thbmmd cortisol ific binding 33:33.95. washing of Etcedures or treatm. ”iextran coated cls~ Incubation of m at4°C reduced the t 4.2. ‘OIHQh affini war measurements ‘0 nifln ‘ vantlv Cm slim-la. e ‘ f r core; a. 700 9 Superna.- ‘ Ecuwi‘ Ronald C. Gorewit heifers bound 413 and 651 molecules of cortisol and dexa- methasone per mammary cell, respectively; dry cows bound 536 and 336 molecules of cortisol and dexamethasone per mammary cell, respectively; and one-month prepartum cows bound 542 molecules of cortisol per mammary cell. All cattle bound approximately 2.0 times as many molecules of dexamethasone as cortisol in mammary tissue slices. Mammary tissue slices from cattle in all physiological states examined contained a major nonspecific component which bound cortisol in both 700 x g tissue fractions. Nonspecific binding could not be completely reduced by repeated washing of tissues, biochemical fractionation procedures or treatment of tissue fractions with florisil or dextran coated charcoal. Incubation of mammary tissue slices from lactating cows at 4°C reduced the total number of molecules of cortisol bound to high affinity binding sites by 82% compared with similar measurements made at 37°C. However, the dissocia- tion constant for the high affinity binding component was not significantly changed by lowering the temperature. Thin—layer chromatography of tritiated cortisol bound in 700 x g supernatant, indicated that the majority of radioactivity was authentic cortisol. Macromolecules which specifically bound cortisol in 700, 15,000 and 100,000 x g snarnatants and 700 are slices from la filtration chromatog: Litreatnent with p. 113: the nacromol cu straining sulfnydry ;:::ein(s) was not i sated that transpor :2: linked to l§a+/K+ Approximate mol ., 'go 5 i. re corticoid b tapgroximate molec ulxlOs. In con: Hood had an appr l16tol3x104. T“ Emery tissue and l incense for the uni 13:31:19 protein(S) V iesigzed to compare nus to bovine s '5: on" hr- I ~2qu approxima :55tél‘one per mg pro 13.125:- ' 2’ tissue slice was“ . w e tnan corti. Ronald C. Gorewit supernatants and 700 x g precipitate tissue fractions of tissue slices from lactating cows were isolated by gel filtration chromatography. Enzyme digestion experiments and treatment with parachloromercuribenzoate indicated that the macromolecules binding cortisol were protein(s) containing sulfhydryl groups. Uptake of cortisol by these protein(s) was not inhibited by 10"5 mM Ouabain, which sug- gested that transport of cortisol into mammary tissue was not linked to Na+/K+ active transport. Approximate molecular weight determinations suggested that the corticoid binding protein(s) of mammary tissue had an approximate molecular weight in the range of 2.5 x 105 to 3 x 106. In contrast, the binding protein for corticoids in blood had an approximate molecular weight in the range of 6 to 8 x 104. Thus, the proteins binding cortisol in mammary tissue and blood sera were dissimilar. Further evidence for the unique character of the mammary cortisol binding protein(s) was indirectly provided by experiments designed to compare the amount of binding of tritiated steroids to bovine sera and mammary tissue slices. Bovine sera bound approximately five times more cortisol and pro- gesterone per mg protein than dexamethasone. In contrast, mammary tissue slices bound more total molecules of dexa- methasone than cortisol. L714 Cell-free prepa: :iaitate fractions 53 :eLL-free preparatio: . 3 , .arge amounts of h-: :ezause addition of ‘ :::sistently failed Unlabelled cortl ;:IuIQ;‘k .aemnasone inhibi litany tissue slice :ation of corticoid like increased in :3: analyses showed O"..- I ‘0'» ability of corti issue slices. When- 'EI‘= Simultaneously but: ‘I..1L* 39 cows glucc waxed with non‘ ROI inflicted with cor: “5‘ results furtlie “.le to lactatihn on ."“ Mological action 4.16 EXternal p1 ‘ u \\§:e:enc :26 i ' Ronald C. Gorewit Cell—free preparations of 700 x g supernatant and pre— cnipitate fractions specifically bound cortisol. Although (zeall-free preparations of 100,000 x g precipitates bound large amounts of 3H-cortisol, this binding was not specific because addition of unlabelled cortisol or dexamethasone czcxnsistently failed to reduce binding of 3H-cortisol. Unlabelled cortexelone, cortisol, triamcinolone and dexamethasone inhibited 14C-glucose incorporation into ITLaunmary tissue slices from lactating cows. As the concen- tration of corticoids increased, inhibition of l4C-glucose uptake increased in a dose response relationship. Correla- tZJixan analyses showed that corticoid binding was related to tillee ability of corticoids to inhibit l4C-glucose uptake in tIlssue slices. When unlabelled cortisol and cortexelone Were simultaneously added to mammary tissue slices from JLiixztating cows glucose uptake was reduced by only 4% when <=<>mnpared with nonhormone treated controls. Cortexelone interacted with cortisol to increase l4C-glucose uptake. These results further suggested that specific corticoid binding to lactating mammary tissue was related to the IPhysiological action of corticoids on glucose uptake. The external pudic artery-mammary vein concentration differences for corticoids were 5.81 ng/ml and 2.89 ng/ml at 6 and 12 minutes after the start of milking. These F— .’ - -,._ z: . . or i 3.“. Ronald C. Gorewit intervals corresponded to the times when serum cortisol concentrations were maximal after the milking stimulus, and provided further indirect evidence that cortisol was associ- ated with a lactational event. In summary, evidence presented in this dissertation suggested that mammary tissue slices possessed receptor molecules which were capable of specifically binding corti- coids. The fact that tissue slices from lactating cows bound more molecules of corticoids than tissue from cattle in other physiological states and that binding and uptake of corticoids appeared related to events associated with milk secretion, may form the basis for further studies to determine the specific role which corticoids play in lacta- t1.011. CORTICOI Mich in Partial f CORTICOID BINDING IN BOVINE MAMMARY TISSUE SLICES AND SERA BY o‘0A . Ronald C. Gorewit A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements ‘ for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1975 To My Parents and Beloved Sister ii Iwish to express Lien Tucker, for serv icing my doctoral st: raggreciation to DIS filterink, H. Wells an 3;; in preparing the Iwish to express 3e;:zeland D. L. Nortc 52535 data. The aut‘: §Iazitude to Ms. Conni Stale high during the Elli; all rough draft Iwish to acknowl “‘41 M difference se “ethical assistance 1 Finally. the “ti 9'1“ M Near-19 Dr. R. L. W. ““9 Preparation of «his work was su “Mn research grant ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. H. Allen Tucker, for serving as major professor and friend _'?'t during my doctoral studies. I would also like to extend my appreciation to Drs. D. W. Collings, J. L. Gill, L. F. Wolterink, W. Wells and W. Frantz for their invaluable help in preparing the final copy of this dissertation. a; I wish to express a special thank you to Drs. Roger ISteitzel and D. L. Norton for their help in statistical analy- 398 of data. The author wishes to offer his fondest gratitude to Ms. Connie Norton for keeping the author's lT‘CZ'JI‘ale high during the writing of this dissertation and tYPing all rough drafts. I wish to acknowledge N. F. G. Beck for providing me ‘“'jL1:h A-V difference samples and Ms. Jean Hornshue for her technical assistance throughout this work. Finally, the writer wishes to recognize his friend and e'Dll.league Dr. R. L. Walker for his constant encouragement during preparation of this manuscript. This work was supported by National Institutes of Health research grant #HD-05750 awarded to Dr. H. A. Tucker. iii LISTS? TABLES. - - LEOF FIGURES . . .‘.:.::":C:ION o o E'Iih‘ OF LITE ‘TL'RI Panzary Developzm in Vivo Exper ' 33- Vitro Expe .nitiation of La >le3 32"“ v 0 me If \ ‘ «.1... enance of L En Vivo Exper ,1 . a: .4. Vitro Ex [5'19 4 J (f TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . . IRIS‘IIEW OF LITERATURE. . . . . . . . . . . Mammary Development. . . In Vivo Experiments . In Vitro Experiments. In Vivo Experiments . In Vitro Experiments. Initiation of Lactation. . .Mbintenance of Lactation In Vivo Experiments . Ln Vitro Experiments. IBind1ng of Corticoids to lBinding of Corticoids to Blood . . . . . . . . MATERIALS AND METHODS . . . Ln Vitro Experiments . . fl Routine Procedures. . IHormone Binding. . . . . Tissue and Cells. Liver, Preliminary Competition Experiments Specificity of Corticoid Binding in Mammary Tissue Slices from Lactating Cows. . . . 1) Competition Experiments . . . 2) Scatchard Analysis. . . . . . Brain and Corticoid Binding in Mammary Tissue Slices from Virgin Heifers, l-Month Prepartum, Lactating and Dry Cows (nonpregnant, nonlactating) . 1) Competition Exper1ments . . . 2) Scatchard Analysis. . . . . . Metabolism of 3Hrcortisol . . . . . O o 9 Page vii sooxmcnoiw u) id x 18 22 22 22 23 24 24 24 25 25 26 27 ‘wamr .3; or COIlTENTS--CO! Etn'sicochemical Ch ‘Receptors in Ti Gel Filtration 1) Mammary C 2) Mammary )4 Potential Hindi I) Enzymes . 2) p-Chloro: 3) Oubain. Comparison of Cort and Blood Seru: Approximate Mo] 1) Gel Filti 2) Sucrose I Polyacrylamide DEA}: Cellulose Binding of TM“ and Progest. Cell Free EXper‘m 700 X g Supern ., 790 x 9 and 10 Fri’lSlOIOgical Eff Tissue Slices Triancinolone- , CL. ”Glucose [3: 3 M Experimer flammary Uptake '91-. :.MT" «$31an ANALYSES RE SULTS O O O O O O O O O I O O O I TABLE OF CONTENTS-~continued Physicochemical Characterization of Corticoid Receptors in Tissue From Lactating Cows . Gel Filtration Chromatography . . . l) Mammary Cytosol Fractions . . 2) Mammary Nuclear Fractions . . Potential Binding Inhibitors. . . . l) Enzymes . . . . . . . . . . . 2) p-Chloromercuribenzoate (PCMB). . 3) Oubain. . . . . . . . . . . . . . «Comparison of Corticoid Binding in Mammary Ti and Blood Serum From Lactating Cows . . . Approximate Molecular Weight Determination 1) Gel Filtration. . . . . . . . . . . 2) Sucrose Density Gradient Analysis . Polyacrylamide Disc Gel Electrophoresis . DEAE Cellulose Chromatography . . . . . . . Binding of Tritiated Cortisol, Dexamethasone and Progesterone to Bovine Serum . . . . (Cell Free Experiments. . . . . . . . . . . . . 700 x g Supernatant . . . . . . . . . . . . 700 x g and 100,000 x g Precipitates. . Physiological Effects of Corticoids on Mammary Tissue Slices from Lactating Cows . . Triamcinolone-Cortexelone Competition C14—Glucose Uptake. . . . . . . . . . .;§_Vivo Experiments. . . . . . . . . . . Mammary Uptake of Corticoids. . . . . SS 8 STATISTICAL ANALYSES. . . . . . . . . . . . . . . 'ln_Vitro Experiments . . . . . . . . . . . . . Hormone Binding Preliminary Competition Experiments . . . . Specificity of Corticoid Binding in Mammary Tissue Slices from Lactating Cows. . . . 1) Competition Experiments . . . . . . . 2) Scatchard Analysis. . . . . . . . . . 00000050000000. Corticoid Binding in Mammary Tissue Slices from Virgin Heifers, l-Month Prepartum, Lactating and Dry Cows . . . . . . . . . . . . . . . . 1) Competition Experiments . . . . . . . . . 2) Scatchard Analysis: Virgin Heifers, l-Month Prepartum, Lactating and Dry Cows (nonpregnant and nonlactating). . . . . . Metabolism of 3H-cortisol . . . . . . . . . . . 42 42 42 44 44 46 57 57 66 76 3;; or CONTESTS-4 :sicochemical C _. « ’l npo' O Receptors in ii Gel Filtration 1) Mammary 2) Mammary Potential Bind l) Enzymes 2) p-Chlorc 3) Oubain Cczparison of Co: and Blood Ser' Approximate .V. 1) Gel Fil 2) Sucrose Polyacrylamid DEAF. Cellulos Binding of Tr and Proges tell-Free Expe ri I ,‘n -~:Siological E: All 700 x 9 Super 700 X g and 1 Tissue Slices TE'i<'11l1C-‘irlolor‘.¢e f ‘Glucose L ”0 Experime ”‘3er UptaI-c '-~~JSSIo:i , . "(any ““‘3GPv‘aPHY AJICES. . ;- ScintillatiOr c: “Peach Correé E gospecific 0] F of? MethOd 1 3 C031 Gel EIECt :2 L c ‘ “ Stati 01.418 TABLE OF CONTENTS--continued Physicochemical Characterization of Corticoid Receptors in Mammary Tissue From Lactating Cows (Comparison of Corticoid Binding in Mammary Ti 1on Cell- -Free Experiments. . . . . . . . . . Gel Filtration Chromatography . . . . l) Mammary Cytosol Fractions . . . 2) Mammary Nuclear Fractions . . . Potential Binding Inhibitors. . . . . l) Enzymes . . . . . . . . . . . . 2) p-Chloromercuribenzoate (PCMB). 3) Oubain (G-Strophanthin) . . . . (30.0000 and Blood Serum From Lactating Cows . . Approximate Molecular Weight Determinati 1) Gel Filtration. . . . . . . . . . 2) Sucrose Density Gradient Analysis Polyacrylamide Disc Gel Electrophoresis DEAE Cellulose Chromatography . . . . . . . Binding of Tritiated Cortisol, Dexamethasone and Progesterone to Bovine Serum . . . . SS ns 700 x g Supernatant . . . . . . . . . . . 700 x g and 100, 000 x g Precipitates. . Physiological Effects of Corticoids on Mammary In Vivo Experiments. . . . . . . . . . . Tissue Slices From Lactating Cows . . Triamcinolone-Cortexelone Competition Cl4-GIUCOSG Uptake o o o c o o o o o o Mammary Uptake of Corticoids. . . . . DI SCUSSION. O O O O O O O O O O O O O O O O O O I O S UMMRY O O O O O O O O O O O O O O O O O O O O O O B IBLIOGRAPHYO O O O O O O O O O O O O O O O O O 0 C APPENDICES . A" m Clwtd UC3U1 Scintillation Fluid . . . . . . . . . . . . . Quench Correction . . . . . . . . . . . . . . DNA Assay . . . . . . . . . . . . . . Scatchard Plot Transformation Correction for Nonspecific Binding . . . . . . . . . . . . . Lowry Method for Protein Determination. . . . Disc Gel Electrophoresis. . . . . . . . . . . Competitive Protein Binding Assay for Serum Corticoids. . . . . . . . . Statistical Analysis of Scatchard Plot Slopes and X Intercepts. . . . . . . . . . . . . . . vi Page 102 102 102 105 105 105 109 114 114 117 135 139 152 152 153 155 157 160 162 163 165 ‘mamv "-0-- O. . “yd- . .i " 3 .. aiming 0f H'CO 5' IA. u- (’V ' . Hormonal inhibit methasone bindin actating cows. and preciiJitate slices from lact. . Hormonal inhibit dexamethasone bii from lactating c: .Smary of Scatcl to mammary tissu' O .Srrrary of Scatcl binding to mama. cows at 37°C. Hormonal inhibit methasone bindin: Virgin heifers. Hormonal inhibit methasone bindin month prepartu LIST OF TABLES GIAJELE Binding of 3H-cortisol to 700 x g supernatant and precipitate fractions of mammary tissue slices from lactating cows. . . . . . . . . . . Hormonal inhibition of 3H-cortisol and 3H- dexamethasone binding to mammary tissue slices from lactating cows . . . . . . . . . . . . . . Summary of Scatchard plots of cortisol binding to mammary tissue slices from lactating cows at 4°C 0 O O O O C O O O O O O O O O O O I O O O 0 Summary of Scatchard plots of dexamethasone binding to mammary tissue slices from lactating cows at 37°C. 0 O O O O O O O O O O O O O O I 0 Hormonal inhibition of 3H-cortisol and 3H-dexa- methasone binding to mammary tissue slices from virgin heifers. . . . . . . . . . . . . . . . . Hormonal inhibition of 3H-cortisol and 3H-dexa- methasone binding to mammary tissue slices from l‘mOnth prepart‘m COWS o o o o o I o o o o o o 0 Hormonal inhibition of 3H-cortisol and 3H-dexa- methasone binding to mammary tissue slices from lactating cows 0 O O O O O O O O O O O O O O O 0 Hormonal inhibition of 3H-cortisol and 3H-dexa- methasone binding to mammary tissue slices from dry cows (nonpregnant, nonlactating). . . . . . Summary of Scatchard plot dissociation constants (Kd's) for cortisol binding to mammary tissue slices from virgin heifers, l-month prepartum, lactating and dry (nonpregnant, nonlactating) cows 0 O O O O O O O O O O O O O O O O O O O O 0 vii Page 43 45 56 58 60 61 63 65 67 .a R. 8" a." t?" ‘v IJST 0F TABLES--continued NUHE 10. 11. 12L 1.3. 1.4. 3.5. 3L6. l]. 18. 19. Summary of Scatchard plot x-intercepts (number of molecules of cortisol bound in mammary cell i SE of mean) of mammary tissue slices from virgin heifers, l-month prepartum, lactating and dry (nonpregnant, nonlactating) cows. . . . . Summary of Scatchard plot dissociation constants (Kd's) for dexamethasone binding to mammary tissue slices from virgin heifers, lactating and dry (nonpregnant, nonlactating) cows. . . . . . . Summary of Scatchard plot x-intercepts (number of molecules of dexamethasone bound in mammary cell :.SE of mean) of mammary tissue slices from virgin heifers, lactating and dry (nonpregnant, nonlactating) cows. . . . . . . . . . . . . . . . Statistical comparison of Scatchard plot slopes (Kd's) for cortisol and dexamethasone in mammary slices from animals in various physiological states. . . . . . . . . . . . . . . . . . . . . . Statistical comparison of Scatchard plot xvinterv cepts (number of molecules of corticoid bound in mammary cell) for cortisol and dexamethasone in mammary slices from animals in various physio- logical states. . . . . . . . . . . . . . . . . . Enzymatic inhibition of 3H—cortisol binding to 700 x g supernatant protein fractions of mammary tissue slices from lactating cows . . . . . . . . Effects of p-chloromercuribenzoate on 3H-cortisol binding to mammary tissue slices from lactating cows 0 O O O O O C C C O O O O C O O O O O O O O 0 Binding of 3H-cortisol to cell-free isolates of 700 x g precipitates of mammary tissue slices from lactating cows . . . . . . . . . . . . . . . Binding of 3H-cortisol to cell-free isolates of 100,000 x g precipitates (microsomes) of mammary tissue slices from lactating cows . . . . . . . . Triamcinolone and cortexelone inhibition of 3H- cortisol and H-dexamethasone binding to 700 x g supernatant and precipitate fractions of mammary tissue slices from lactating cows . . . . . . . . viii Page 69 71 72 74 75 85 87 106 107 108 1- .—.—.._—-.- '1 External tration :ilking. LIST OF TABLES-~continued TABLE Page 20. Cl4-glucose uptake into mammary tissue slices from lactating cows. . . . . . . . . . . . . . . 113 21. External pudic artery and mammary vein concen- tration of serum corticoids before and after milking O O O O O O O O O I O O O I O O O O O O O 115 ix 3' 1mm... ‘I L". {I} L . -.Scatcha 4 x 10' tions 0 cows. . Scatcha 4 x 10' tions 0 CO'u'S, . Transfo ing (0. 9 Super Slices 'Transfc 139 (0. 9 preci slices Thin‘le tractec tiOns ( SllCes Gel ii; of 700 SliCes Sol Pl: URIabel FIGURE 1A. 13. 2A. 2B. LIST OF FIGURES Scatchard plot of 3H-cortisol binding (0.005 to 4 x 10’8 M) at 37°C to 700 x g supernatant frac- tions of mammary tissue slices from lactating cows 0 I O O O O O O C O O O O O O O O I O O C O Scatchagd plot of 3H-cortisol binding (0.005 to 4'x 10' M) at 37°C to 700 x g precipitate frac- tions of mammary tissue slices from lactating cows 0 I O O O O O O O O O C O O O O O O O O C O Transformed Scatchard plot of 3H-cortisol bind— ing (0.005 to 0.041 x 10-8 M) at 37°C to 700 x g supernatant fractions of mammary tissue slices from lactating cows. . . . . . . . . . . Transformed Scatchard plot of 3H-cortisol bind- ing (0.005 to 0.041 x 10"8 M) at 37°C to 700 x g precipitate fractions of mammary tissue slices from lactating cows. . . . . . . . . . . Thin-layer chromatography of 3H-cortisol ex- tracted from "protein-bound" and "free" frac- tions of 700 x g supernatants of mammary tissue slices from lactating cows. . . . . . . . . . . Gel filtration (Sephadex G-25) elution profile of 700 x g mammary supernatant from tissue slices of lactating cows treated with 3H-corti- sol plus ethyl alcohol or 3H-cortisol plus unlabelled cortisol . . . . . . . . . . . . . . Gel filtration (Sephadex G-25) elution profile of 0.3 M KCl extract of 700 x g mammary precipi- tate, isolated from tissues incubated with 3H- cortisol plus ethyl alcohol or 3H-cortisol plus unlabelled cortisol . . . . . . . . . . . . . . Page 48 50 53 55 78 81 84 “I; 'i u- . lar weig Sucrose 501 01nd fraction 1,, ' ' PC‘Iacrl 0f H-cc SL‘pernat from lac Polvacr3 0f H-cc sera 3303 ce] binding sue Slir LIST OF FIGURES-~continued FIGURE 6. 10. 11. 12. 13. A1. A2. Gel filtration (Sephadex G-200) elution volumes of 700 x g supernatant, mammary cortisol bind- ing proteins, serum cortisol binding proteins and various standard proteins of known molecu- lar weight. . . . . . . . . . . . . . . . . . . Sucrose density gradient patterns of 3H-corti- sol binding to 100,000 x g mammary supernatant fractions and bovine sera . . . . . . . . . . . Pol acrylamide disc-gel electrophoretic profile of H-cortisol binding to protein in 700 x g supernatant fractions of mammary tissue slices from lactating cows . . . . . . . . . . . . . . Polyacrylamide disc-gel electrophoretic profile of H-cortisol binding to proteins of bovine sera. O O O C O I O O O O O O O O O O O O O I O DEAE cellulose elution profile of 3H-cortisol binding to 700 x g supernatant of mammary tis- sue slices from lactating cows. . . . . . . . . DEAE cellulose elution profile of 3H-cortisol binding to bovine sera. . . . . . . . . . . . . Sephadex G—25 elution profile of 700 x g mam- mary cortisol receptor protein(s), after cell- free incubation with H-cortisol or 3H-cortisol plus unlabelled cortisol. . . . . . . . . . . . Effects of various unlabelled corticoids on C14-glucose uptake into mammary tissue slices from lactating cows at 37°C . . . . . . . . . . Scatchard plot unadjusted for nonspecific bindv ing 0 O O O O O O C O O O O O O I O O O O O O O Scatchard plot adjusted for nonspecific binding xi Page 90 92 95 97 99 101 104 111 157 159 ' ~ 3"“.11- View:~ -i ' A Hormones Eteloprent a: menace o: -1=:t their pi Levee that t :e:e;t0r mole 2:03 on deo Corticoi 139! of mam liblood. '1 ZiSS'JQ and Cl; 391102158, 5 Either rad iOé Iiiis, Rat; tired ll Viti bl \ lazing was < IIIII INTRODUCTION Hormones of the adrenal cortex stimulate mammary development and are associated with the initiation and maintenance of lactation. The mechanism by which corticoids exert their physiological action is obscure, but it is be- lieved that the initial event requires binding of hormone to receptor molecule(s) within the mammary cell with eventual action on deoxyribonucleic acid (DNA). Corticoid hormone receptors have been described for a number of mammalian tissues including thymus, liver, brain and blood. They have only recently been found in mammary tissue and cultured mammary cells. For example, lactating rat, mouse, and vole mammary tissues were capable of binding either radioactive cortisol or radioactive synthetic corti- coids. Rat mammary tumors and bovine mammary cells cul- tured in_yitrg_also bound radioactive corticoids. This binding was considered specific because it was reduced by unlabelled corticoids and binding site(s) were saturated at physiological hormone concentrations. The primary objective of the research described in this dissertation was to determine whether corticoid hormones would specifically bind to fresh mammary tissue slices from grazing catt t0 demons :azizals in e:e made to < 223010 rec: sizes with c. 15 this war :an regardi 1.5538. lactating cattle. Once this was established, attempts were made to demonstrate specific corticoid binding and uptake in animals in other physiological states. Further attempts were made to compare some physicochemical characteristics of corticoid receptor molecules in lactating mammary tissue slices with corticoid receptors in blood serum. It is hOped that this work will help resolve certain unanswered ques- tions regarding the mechanism of corticoid action on mammary tissue. " I! ~ - REVIEW OF LITERATURE Mammary Development It is generally recognized that hormones of the adrenal cortex stimulate mammary develoPment secondarily through synergism with pituitary, ovarian, thyroid and pancreatic hormones. Evidence for this concept is given below. Lg 211g Experiments ln_yiyg_experiments designed to determine the effects of hormones on mammary development and growth have been carried out by one of two methods. The first method involved measurement of mammary growth after exogenous administration of hormones to animals with intact endocrine glands. The other experimental method involved observing changes in mammary gland development and growth after remov- al of various endocrine glands, with or without hormone replacement therapy. meareson et a1. (1967) and Griffith and Turner (1963) examined the effects of exogenously administered corticoids on mammary growth in intact animals. Corticosterone given throughout pregnancy in rats stimulated mammary cell numbers (DNA content) 23% and metabolic activity (ribonucleic acid, :l U "- 190' rag c... “-.v .. a "i' “I ‘ . ‘DA — a. ."‘b ‘.\ , . ~ . a." nu ‘. ' - f." ..... . ‘is ' “. Q -- . fi “-53.! a». ‘ .n ‘ 'H ng‘..:u - "A g 'ut \"H ~'. ‘ Q . i " ~' A ‘3’“. vv‘ .‘CC ‘- ‘- ~“ \H ‘ s n ”3“ _ ‘s‘gh 5» n“ ~‘ g; “o ‘ . s .‘..f,‘ ~-‘ 5“ .. "-3. 1 fi ,. \‘ ‘ ‘ v d#!"‘. 1 ‘V RNA) 52% at the 19th day of pregnancy. Mammary growth equivalent to that observed during lactation was accom- plished in intact rats with estradiol benzoate and progester- one treatment for 19 days followed by injection of growth hormone and cortisol. Anderson and Turner (1962a) demonstrated the synergis- tic effects of corticoids and ovarian hormones on mammary growth of adrenalectomized-ovariectomized rats. Predniso- lone and cortisol enhanced mammary growth in operated rats when administered simultaneously with estrogen and progester- one. These corticoids given alone did not enhance mammary growth in adrenalectomized-ovariectomized rats previously treated with ovarian steroids. These results were substan— tiated further by Hahn (1967) who reported that corticoster- one synergized with estradiol and progesterone to promote mammary growth in ovariectomized rats. Lyons et al. (1958) and Nandi (1958) demonstrated that corticoids synergized with ovarian and pituitary hormones to enhance mammary duct growth and lobule-alveolar develop- ment. This work was carried out on hypophysectomized- ovariectomized-adrenalectomized (triply-operated) rats and mice. In these studies, replacement therapy with estrogens, growth hormone and deoxycorticosterone or prednisolone ace- tate led to full duct growth. However, neither estrogen, deoxycorticosterone acetate nor prednisolone acetate - ‘ mm o "“ aié :wd- .nv-«o: _-.".'II' .7... v“ .-- a... .. V 39-. 'o-‘vnn a tab. 5..t .~. , : c?‘~_ . l—l . " ‘o-.._ “v._ 1‘! id’s I“ b,.“ N‘-: . ~.' “ C +3 54 1'... ‘-.:' F,‘ r. 'V M“ '4 ~“ -l~:. ".. Cr ~ V- a” . I 1‘. Q h .“ U! ‘ 1 7. \ tab ‘5 stimulated ductual growth when administered alone. Growth hormone plus estrogen stimulated ductual growth, but corti- coid was required before ductual growth typical of pre- puberal and puberal ages was duplicated. Treatments with estrogen, progesterone, prolactin, growth hormone and corti— coid resulted in full lobule-alveolar develOpment. In contrast to these results, Hahn and Turner (1967) showed that the depression of mammary growth in triply-operated rats was not significantly greater than the depression in mammary growth observed for hypophysectomized rats. Further experimental evidence for the synergism between corticoids, ovarian and pituitary hormones on stimulating mammary de- velopment was provided by Cowie et al. (1966a). These workers demonstrated lobule-alveolar development, comparable to mid-pregnancy in hypophysectomized-ovariectomized goats after treatments with hexestrol, progesterone, growth hor- mone, prolactin, and adrenocorticotropic hormone (ACTH). Wbrk in other laboratories has shown the synergism be- tween corticoids, thyroid and pancreatic hormones on enhanc- ing mammary development. For example, Hahn (1967) showed that corticosterone, thyroxine, bovine growth hormone and ovine prolactin stimulated mammary growth in hypophysectom- ized rats to a level 97% above control animals. Moreover, Ahren and Jacobsohn (1957) presented histological evidence that glucocorticoids stimulated mammary growth when ”4"“ ;..-~a-c ”.00 O l -l. .e.-_..‘ v I M-.: ‘ ’ "uvu‘.. 0".-. _ — A— .‘!'§v'u . -.A I". v .4d4’ : .‘u .A" . fio ““I .‘-. ' - ““ A H N ‘l \ .5 :q,-_ “t p_‘ u". hfil ‘4: 's \. “‘ ‘- B: i;\ ‘C‘R. -‘.‘: administered with estrogen, progesterone and insulin in hypophysectomized rats. In_yitrQ,Experiments In general, experiments carried out on mammary tissue explants from mice and rats, cultured in yitrg in medium containing hormone supplements, have confirmed the results of in_yiyg_work on hormonal requirements for normal mammary development and growth. Ovarian, pituitary, thyroid, pan- creatic and adrenal hormones were necessary for normal development of mammary explants (Lasfargues and Murray, 1959; Prop, 1966; Bern and Rivera, 1960; Rivera, 1963, 1964a, b,c; Rivera and Kahn, 1970; Ranadive and Chapekar, 1964; Ichionose and Nandi, 1964, 1966; Gadkari et al., 1968; Dilley and Nandi, 1968; El Darwish and Rivera, 1970; Dilley, 1971 and Forsyth, 1971). Initiation of Lactation In Viyo Experiments Serum glucocorticoids in many species remain unchanged or are slightly reduced during most of pregnancy. However, serum concentrations of corticoids increased along with growth hormone, prolactin and estrogens, while progesterone decreased shortly before parturition in cows (Adams and Wagner, 1970; Heitzman et al., 1970; Robinson et al., 1970; ‘ I. -IQ.' f; 3:1. gnu .:'r* H at a \r 'On‘_‘ b.“ _‘ .V P - “"I' we“, ‘ V '.C his. .' l‘ . , T» 0 ~Cm (‘7: 7‘1. 2"} § Jtlex 4 ‘v, y “H Ingalls et al., 1973 and Smith et al., 1973), guinea pigs (Gala and Westphal, 1967 and Rosenthal et al., 1969), dogs (Seal and Doe, 1963), rats (Milkovic and Milkovic, 1963; Kamoun et al., 1965; Gala and Westphal, 1965a,b and Voogt et al., 1969), mice (Gala and Westphal, 1967), rabbits (Gala and Westphal, 1967) and women (Stewart et al., 1961; Friedman and Beard, 1966; Scholz and Huther, 1971). It is believed that increased serum levels of growth hormone, prolactin, estrogens and adrenal cortical hormones and decreased progesterone levels, just before parturition, may provide the stimulus for the initiation of lactogenesis (Cowie and Tindal, 1971). This hypothesis does not apply for corticoids in ewes and monkies, since elevated secre- tion of corticosteroids has not been observed before par- turition in these species (Patterson and Harrison, 1967, 1968; Basset and Thorburn, 1969 and WOlf and Bowman, 1966). Several workers have succeeded in initiating lactation in intact pregnant animals with injections of either pro- lactin or cortisol (Talwalker et al., 1961, rats; Nandi and Bern, 1961, mice; Talwalker et al., 1961, Meites et al., 1963 and Friesen, 1966, rabbits; Delouis and Denamur, 1967, ewes; Tucker and Meites, 1965, cows). Furthermore, Chadwick and Folley (1962) and Chadwick (1971) increased the sensi- tivity of mammary glands by treatment with ACTH and adrenal corticosteroids. 5R _ W .1332: a a .g-a 0.- V. -o:o.' z" « -.. fivv:r .h. q"- ';‘~--~-§A.y6 "”5: .g- . -A .4.‘ "PA ‘V“ “"J . ' . ‘Q.. ‘ 4‘ ""“VEQ "p‘ “"a "fi “'L .. as a Lyons (1958) successfully initiated lactation in triply-0perated virgin rats. Animals were treated initially with ovarian hormones, prolactin and cortisol to maximize lobule-alveolar development. Following this, milk secretion was initiated in 6 days with a combination of prolactin, deoxycorticosterone acetate or prednisolone acetate. Similarly, Davis and Liu (1969) and Meunier (1962) have shown that cortisol, corticosterone or cortisone restored normal milk secretion in adrenalectomized or adrenalectomized- ovariectomized rats. Ben-David and Sulman (1970) provided further evidence that initiation of lactation was dependent upon prolactin and glucocorticoids in rats. Lactation was induced in normal intact rats with the tranquilizer per- phenazine. However, this response did not occur in adrenal- ectomized animals. Since perphenazine elevated prolactin in blood, the failure of lactogenesis was attributed to a lack of glucocorticoid. In contrast, work by Cowie and Watson (1966b) and Denamur (1969) suggested that rabbits may have no absolute requirement for steroids in the initia- tion of lactation. These workers initiated lactation with prolactin alone in adrenalectomized and ovariectomized- adrenalectomized rabbits. Furthermore, Cowie et a1. (1969) reinitiated or maintained lactation in hypophysectomized lactating rabbits while Denamur (1969, 1971) initiated lactation in hypophysectomized or triply-operated pseudo— pregnant rabbits. In Viggg Experiments Hormonal requirements for the in_yit£9_initiation of milk secretion in mice have been examined by several workers. For example, Rivera (1964a,b) found that milk secretion in C3H mice was initiated in yitrg by corticosterone, aldoster- one or cortisol in combination with insulin + prolactin + growth hormone. Similarly, Juergens et a1. (1965) reported that a combination of insulin, prolactin and cortisol in- duced the synthesis of "casein like" phosphoprotein by mid- pregnant C3H mouse mammary explants. Other hormone combina- tions were ineffective. Maximum casein synthesis occurred after 48 hours of culture. Further support of this observa- tion was provided by Topper (1968) who showed that the maximal rate of induced casein synthesis, which occurred after 48 hours, was approximately 50% of that seen in normal mammary tissues for 10 day postpartum mice. Corticoid molecular structural requirements for the initiation of lactation in mice have been investigated by Turkington et a1. (1967). They compared histological and biosynthetic responses to insulin and prolactin with and without corticoids. The five most effective steroids which supported in yitrg_milk secretion were 21-deoxycortisol, aldosterone, cortisone, cortisol and prednisolone. Biochemical evidence has indicated that prolactin, insulin, progesterone and corticoids play roles in the gtzesis 0f mi 5:222:0119d l :;-:sed‘ 0f “0' :53 specific track and ;; :3 Turkint 2. insulin i r: synthes 1; response . 10 synthesis of milk lactose. For example, lactose synthesis is controlled by lactose synthetase, an enzyme which is composed of non-specific galactosyl transferase (A protein) and a specific whey protein (B protein, a—lactalbumin) (Brodbeck and Ebner, 1966). In mice, mammary explant stud— ies by Turkinton and Hill (1969) have indicated that prolac- tin, insulin and cortisol in combination, induced a-lactal- bumin synthesis, but that progesterone selectively inhibited the response. It is believed that the fall in circulating progesterone, which has been observed at the end of pregnancy in a number of mammals, permits stimulation of a-lactalbumin synthesis. Maintenance of Lactation M Exmeriments Several investigators have shown that corticoids are rate limiting to lactation in rats. For instance, Thatcher and Tucker (1968) showed that rat pituitary ACTH decreased 68% between days 20 and 36 of lactation. Additionally, Holzbauer (1957) showed that adrenal corticosterone content, which has been used as an index of secretory activity, decreased linearly between days 16 and 32 of lactation in rats. Furthermore, Hahn and Turner (1966), Johnson and Meites (1958) and Talwalker et a1. (1960) stimulated lacta— tional performance by 12-27% in intact rats by injections :.-----v’ I .. rebu- ,- .aoo:oa 2.3"..- I p .1... .:‘ M.“ -‘b u -2’- “he. - n A... on ‘ . .s..“; uu¢d“‘. “v.‘ A u-s .‘\'v‘ V ‘V “ 0“ . '1‘: a“ a. ‘; ‘ >1 a ' ‘20., ‘.‘~ \ 11 of either corticosterone, cortisone acetate or cortiSol acetate. In addition to this work, Thatcher and Tucker (l970a,b) markedly reduced the decline in lactation seen in rats by treatment with either cortisol acetate or 9 a-fluoro- predinisolone acetate. Emery et a1. (1971) maintained mam- mary cell nucleic acid content for up to 32 days when intact rats were given glucocorticoids alone or in combination with high fat diets. In contrast to the work described above, Hahn and Turner (1966) and Kowalewski (1969) inhibited lac— tational performance in early lactation with high doses of corticoids. Cowie and Tindal (1958) and Anderson and Turner (1962b, 1963a,b,c) examined the effects of adrenalectomy on the lactational performance of goats, rats and mice. These workers found that adrenalectomy impaired milk secretion. Furthermore, Anderson and Turner (1962b, 1963a,b,c) demon- strated that glucocorticoids or mineralocorticoid partially restored milk secretion in adrenalectomized animals. A com— bination of the two steroids was more effective than either given separately. This suggested that the decline in milk secretion in adrenalectomized rats was in part a result of metabolic defects, which were corrected for by exogenous mineralocorticoid. Corticoids seem to be rate limiting to lactation in rats as described above; however, most experimental evidence to date suggests that this is not the case for cows. E: ir.stance . '1‘ :2: lose 93;: at 311:: zaxaticns C ::::;'::out 15 am ' n1: 4 l: I. . Eterisolone' ':~b- a. 333112110n o- ’81" cor S greate 12 For instance, Koprowski and Tucker (1973) observed that cows did not lose their ability to discharge corticoids into serum at milking as lactation progressed. Also, basal con— centrations of corticoids in blood remained unchanged throughout lactation. Moreover, Head et al. (1972) exogen— ously administered flumethazone (6a, 9a-dif1uoro-l6a-methy1 prednisolone) to lactating cows beginning at the 28th day of lactation. The corticoid failed to affect daily milk production over a 305 day lactation. The pituitary seemed unresponsive to exogenously administered corticoid during the first 28 days of lactation, since flumethazone failed to depress endogenous plasma corticoids during this inter- val. Even though corticoids may not be rate limiting to lactation in cows, work by Patterson and Linzell (1971, 1974) suggested that corticoids were utilized by goats and cows during milk secretion. Mammary glands of preparturient and lactating goats and cows removed an average of 1.1 to 1.3 ug of cortisol per minute from the blood. Metabolic clearance rates of corticoids did not change appreciably; however, corticoid secretion rates were approximately four times greater in lactating goats and cows as compared with pregnant animals. The mammary glands of goats and cows removed approximately 3 to 4% of the secreted cortisol and 2/3 of the unbound hormone which entered the gland. in vitro EXPE he maint 4 III a (‘1' 2:11; secretory g .. ..o.actin fail Y? .33 is omitted Bin! It is curren :Les, corticoids 12.-ti: respective action can be 8X9 ‘:5# "9». 7‘0: molecule a! tissue and Cu B: 9W (1969) if; 4- ~ ‘a‘eq C0rtico' {it , 32:14 ‘ Q ‘A. “o~ 13 In_yith_Experiments In Vitro experiments suggesting the importance of corti- coids on the maintenance of lactation have been reviewed by Forsyth (1971). In summary, mammary explants which are pro- ducing secretory products in response to insulin, corticol and prolactin fail to do so when one or more of these hor- mones is omitted from the culture medium. Binding of Corticoids to Mammary Tissue and Cells It is currently believed that, like most steroid hor- mones, corticoids must bind to receptor molecules within their respective target tissues before their physiological action can be exerted. Corticoid binding and corticoid receptor molecules have recently been demonstrated in mam— mary tissue and cells. Emery (1969) and Steyert and Emery (1971) demonstrated tritiated corticosterone binding and receptor molecule(s) in rat and cow mammary gland homogenates. Corticosterone was bound to extranuclear fractions of homogenates which con- tained mammary cytosol along with microsomes and mitochon- dria. Fractionation of homogenates by differential contrifugation and chromatography revealed that tritiated corticosterone was bound to a protein(s). In a series of preliminary experiments, Emery (1969) found that the amount Ls: found the .‘g :33 bound :‘:;e:ate of Prev nti 35 carticoste I! 00. 11k to ra l.‘ -.' !; 4.113139 6C Ii 1.18 C811 1 rel-V ‘ . . ... T511: 13 is . PrI3tc 5:7. “23 Sum. Via. .\ ~_‘ 'Suln s sur 1'” hole ‘kfihfi‘ y“ 14 of radioactive corticosterone bound to rat mammary homogen- ates increased sixfold at 16 days of lactation when compared with rats which were in their 13th day of pregnancy. He also found that mammary homogenate isolated from one lactat- ing cow bound more molecules of corticoid than the mammary homogenate of a pregnant cow. Prevention of suckling in rats lowered the total amount of corticosterone bound per mg of protein. Since addition of milk to rat and cow mammary homogenates had little effect on binding activity, it was concluded that milk precursors in the cell interfered with binding activity or increased the lability of the corticosterone binding protein. Shyamala (1973a) reported specific corticoid binding in cytOplasmic fractions of lactating mouse mammary tissue. Unlabelled triamicinolone, fluorocortisol, corticosterone, cortisol and aldosterone competed with tritiated dexametha- sone for binding sites. Unlabelled 17-5 estradiol, testo- sterone, androstendione and spironolactone were without effect. This suggested that binding was specific for corti- coids. Proteolytic enzymes and mercurials reduced or in— hibited binding, but DNAse and RNAse were without effect, which suggested that the receptor molecule(s) was a protein. Receptor molecule(s) sedimented near 68 in low ionic strength sucrose and at 48 in high ionic strength sucrose. The Kd for the dexamethasone-receptor complex was 6 x 10‘904 54°C. Scatchar 23.1: line with ass :5 specific r .3: fond glucocor :;se ternary tumor dirty for cortic ;s;'.a;.'ed a limited Exes saturated at Gardner and Hi EE-Zrted the prese; 2;:iazed triancinc :5. lactating If n k. ‘93- coefficients < hull.“ weight C< SEE: r . Ltd on sucro: 7:! w ’ “7611 tive hormom 23315 1.. ' g.ucocortic< :Q- ‘.I A! we reduced tr. 11% :5 rnone-receph ‘ ‘ : . A. ParacthU E1;;ested that 501: C; reaction. Acre £31m and pregnanl «1 Wlth the r 15 at 0-4°C. Scatchard plots of binding data revealed a single straight line with high slope, which suggested a single class of specific receptors for corticoid. Shyamala (1973b) also found glucocorticoid receptors in cytosol fractions of mouse mammary tumor tissue. The binding protein(s) had high affinity for corticoid (Kd = 4 x 10.9 M, dexamethasone) and displayed a limited number of binding sites. The binding sites saturated at hormone concentrations of 1 x 10"8 M. Gardner and Witliff (1973a,b) and Turnell et al. (1974b) reported the presence of specific molecules which bound tritiated triamcinolone acetonide in cytosol fractions from normal lactating rat and vole mammary glands and rat mammary tumors. The receptors were protein(s), exhibited sedimenta— tion coefficients of 7 to 88 and dissociated into lower molecular weight components which sedimented at 4 to SS when separated on sucrose gradients containing 0.4M KCl. Competitive hormone binding experiments showed specificity toward glucocorticoids. However, progesterone and aldo- sterone reduced tritiated triamcinolone binding. The Kd for the hormone-receptor complex was in the range of 10-7 to 10.9 M. Parachloromercuribenzoate reduced binding, which suggested that sulfhydryl groups played a role in the bind- ing reaction. Moreover, mammary cytosol fractions from virgin and pregnant rats contained fewer binding sites when compared with the number found in lactating rats. ‘“mnmy 9......3‘39' . .‘Vhb‘ 'EIECC-JIS 1?. Q :;s ’i-CIK , i v 0! V J .R ‘ ‘ c t.‘ uoov C: ‘ J. n 123528330128 :zrlmrcces . J O ‘ an“; . ~. »v&1.rast . " "fiu “2“ .~—““j . s :" 6.’ “13.1035 C 16 Tucker et a1. (1971) demonstrated cortisol binding receptors in bovine mammary cells cultured in zitrg. In this work, 77% of the amount of tritiated cortisol taken up by the cells was present in the cytosol after incubation at 2 or 37°C. The binding of tritiated cortisol was reduced by unlabelled cortisol, corticosterone, deoxycorticosterone, aldosterone and dexamethasone. Progesterone and 17 a-hy- droxyprogesterone also reduced tritiated cortisol binding. In contrast, estradiol-l7-B and testosterone did not affect binding. Scatchard plots revealed that mammary cell cytosol fractions contained three components which bound cortisol. One of the components had almost zero slope and was prac- tically unsaturable. This component was thought to repre- sent nonspecifically bound hormone. The other two binding components had high affinity for cortisol. For example, the major high affinity component had a dissociation constant 8 (Rd) for cortisol equal to 5 x 10- M.> This component bound approximately 5,400 molecules of cortisol per cell. The third component had a Kd = 2 x 10-9 M for cortisol, but it bound only approximately 500 molecules of cortisol per cell. Lowering the incubation temperature from 37 to 2°C had little affect on dissociation constants, but reduced the number of molecules bound by approximately 33%. Most of the radioactivity in the cytosol was in the form of unmetabolized cortisol; however, 18% of the radioactivity ~“ v.- ~\9 17 was associated with metabolite(s). The majority of tri- tiated cortisol was found in cytoplasmic fractions within 15 minutes of incubating the cells with labelled hormone. Radioactive cortisol moved toward the nucleus during the first 60 minutes of incubation at 37°C. Heating the cytosol to 100°C destroyed the binding of tritiated cortisol in cytosol fractions. Tucker et al. (1971) also demonstrated cortisol bind- ing in nuclear fractions of cultured bovine mammary cells. Twenty-one percent of the total tritiated cortisol, taken up by the cells, was associated with nuclear fractions after incubation at either 37°C or 2°C. Unlabelled dexamethazone, cortisol, and corticosterone reduced tritiated cortisol binding to nuclear receptor sites. Like the mammary cyto- sol, Scatchard plots of the nuclear fractions contained three components which bound cortisol. One component was virtually unsaturable and was believed to represent non- specifically bound hormone. The second component had a high affinity for cortisol (Kd = 4 x 10.8 9 M). The third component had a Kd = l x 10- M for cortisol. Heating the cells to 100°C for 10 minutes destroyed binding to nuclear fractions. Thirty-one per cent of the cortisol bound to nuclear fractions was in the form of metabolized hormone. In addition, most of the radioactive cortisol incorporation into the nuclei occurred within 15 minutes of incubation. ‘52.” 18 These workers also observed minimal uptake of tritiated cortisol into microsomal and mitochondrial fractions of cul- tured mammary cells. The uptake in these fractions accounted for only 0.7 and 9.7%, of the total amount of radioactivity incorporated per cell respectively. Binding of Corticoids to Thymus, Liver, Brain and Blood Glucocorticoids play important roles in regulating the physiology and biochemistry of the thymus, liver and brain. For instance, glucocorticoids cause atrophy of the thymus and other lymphoid tissues (Yates, 1974; Ganong, 1969 and Litwack and Singer, 1972), regulate carbohydrate and fat metabolism in the liver (Heber et a1. 1966, 1968) and affect carbohydrate metabolism, growth and excitability of brain tissue (Frieden and Lipner, 1971; Ganong, 1969; Axelrod, 1971; Vernadakis and Whodburg, 1971 and DeVellis et al., 1971). Furthermore, corticoids are transported to these target tissues via blood. Corticoids are bound to blood proteins which provide for corticoid transport. The primary transport protein for corticoids in blood is corticoid binding globulin (CBG) or transcortin (Westphal, 1971). Corticoid binding mechanisms have been extensively examined in all the tissues described above (Munck and Brinck-Johnsen, 1968; Litwack and Singer, 1972; Koch et al., 1972; Turnell 511.,19743; Hunck a :2: 232115), (Snart et 2‘. Baxter and Tomlin 543.2293. 1970; McEw .3’3; rat brain) , and ‘t" .1 blood). The re Unlabelled corti tizortexelone (ll-c arse: radioactive C: :: triamcinolone ace rain. In addition ..ucr‘.nated corticoi ~.-;nosphate, 9 u-fl =i~510nide reduced th 3*“ ' “N.“g sl ' ‘1 tes in th, 1““ ‘. S.“~I- “lied fluorinat “-lCOId binding tc Wis, liver, and t rEEiS et al., 1969? ..al., 1966). [ml ( .w-eesterone also r ““5 arid brain 19 et al., 1974a; Munck and Wira, 1971; Schaumburg, 1970, 1972; rat thymus), (Snart et al., 1970; Gardner and Tomkins, 1969 and Baxter and Tomkins, l97la,b; rat liver), (McEwen and Plapinger, 1970; McEwen et al., 1972 and Grosser et al., 1973; rat brain), and (Sandberg et al., 1966; Seal and Doe, 1961, 1962, 1963 and 1966 and Muldoon and Westphal, 1967; human blood). The results of this work are summarized below. Unlabelled cortisol, corticosterone, deoxycorticosterone and cortexelone (ll-deoxycortisol) were capable of reducing either radioactive cortisol, corticosterone, dexamethasone or triamcinolone acetonide binding in thymus, liver and brain. In addition to these hormones, various unlabelled fluorinated corticoids such as dexamethasone, dexamethasone- 21-phosphate, 9 a-fluoroprednisolone and triamcinolone acetonide reduced the binding of radioactive corticoids to binding sites in thymus, liver and brain. In contrast, unlabelled fluorinated corticoids did not reduce radioactive corticoid binding to CBG of blood to the extent seen in thymus, liver, and brain (Kolanowski and Pizarro, 1969; Peets et al., 1969; Baxter and Tomkins, 1971a and Sandberg et al., 1966). Unlabelled estradiol 178 and l7a-hydroxy progesterone also reduced corticoid binding in liver, thymus and brain. This reduction in binding, however, wasrxn:to the degree seen for unlabelled corticoids. Testosterone and progesterone had no effect on radioactive ::;::id binding in N {gesterone greatly re i":'.":en compared to ( radial-175 and tes :33, but not to the : 3::ge5terone . Dissociation con: 11:;Lated from Scatc 2 Liver and brain 32:;fied C30 from hurt 3113-8 ’4 . . and 2 x l( 23:15, liver and by xlE-C‘i‘ . £5 0f cortico 2.5-{3‘41 e of Steroid 23:1 .?.0rs from thym~ swinger, 1972. B 3' Ton ' L- W's-”'5‘ n Drain; and Seal anc “Essex 20 corticoid binding in liver, thymus and brain tissue. Progesterone greatly reduced corticoid binding to CBG at 37°C when compared to experiments carried out at 4°C. Estradiol-l78 and testosterone reduced corticoid binding to CBG, but not to the extent seen with either corticoids or progesterone. Dissociation constants for corticoid-receptor complexes, calculated from Scatchard plots of corticoids bound to thy- 8 10 mus, liver and brain were in the range of 10— to 10- M. Purified CBG from human blood sera had a Kd for cortisol of 3 x 10"8 M and 2 x 10-9 M at 37° and 4°C, respectively. Thymus, liver and brain bound approximately 2400-5000 total molecules of corticoid per cell. Purified CBG bound one molecule of steroid per molecule of protein. Several investigators have isolated the glucocorticoid receptors from thymus, liver, brain and blood (Munck and Wira, 1971 and Schaumberg, 1970, 1972, rat thymus; Litwack and Singer, 1972; Beato et al., 1969, 1970a,b, 1971; Gardner and Tomkins, 1969 and Baxter and Tomkins, l97la,b, rat liver; McEwen and Plapinger, 1970 and McEwen et al., 1972, rat brain; and Seal and Doe, 1961, 1963,.1966 and Muldoon and Westphal, 1967, human blood). In summary, the receptors all appeared to be proteins since they were destroyed by proteolytic enzymes, mercurials and heating at 100°C for 10 minutes. The sedimentation coefficients of the binding 329.25 ranged from . axis; to the ioni :Le:‘.".ar weights of 31.530, again depend :::;t'.0ns utili zed . 21 proteins ranged from 4 to 88 on sucrose gradients and varied according to the ionic strength of the buffers used. The molecular weights of the binding proteins ranged from 50- 250,000, again depending upon the particular experimental conditions utilized. 51223.8 Procedu: Emery ti are randomly I 335213913 he: a ...... ‘ ““nih a.g ;;«e p- leC 5.. ‘se n b.) Quotes-e. ‘ Q .75., s‘ 0‘ .103 a ‘r‘flx at: \i‘,‘ ’ 3 I: . ‘1 ‘ Q‘C? ‘ MATERIALS AND METHODS £3_Vitro Experiments Routine Procedures Mammary tissue samples weighing approximately 500 mg were randomly removed from eight areas of the mammary glands of Holstein heifers or cows within 15-30 minutes after slaughter. The samples were immediately transferred to either ice cold 0.01M Tris-EDTA buffer (0.01M Trizma base, and 0.01M ethylenediamine tetracetic acid, Sigma Chemical, adjusted to pH 7.4 with 6N HCl) or to a 1:1 mixture of medium 199:Eagle's minimal essential medium (MEM) at pH 7.4. Within 1-2 hours, the tissue samples were further cut into approximately 0.2-0.4 mm3 pieces and dispersed into tubes containing either 3 ml of Tris-EDTA buffer or medium 199:MEM. Single pieces from each of the original eight tissue samples were placed in each assay tube. Each treatment usually was conducted in quadruplicate on a given animal, and no more than one animal was used per day. Treatments consisted of incubating tissue with 40 ul of 100% ethyl alcohol contain— ing various radioactive and/or unlabelled steroids. Incuba- tion was carried out for 1 hour at 37°C with intermittent 22 Wee 5 second ;',5 iii-.5 routine 3-..? ‘- _._,es were CC :3. Spectromel :rrect for 911‘ ::.e:};ed for ra: freer et a1. 2:: of the et .: quantified the data u Stations per MEI REpt w: “mated for 23 shaking. Tissues were then washed 5 times with 5 ml of either Tris-EDTA buffer or Medium 199:MEM at 4°C, and homo- genized (Polytron homogenizer, Brinkmann Instruments) in three 5 second bursts at 10 second intervals. The homogen- ate was routinely centrifuged at 700 x g for 10 minutes at 4°C, and the supernatant decanted into scintillation vials containing 10 ml of scintillation fluid (Appendix A). Samples were counted in a Nuclear-Chicago liquid scintilla- tion spectrometer, and external standardization was used to correct for quench (Appendix B). Isotopic steroids were checked for radiochemical purity as previously described by Tucker et al. (1971). The DNA (Deoxyribonucleic acid) con- tent of the ethyl alcohol extracted 700 x g precipitates was quantified according to the procedures in Appendix C, and the data usually were expressed as DPM/ug DNA (disinte— grations per minute per microgram DNA). Hormone Binding Preliminary Competition Experiments In preliminary experiments, tissue slices weighing 500 mg were taken from four lactating cows. The tissues were either kept whole, halved, quartered or scissor minced and incubated for 1 hour at 37°C in Medium 199:MEM containing 9 either 2.7 x 10- M 3H-cortisol (1,2 3H-cortisol, 44 ci/mmole, 24 New England Nuclear) plus ethyl alcohol or 2.7 x 10-9 M 3H- cortisol plus 6.7 x 10-8 M unlabelled cortisol (Sigma Chemi- cal). After incubation, tissues were homogenized, centri- fuged at 700 x g and radioactivity determined as described above. Specificity of Corticoid Binding in Mamma§y_Tissue Slices from Lactating Cows 1) Competition Experiments Specificity of 3H-cortisol binding in mammary tissue slices from four lactating cows was determined by adding 8 M of various unlabelled steroids simultaneously 9 6.7 x 10" with 2.7 x 10- M 3H-cortisol. The unlabelled hormones were cortisol, dexamethasone, progesterone, 17-8 estradiol and testosterone (Sigma Chemical). Control tubes received ethyl alcohol plus 2.7 x 10.9 M 3H-cortisol. After 1 hour incuba- tion at 37°C, the 700 x g supernatant and precipitate frac- tions were isolated and counted for radioactivity. A simi- lar experiment was conducted in four additional lactating cows except that 2.7 x 10"9 M l, 2, 4-3H-dexamethasone (Schwartz/Mann; 5.2 ci/mmole) was used in place of 3H- cortisol. 2) Scatchard Analysis Results of the above experiments showed binding of 3H-cortisol and 3H—dexamethasone was decreased by unlabelled :::;501 and :racteri s t 1 11’s) and nu In. .. 25 cortisol and dexamethasone. In order to determine further characteristics of this inhibition, dissociation constants (Kd's) and number of binding sites were determined in mam- mary slices from four lactating cows incubated at 37°C or 4°C with 0.005 to 4.0 x 10-8 M of a combination of 3H- cortisol and unlabelled cortisol. Other mammary slices were collected from four additional cows and incubated at 37°C with 0.005 to 4 x 10"8 M of a combination of 1, 2-3H— dexamethasone (Amersham/Searle;Jr7ci/mmole) and unlabelled dexamethasone. The amount of cortisol and dexamethasone bound in the 700 x g supernatant and precipitates was ana- lyzed from Scatchard plots (Scatchard, 1949). Scatchard plots were corrected for nonspecific binding by the trans- formation given in Appendix D. Corticoid Binding in Mammary Tissue Slices from Virgin Heifers, l-Month Prepartum, Lactating_and Dry Cows (nonpregnant, nonlactating)? 1) Competition Experiments Virgin Heifers Specificity of 3H-cortisol binding in mammary tissue slices from four virgin heifers was determined by adding 8 6.7 x 10- M of various unlabelled steroids simultaneously with 2.7 x 10"9 M 3H-cortisol. The unlabelled hormones were cortisol, dexamethasone, progesterone, 17-8 estradiol and testosterone (Sigma Chemical). Control tubes received ethyl 51.231 plus 2'7 x :2: at 37°C: the were isolate iaszribed pIEViOU salarly on th . fl 3,, c, 4- h-dexarne 1- at: in place of 1314.03th \- EXperiments :53 Gilt us ing .55; arld f Our ’ a Scatchi \H In order nude from CE at: constant ‘ICErT-AiHEd f0 5“!“ N, L ' “In prEPE 26 alcohol plus 2.7 x 10.9 M 3H-cortisol. After 1 hour incuba- tion at 37°C, the 700 x g supernatant and precipitate frac- tions were isolated and counted for radioactivity as described previously. Additional experiments were conducted similarly on the same heifers, except that 2.7 x 10"9 M l, 2, 4-3H-dexamethasone (Schwartz/Mann; 5.2 ci/mmole) was used in place of 3H-cortisol. l-Month Prepartum, Lactating and Dry Cows Experiments similar to those described above were car- ried out using six, l-month prepartum cows; five, lactating cows; and four, dry cows (nonpregnant, nonlactating). 2) Scatchard Analysis In order to quantify corticoid binding in mammary tissue from cattle of various physiological states, dissocia- tion constants (Kd's) and number of binding sites were determined for cortisol and dexamethasone in virgin heifers, l-month prepartum, lactating and dry cows. Vipgin Heifers Mammary slices from four virgin heifers were incubated 8 at 37°C with 0.005 to 4.0 x 10- M of a combination of 3H—cortisol and unlabelled cortisol. Other mammary slices were collected from these heifers and incubated at 37°C with 8 0.005 to 4.0 x 10— M of a combination of l, 2-3H-dexametha— sone (Amersham/Searle; 27 ci/mmole) and unlabelled arenasone. T. 273-3 x g super 5:21;.2ard plots :::e:ted for no :eixes given in l-Montn “— EXPerime nt iirried out us: 2'!‘ H..- 3‘th COWS E‘.s was Omitte ’II. :{&~mar‘£ th'iatated "if ti he“‘°§‘ém ..4 ~11 0i A =. eruted 1131:1132 15;. -“‘%¥‘ ‘ d l E. ;-‘\“ i“ c a“. “X;Vl a1 T-m \‘Lr“‘ «La- < is 27 dexamethasone. The amount of cortisol and dexamethasone in the 700 x g supernatant and precipitates was analyzed from Scatchard plots (Scatchard, 1949). Scatchard plots were corrected for nonspecific binding according to the pro- cedures given in Appendix D. l-Month Prepartum, Lactating and Dry Cows Experiments similar to those described above were carried out using four, l-month prepartum cows; four, lactating cows; and 4, dry cows, except that Scatchard analy- sis was omitted for 3H-dexamethasone binding in l-month prepartum cows. Metabolism of 3H-cortisol Mammary tissue slices from four lactating cows were incubated with 1.3 x 10-7 M 3H-cortisol and after washing and homogenization, 0.6 ml of the 700 x g supernatants plus 0.4 m1 of 40% sucrose were applied to Sephadex G-200 columns and eluted with Tris-EDTA buffer pH 7.4. Those fractions containing the 3H-cortisol-receptor complex and those con— taining free 3H—cortisol were extracted with methylene chloride. This removed over 90% of the radioactivity in both fractions. Extracts were dried under N dissolved in 2! ethyl alcohol and 50 ul samples were placed on silica—gel thin-layer chromatograms (Eastman 6060 plates) and developed as previously described by Tucker et a1. (1971). "U 21’ L4: '—" Filtration 35 'E-cortisol iii-relied cor :: centrifuga Q7136 was a; 28 Physicochemical Characterization of Corticoid Receptors in Tissue From Lactating Cows Gel Filtration Chromatography l) Mammary Cytosol Fractions 700 x_g supernatant Fresh mammary tissue slices from four lactating cows were incubated for 1 hour at 37°C with either 2.7 x 10-9 M 9 M 3H-cortisol plus 6.7 x 10-814 of 3H-cortisol or 2.7 x 10- unlabelled cortisol. After routine washing, homogenization, and centrifugation, a 0.6 m1 sample of the 700 x 9 super- natant was added to 0.4 m1 of 40% sucrose solution and the mixture was applied to 1.5 x 20 cm columns of Sephadex G-25 (fine, A.B. Pharmacia). The columns were eluted with Tris- EDTA buffer pH 7.4. Column operations were carried out at 4°C, and 3 ml fractions were collected. One ml aliquots were counted for radioactivity, and the remaining aliquots were assayed for protein by the method of Lowry et a1. (1951), modified by Oyama and Eagle (1956) (Appendix E). 15,000 and 100,000 x g supernatants To compare 3H-cortisol binding in 700, 15,000 and 100,000 x g supernatant fractions the following was done. Mammary slices from four lactating cows were incubated, as 7 above, with 1.3 x 10- M 3H-cortisol, washed, homogenized and centrifuged at 700 x g for 30 minutes. A 0.6 ml sample m :fzze 7-30 x g iii-£- Sephadex c jzrtion of the s 5:: 30 minutes a :gragned on Se; 53:10:} was Ce: '~ siris: of th. 29 of the 700 x g supernatant was applied to and eluted from G-200 Sephadex columns as described above. The remaining portion of the supernatant was centrifuged at 15,000 x g for 30 minutes and a sample of this supernatant was chroma- tographed on Sephadex G-200. The remaining supernatant fraction was centrifuged at 100,000 x g for 1 hour. A 0.6 ml sample of this supernatant was chromatographed. 2) Mammary Nuclear Fractions 700 x_g precipitate To determine if binding of 3H-cortisol occurred in pro— tein fractions of 700 x g mammary tissue precipitates (nuclei), the following experiment was conducted. Mammary slices from four lactating cows were incubated with 1.3 x -7 M 3H-cortisol plus ethyl alcohol or 3H-cortisol plus 6 10 unlabelled cortisol (2.4 x 10- M). After washing, homo- genization and centrifugation at 700 x g for 30 minutes, the pellet was extracted with 2 ml of 0.3 M KCl for 1 hour at 37°C. The extract was centrifuged at 50,000 x g for 20 minutes, 1 ml of the supernatant was layered on a Sephadex G-25 column and eluted with Tris-EDTA buffer pH 7.4. Potential Bindinglnhibitors 1) Enzymes Tissue slices from four lactating cows were incubated 7 with 3H-cortisol (1.3 x 10- M) alone or 3H—cortisol plus Labelled cc I 1 $3318 51 ice 5 .:e cold Tris 3113/31 0 f O O . LII n;—-P'o'q-I-' “‘1‘!“ Urn-4‘." hJC-o \' ‘ ' ...,. The no ".‘Er' ; - \- 401‘ an a his. ‘- -..'O X g, a n .9- ‘ 5:35:15)! G- 2 0 «ram buf {~- 7.4 Conta‘ 30 unlabelled cortisol (2.4 x 10-6 M) for 1 hour at 37°C. The tissue slices were then washed 5 times, and homogenized in ice cold Tris-EDTA buffer pH 7.4 containing no enzyme or 0.5 mg/ml of either trypsin, lipase, ribonuclease (RNAse) deoxyribonuclease (DNAse) or hyaluronidase (Sigma Chemical Co.). The homogenates were incubated with the enzymes at 37°C for an additional hour. Homogenates were centrifuged at 700 x g, and the supernatant fractions chromatographed on Sephadex G-200. 2) p—Chloromercuribenzoate (PCMB) Tissue slices from four lactating cows were incubated with 2.7 x 10"9 10"9 M 3H-cortisol plus 6.7 x 10- M 3H-cortisol plus ethyl alcohol or 2.7 x 8 M unlabelled cortisol in Tris—EDTA buffer pH 7.4 alone (controls) or Tris-EDTA buffer pH 7.4 containing 5 x 10-3 M PCMB (Sigma Chemical), a sul— fhydryl group antagonist. Tissue slices were then washed, homogenized, centrifuged and radioactivity was quantified in the 700 x g supernatant and precipitate fractions as described under routine procedures. 3) Oubain (G-Strophanthin) Tissue slices from four lactating cows were incubated with 20 ul of 5 x 10-5 mM oubain for 30 minutes at 37°C. The tissues were further incubated for 1 hour at 37°C with 9 9 2.7 x 10' M 3n-cortisol plus ethyl alcohol or 2.7 x 10' M :E~:::tisol pl‘d :"~es were the i:'.'£1t‘j was q: ::e:::itate fr: proximate Mo N I) m Gel filtr l'I‘e'3ulc‘1r weig is“ binding 5:“ laetatins “"501! wash ’- :35? ”t 158d . A am of Sep‘h :f‘a sabr’ pH 7 . 4 "MC Pressur .35 run and rad 31 3H-cortisol plus 6.7 x 10-8 M unlabelled cortisol. Tissue slices were then washed, homogenized, centrifuged and radio- activity was quantified in the 700 x g supernatant and precipitate fractions as described under routine procedures. Comparison of Corticoid Binding in Mammary Tissue and Blood Serum FrOm Lactating Cows Approximate Molecular Weight Determina- tions 1) Gel Filtration Gel filtration was used to estimate the approximate molecular weight of the 700 x g mammary supernatant and serum binding components of 3H-cortisol. Tissue slices from four lactating cows were incubated with 1.3 x 10-7 M 3H— cortisol, washed, homogenized and centrifuged as previously described. A 0.6 m1 sample was applied to a 1.5 x 25 cm column of Sephadex G-200 and eluted at 37°C with Tris-EDTA buffer, pH 7.4 at a flow rate of 0.25 ml/min using a hydro- static pressure head of 6 to 10 cm. Three m1 fractions were collected, protein concentrations were monitored at 280 nm and radioactivity was counted from each fraction. The relative elution volume for the protein component binde ing 3H-cortisol in the 700 x g supernatant was noted. In other experiments, whole bovine serum was incubated 7 with 1.3.): 10- M 3H-cortisol under the same experimental :11tions as c :22: incubatic :e;:.aiex G-Z 0 0 12 Standard 5c ‘1 r“ «aner, The c N f]; 2:"); oval; '2 4 "" X 10 MW; J. x g mamma; ““33 Volumes . 2) Su & Fresh ti :23‘3’:oyli a Zed ' - g I'D 451‘ .r ‘ lOOIOf' <2 .1 Of 1 3 nkblon was 32 conditions as described above for mammary tissue slices. After incubation a 0.6 m1 sample of serum was eluted on Sephadex G-200 as described above. The relative elution volumes for the protein components binding 3H-cortisol in blood serum were noted. Protein fractions of 700 x g mam— mary supernatants and bovine serum which bound 3H-cortisol were compared, in terms of elution volume, with a series of standard solutions which were chromatographed in the same manner. The standard solutions were Dextran 2,000, 2 x 106 MW; 2.0% ovalbumin, 4.3 x 104 MW; 2.0% B-lactoglobulin, 1.8 x 104 MW; and 2.0% ribonuclease A, 1.5 x 104 MW. The 700 x g mammary supernatant and serum binding proteins were fitted on a standard curve based upon their respective elu- tion volumes. 2) Sucrose Density Gradient Analysis Fresh tissue slices from two lactating cows were homogenized, and centrifuged at 700 x g for 10 minutes. The remaining supernatant fractions were then centrifuged at 45,000 x g to remove other particulate materials. The remaining supernatant was then centrifuged at 100,000 x g for two hours to isolate the cytosol fraction. Three m1 of the 100,000 x g cytosol fractions were incubated with 20 ul of 1.3 x lo‘7 M 3H-cortisol for 1 hour at 37°C. This fraction was adjusted to contain 25 mg of protein/ml of cytosol. Stcvine Seru 1.1' Gent-Ii fug 1..» " 3 .n-‘ Singles Of ;::;.e serum: 9‘ .agered on 4-5 11 $2,030 x g in .- :‘:es were pier= Here collected « Election, 3 m is vials were czve. The app iezernined by u :~:Sv. alcohol d ‘\’4VaaOl dehYdrC 'a‘: L sone “» fiv We tin “ans . 33 Bovine serum was centrifuged at 100,000 x g for 2 hours. After centrifugation, 3 ml of sera were treated with the same amount of 3H-cortisol described above, and incubated under the same experimental conditions. Protein of sera was adjusted to 25 mg/ml of sera. Samples of 0.3 ml of 100,000 x g mammary cytosol or bovine serum, previously incubated with 3H-cortisol, were layered on 4.5 ml gradients of 10-30% sucrose in Tris-EDTA buffer pH 7.4 and were centrifuged at 4°C for 18 hours at 100,000 x g in a Beckman Model L-2 ultracentrifuge. The tubes were pierced after centrifugation and 32 fractions were collected directly into scintillation vials. After collection, 3 ml of ethyl alcohol was added to the vials. The vials were then counted for radioactivity as described above. The approximate sedimentation coefficients were determined by using various standards. The standards were yeast alcohol dehydrogenase (7.6 8, Sigma Chemical); liver alcohol dehydrogenase (5.0 8, Sigma Chemical); and catalase (11.0 8, Sigma Chemical). The migration of standards was estimated by optical density determinations at 280 nm. Polyacrylamide Disc Gel Electrpphoresis Mammary tissue slices from three lactating cows were 7 M 3H-cortisol for 1 hour at 37°C, incubated with 1.3 x 10’ washed five times, homogenized and 700 x g supernatant frac- tions were electrophoresed on 7% polyacrylamide gels .5.- Cellulose Ion excha i§§roach to cc issue and blc li‘bated for ;i;:s;hate buf 1 u ._‘_ a.:: .e ”as has 2: X g! and 1 11:3th Sephac Lg. .EQlDSt 100 vc 13‘ h p In . 1271‘ 5‘ ulos e COl U 34 (Appendix F). Bovine serum from three lactating cows was incubated with 1.3 x 10-7 M 3H-cortisol then diluted 1:5 with 0.85% NaCl and electrophoresed under the same experi- mental conditions as the mammary 700 x g supernatants. DEAE Cellulose Chromatography Ion exchange chromatography (DEAE) was used as another approach to compare cortisol binding proteins in mammary tissue and blood. Thus, mammary slices from three cows were incubated for 1 hour at 37°C in 2 ml of 0.01 M potassium phosphate buffer, pH 8.0, containing 1.3 x 10—7 M 3H-cortisol. Tissue was washed five times, homogenized and centrifuged at 700 x g, and the supernatant fluid was desalted by passage through Sephadex G—25. The fractions containing protein bound 3H-cortisol were combined and applied to a DEAE cellu- lose column (Whatman DE-52, Whatman Co.) which had been pre- equilibrated with the 0.01 M potassium phosphate buffer, pH 8.0. The mammary cytoplasmic proteins were eluted by a stepwise 0.01 M to 2.0 M potassium phosphate gradient, pH 8.0. Ten m1 of bovine serum from three cows was dialyzed against 100 volumes of 0.01 M potassium phosphate buffer for 18 hours, then clarified by centrifugation at 2,000 x g for 10 minutes. The supernatant was incubated with 1.3 x 10.-7 M 3H—cortisol for 1 hour at 37°C and then applied to a DEAE cellulose column and eluted as described above for mammary cytoplasmic proteins. 5‘ 35 Binding of Tritiated Cortisol, Dexamethasone and Progesterone to Bovine Serum In order to determine the relative degree and order of binding of tritiated steroids to bovine sera at 37°C, the following experiment was carried out. Three separate tubes containing 3 ml each of bovine serum from three cows were 9 M 3H-cortisol incubated with 10 pl of either 2.7 x 10- (48 ci/mmole), 3H-dexamethasone (28 ci/mmole) or 3H-proges- terone (48 ci/mmole) for 1 hour at 37°C. After incubation, the tubes were placed in an ice bath at 4°C and samples containing 0.6 ml of serum plus 3H-steroid and 0.4 m1 of 40% sucrose were applied to Sephadex G-200 columns. Three ml fractions were collected, counted for radioactivity and assayed for protein as previously described. The tubes which contained maximal quantities of radioactive cortisol and protein eluting in the region of CBG (MW. 52,000:1500) were pooled. The data were expressed as umole steroid bound per mg protein. Cell Free Experiments 700 x g Supernatant Fresh unlabelled mammary tissue from four lactating cows was homogenized and centrifuged at 700 x g. The super— natant fluids were chromatographed on Sephadex G—25. The fraction containing the cortisol binding receptor was slated. and div} 3. - raved d-cortlS :. other received 221501 (6-7 X 1C (2:: at 37°C. 1 erranatographed E-zcrtisol bou. d e... Ii. protein as pr “M- and 100,: T a: esh marina tie lSOlated a: den“ ““9 199-1134 ‘ I "frr‘atant. 10' 1231'“ - "9.1501 Or 3 § V ‘ i, we at: yLLeCt of U 32:11 . 501 Into t lied . ‘KraCts. Mam 36 isolated, and divided into two aliquots. One aliquot received 3H-cortisol (2.7 x 10_9 M) plus ethyl alcohol while 9 the other received 3H-cortisol (2.7 x 10- M) plus unlabelled cortisol (6.7 x 10-8 M). The fractions were incubated for 1 hour at 37°C. After incubation, these fractions were rechromatographed on Sephadex G-25, and the quantity of 3H-cortisol bound was determined. Each fraction was assayed for protein as previously described. 700 x g and 100,000 x g Precipitates Fresh mammary tissue from four lactating cows was homogenized, and centrifuged at 700 x g. The precipitates were isolated and incubated with 5 ml of the following media: Medium l99-MEM, Tris—EDTA buffer pH 7.4, 700 x g mammary supernatant, 10% bovine serum albumin (Sigma Chemical) and 100% bovine serum. Each medium contained 1.6 x 10"9 M 3H- cortisol or 3H-cortisol plus 4 x 10"8 M unlabelled cortisol. The effect of unlabelled cortisol on the binding of 3H- cortisol into the isolated 700 x g precipitate was deter- mined by measuring the radioactivity in the ethyl alcohol extracts. Mammary slices from four lactating cows were homogen— ized, centrifuged at 15,000 x g for 15 minutes to remove cell debris, nuclei and mitochondria. The 15,000 x 9 super- natant was centrifuged at 100,000 x g for 1 hour. VThe 100,000 x g precipitate was resuspended in Tris-EDTA buffer :14 using a 9'1 stated for 1 l” .V_ ~,,1 "‘1" '1”; 31:11; alCO-A if x 10—8 3‘1) O] 1;, the superna aired once , ani r; precipitate . Phys: A series 0 ”i ”,1 V ~~ s. abelled t sci-ting the bi W: lactating lire designed 1 :r. :1». ..e L1Ptake ‘v. all lactating 37 pH 7.4 using a glass hand homogenizer. The suspensions were incubated for 1 hour at 37°C with 3H-cortisol (2.7 x 10—9 M) plus ethyl alcohol, or 3H-cortisol plus unlabelled cortisol (6.7 x 10-8 M) or 3H-cortisol plus unlabelled dexamethasone (6.7 x 10.8 M). The suspension was recentrifuged at 100,000 x g, the supernatant was discarded, the precipitate was washed once, and radioactivity was measured in the 100,000 x g precipitate. Physiological Effects of Corticoids on Mammary Tissue Slices From Lactating Cows A series of eXperiments were carried out to determine if unlabelled triamcinolone and cortexelone were capable of reducing the binding of 3H-cortisol in mammary tissue slices from lactating cows. Once this was established, experiments were designed to determine the effects of several corticoids on the uptake of C14-glucose into mammary tissue slices from lactating cows. These experiments were carried out in an attempt to correlate physiological activity, such as glucose uptake, with corticoid binding in the mammary tissue slice. Triamcinolone-Cortexelone Competition Tissue slices from four cows were incubated for 1 hour at 37°C in Medium 199:MEM containing either 2.7 x 10.9 M 3anortisol (l, 2 3H-cortisol, 44 ci/mmole, New England izzlear) or 2.7 x '_; K :4 unlabelled 5:;1. cortisol, 5. 2:1. After incu :eztrifuged at 70 . zeszri‘oed above . M n.. P -- Tissues (SO e, “‘4‘“ divided “Elma either “Ough 5 recei 5:3 with ( C‘jes ‘ in ice < ~Rt.‘ «l fuged a 38 Nuclear) or 2.7 x 10-9 M 3H-dexamethasone (l, 2, 4-3H-dexa- methasone; 5.2 ci/mmole, Schwartz/Mann) plus ethyl alcohol or 2.7 x 10.9 M 3H-cortisol or 3H-dexamethasone plus 6.7 x 10“8 M unlabelled triamcinolone, cortexelone (ll-deoxycorti- sol), cortisol, dexamethasone, progesterone and 17-8 estra- diol. After incubation, the tissues were homogenized, centrifuged at 700 x g and radioactivity was determined as described above. Cl4-Glucose Uptake Tissues (500 mg) from four lactating cows were divided into five experimental groups. Each experimental group was further divided into five treatments. Tissues in group 1 received either 1.0--8 M of unlabelled cortexelone, cortisol, triamcinolone dexamethasone, or progesterone. Groups 2 through 5 received either 10'7, 10'6, 10"5 or 10'4 M of the five corticoids listed above or progesterone. Tissues were incubated with unlabelled steroids for 1 hour at 37°C. After 1 hour of incubation, the tissue samples were labelled with 0.5 u ci of 14C-glucose and reincubated for 30 minutes at 37°C. Controls were incubated with 20 ul ethyl alcohol for 1 hour at 37°C, then reincubated for an additional 30 minutes with 0.5 u ci 14C-glucose. Tissues were washed 3 times in ice cold Tris-EDTA buffer pH 7.4, homogenized and centrifuged at 700 x g. The supernatant fractions were counted for radioactivity. Data were expressed as per cent Lbiticn Of tOt 213.115: “Sing th two lactatir 8" H ".azelled cor ti SO1 .,.vn ..-.-.l tissues 1 2:13;, tissues \ -.::' reincubated lfiun eigenized and Six lactati ~-.:.na1 pudic a 39 inhibition of total Cl4-glucose uptake into 700 x 9 super- natants, using the ethyl alcohol controls at 0%. In a series of preliminary experiments, tissue slices from two lactating cows were incubated with 10.6 M, un- labelled cortisol plus 10-4 M cortexelone for 1 hour at 37°C. Control tissues received 20 ul of ethyl alcohol. After incu- bation, tissues were labelled with 0.5 u ci of l4C—glucose and reincubated for 30 minutes at 37°C. The slices were homogenized and treated as described above. En Vivo Experiments Mammary Uptake of Corticoids Six lactating Holstein cows were cannulated at the external pudic artery and the mammary vein. Ten ml blood samples were collected at the following intervals prior to and after milking: -30, -15, -6, 0, +6, +12, +16, +20, +30 and 60 minutes. Arterial and venous blood samples were assayed for total glucocorticoids, as described by Smith et a1. (1972, 1973) and Appendix G. Data were expressed as ng/ml differences, between arterial and venous samples. Statistical ere carried out :di'.'idual an ima kmet's 'T" tes :31 means (3H-c 1.331201) with i: 3:; . texanethasone Cine analyses in .t 3. least squares ~9r0duce a six 33:: ~ - . esenting the STATISTICAL ANALYSES Statistical analyses of hormone competition experiments were carried out using a two-way analysis of variance with individual animals as blocks and hormones as treatments. Dunnet's ”T" test (Dunnett, 1955) was used to compare con- trol means (BB-cortisol or 3H-dexamethasone plus ethyl alcohol) with individual treatment means (3H-cortisol or 3H-dexamethasone plus unlabelled hormones). Two component curve analyses were done on Scatchard plots using the method of least squares regression. The data were then transformed to produce a single "best fitting" linear regression line representing the high affinity (specific) corticoid binding component (Appendix D). Statistical analysis of regression line slopes (Kd's) and x intercepts (number of molecules of corticoid bound per mammary cell) from animals in various physiological states were performed using Scheffe's test for multiple comparisons (Appendix H). A Student's "t" test was used for comparing means of 3H-cortisol bound to components within 700, 15,000 and 100,000 x g mammary supernatants after chromatography on Sephadex G-200 columns. 40 Corticoid bin: 5:35 by standard 1 33.3.1969). The 13.13; time) in A- iere performed wit} ..;':e:t1 program‘s awe»; C W .rom a CDC . 41 Corticoid binding and Cl4 glucose uptake were corre- lated by standard product-moment procedures (Sokol and Rohlf, 1969). The significance of treatments (i.e., milking time) in A-V difference experiments was examined by applying the Student's "t" statistic on the null hypothesis: Ho = A:V = 0, at each interval of milking. All analyses were performed with the aid of a CDC 6500 computer and an Olivetti programable calculator. Scatchard plots were ob- tained from a CDC calcomp plotter. RESULTS £11 VITRO EXPERIMENTS Hormone Binding Preliminary Competition Experiments Unless otherwise stated, mammary tissue slices were incubated with hormones, and the binding subsequently determined in 700 x g supernatant or precipitate fractions. In the absence of unlabelled cortisol, uptake of 3H-cortisol (DPM/ug total DNA) was 35 to 63 times greater in 700 x g supernatants than in 700 x g precipitates of mammary tissue (Table 1). Addition of unlabelled cortisol significantly reduced (P < 0.05) the uptake of 3H-cortisol in 700 x g supernatant and precipitate fractions, but quantitatively this reduction ranged from only 5-17% in the supernatant and 30-35% in the precipitate fractions. Surface area of mam- mary slices did not alter the degree of competition between 3H—cortisol and unlabelled cortisol; thus, subsequent ex- periments used eight whole tissue slices averaging 0.2-0.4 mm3 per tube as described in routine procedures. 42 43 . . 3 . . . Table 1. Binding of H-cortisol to 700 x g supernatant and prec1p1tate fractions of mammary tissue slices from lactating cowsa DPM/ug Total DNAC Whole Halved Quartered Minced 700 x g supernatant 3H-cortisol + ethyl alcohol 227 223 270 153 3H-cortisol + cortisolb 188 211 255 135 700 x g precipitate 3H-cortisol + ethyl alcohol 4.3 4.6 5.0 5.8 3H-cortisol + cortisolb 2.9 3.2 3.5 3.8 aApproximately 0.5 9 tissue was suspended in 3 ml MEM-l99 medium pH 7.4. 3H-cortisol concentration was 2.7x10'9 M in ethyl alcohol. Unlabelled cortisol concentration was 6.7x10‘8 M in ethyl alcohol. bAll values significantly less than respective ethyl alcohol controls (P< 0.05). cTotal DNA was measured in 700 x g precipitate after ethyl alcohol extraction of 3H-corticoids, and was used to adjust data in 700 x g supernatant and precipitate fractions. z-wne 0 I‘D-1 “v 44 Specificity of Corticoid Binding in Mammary TiSsue Slices from Lactating»Cows 1) Competition Experiments Uptake of 3H-cortisol (DPM/ug total DNA), in the absence of unlabelled cortisol, was 61 times greater in 700 x g supernatants than in 700 x g precipitates (Table 2). When compared with ethyl alcohol controls, unlabelled corti- sol and dexamethasone reduced the binding of 3H-cortisol in 700 x g supernatant fractions by 7.8 (p< 0.05) and 33.6% (p< 0.01), respectively; and by 47.4 (p<:0.01) and 57.9% (p<=0.01), respectively, in 700 x g precipitates (Table 2). In contrast, progesterone, l7B-estradiol and testosterone did not significantly (p<=0.05) reduce binding of 3H-cortisol in either fraction. When 3H-dexamethasone was used in place of 3H-cortisol and the various unlabelled hormones were added as described previously, cortisol and dexamethasone reduced the binding of 3H-dexamethasone in 700 x g supernatants by 10.9 and 10.3% (p<=0.05), respectively; and by 27.5 and 50.0% (p<<0.01), respectively in 700 x g precipitates (Table 2). Again, the other unlabelled hormones were without signifi- cant effect on reducing 3H—dexamethasone binding. Uptake of 3H-dexamethasone, in the absence of unlabelled dexa— methasone, was 39 times greater in 700 x g supernatants than in 700 x g precipitates (Table 2). if .v-u-unv Oat- ’unc - w .G .11... .ne'... a .- pal-xviu«))-v ‘4'... ~a-ua 3.)..3A.d..d Inn... 5 5.4.! V, ,0 d.- ‘e‘Ifl-\-h . was- a u n...~. nth-)1 .~ V 4... I o 4“ ‘ r-.-..~. 45 .msoauomuw oumuflmwooum pom ucmumcuomsm m x 005 :H cusp umsnnm o» poms mm3 use .mcfioofiuuooumm mo cowuomuuxo Hosooam Hwauo umumm oumuflmfiooum m x con cw vouommoe was «20 Hmuoav .Aao.ouymv Houusoo Hocooam Hmnuo can» mquo .Amo.ouvmv Houusoo Honooam Hanuo can» mmoqa .Honooam Hmnuo CH 2 mloaxh.m mmz ocommsuosmxoolmm new HomHDHOUImm mo cofiumuucoocoo onp oafl£3 .Honooam dunno :w z m oaxh.m mm3 mvwououm uoaaonmacd mo coaumuucoocoom oé m3 m4 e2 Hoficoo 3:81.. durum n.m Hod m.a mmH osououmoumoa ma 3: m4 m3 H3333 m: a . m mm: a . H a: accumummmoum oo.m nmma om.o own ocommsuofimxoo om.m amma oo.a ghoa Homflunoo manuamflooum ucmumsuomsm oumuflmflooum usuamcuomsm ososuon ooaaoansD are? mxooa exoon mxooa eazo H33 933a MU$5 H33 magma ocommnuosmxoolmm Homfiuuooumm m.m3oo msaumuoma Scum mooflam osmmflu xumEEoE ou mCHUcen oncomnuofimxoplmm 6cm HOmHuuoolmm Mo coauwnwncw Honeshom .N magma (r) .n- v - var. - -A - O-D “ 0“..- I‘ .— 'Ciciu *A.. .. ‘4 - 'V-o.‘ . ' I 2" A . w.. c . : ‘P-Q“ \ ' en, § I § V ‘> ‘ ‘ .. "Uav h ”C. . ‘ N 1 \ -.Q . . : w . . N“§. 46 2) Scatchard Analysis Scatchard plots obtained after incubation of mammary tissue slices from lactating cows with various levels of 3H-cortisol-cortisol at 37°C are given in Figures 1A and 1B. The 700 x g supernatant and precipitate fractions of mammary tissue each had two components with affinity for cortisol. One component had a high affinity for cortisol, with a dis- sociation constant Kd = 5.0 x 10-10 M, as calculated from the slope, in 700 x g supernatant and 0.39 x 10.10 M in 700 x g precipitate fractions. These components were satur- able at low hormone concentrations and believed to represent specifically bound cortisol. Figures 1A and 1B illustrate saturation of the high affinity binding component as the concentration of 3H-cortisol was increased. The number of molecules of cortisol bound per cell by the high affinity component, as calculated from the intercept at the X axis (Figures 1A and 1B) was estimated to be 2837 in 700 x g supernatant and 25 to 700 x g precipitate fractions. The second component had almost zero slope (Figure 1A, and 18), had low affinity for cortisol and appeared unsaturable. For example, this component bound cortisol at concentrations up to 4 x 10_8 M. Binding of cortisol to this component was also nonspecific, since 3H-cortisol binding could not be eliminated by a fivefold excess of unlabelled cortisol. 47 .sofimmoumou moumovm unmoa mo ponuofi oz» mafims ocow ouo3 momxamcm m>uso .oGOEHo: mo coaumnucoosoo owmwoomm o no 300 moumH Hoasofluumm o How moamfiom mucosamoupmsw mo Hosdom m> mmmmm ooam> some m unomoumou m.x one .m3oo mswumuooa Eoum mooflam momma» humEEmE mo mcowuomum ucmumcuomsm m x com on Oohm no nos x e on moo.ov manages acmAUHooum mo poem eharoumom A: m w .fia ousmflm i as: $.70. x <20 9: $.22. 28m com. 80. com com 1 u u — _ u q q H u u _ - _ - u q q - ————— ———'---‘ 48 quEzmmeaw $4.22; 382. A u “ 2070. x on a 3. l. n 7‘ “A l [‘0 OON 00¢ O O (D IXVNG 5W 5191!“ a91:1/DUWB 0 Dow . (6 000. DON. *5. 1' 49 .c0ammoumou moumsvm ummoa mo ponuos on» moans ocoo ouo3 mommamsm o>nso .oGOEHon mo coaumuucoocoo oamaoomm o no Boo umaoofluumm m “0m monEMm oumowamsnomsv mo Hocsom m>.mmmmwa moam> some m usomoumou m.x one .mBOo msflumuomH Eouw woOHHm mommau mumEEmE mo macauomum oumuflmfloonm m x con ou Oohm um A2 mnoa x v 0» moo.ov msflosfln HOmHuHOOImm mo poam oumcoumom .ma ouomflm 50 ma ouswfih 3.70. x <20 033.05 ecsom 0.2 or. o.~_ 0.0. or 0.6 or ed 0 1))4 _ s a))J 4 _ _ 1 _ _ _ _ _ ))+ t“ O u)! rift. kill») )lkllxu . om oe om 5556me >m<22<2 382 or x . no Illv .2070. $0 3. oo_ om. OE (5.0l X VNO fifl/ 519111) 9913/ punog Figures ~: :czoonen1 :2 grecipit. :25 after t It adjustec -; t f‘ CortisQl 22301ecu1e tr3~2$format 1.3 > O. 05) a 33th tiSSuE the tOtal ch‘ “Ll! rE< the}: . d in 1 all]; 51 Figures 2A and 2B show the specific high affinity bind- ing components for cortisol at 37°C in 700 x g supernatant and precipitate fractions of mammary tissue from lactating cows after transformation and adjustment for nonspecific binding (Appendix D). After adjustment and transformation, the Kd for cortisol was 10.5 x 10-10 10 M in 700 x 9 super- natant and 0.5 x 10- M in 700 x g precipitate fractions. The adjusted high affinity components bound 1277 molecules of cortisol per cell in 700 x g supernatant fractions and 22 molecules of cortisol in 700 x g precipitate fractions. Transformation of Scatchard data did not significantly (p>'0.05) affect the dissociation constants for cortisol in both tissue fractions, but significantly (p‘=0.05) reduced the total number of molecules of cortisol bound per cell by approximately 55% when compared with the values obtained with non-transformed data. The dissociation constants from adjusted and unadjusted Scatchard plots, of cortisol bound to mammary tissue slices from lactating cows incubated at 4°C, were not significantly different (p>’0.05) from those for tissue slices incubated at 37°C (Table 3). The lower temperature, however, signifi- cantly reduced (p<:0.01) the number of molecules of cortisol bound in mammary cells by approximately 82% (calculated from adjusted‘values). Figure 2A. 52 Transformed Scatchard plot of 3H-cortisol bind- ing (0.005 to 0.041 x 10’8 M) at 37°C to 700 x g supernatant fractions of mammary tissue slices from lactating cows. The A's represent Bound 15%;; (corrected) vs Bound] of quadrupli- means [ cate samples for a particular cow at a specific concentration of hormone. Curve analyses were done using the method of least squares regres- sion. Bound/Free (Liters /yg DNA x 10‘9 J 360 320. 280 m 4: O I m C) O I O I IGOF [20)- (I) 0 fi 4:. O f I O Bound /Free (Liters lpg DNA x l0'9) 360 320 280 N A 0 PO C) CD l60 |20 00 C) A C) CD I J l l l 1 J 0 8.0 16.0 24.0 32.0 40.0 53 700 X g SUPERNATANT <—— KdEIo.5on"°M L l l _1 Bound (Moles/pg DNAXIO‘W) Figure 2A Figure 2B. 54 Transformed Scatchard plot of 3H-cortisol bind- ing (0.005 to 0.041 x 10‘8 M) at 37°C to 700 x g precipitate fractions of mammary tissue slices from lactating cows. The A's represent means [23:23 (corrected) vs Bound] of quadruplicate samples for a particular cow at a specific concentration of hormone. Curve analyses were done using the method of least squares regres- sion. Bound /Free (Liters/pg DNA x IO_9) cu 4s (n o» C) __:_;3 _g) (3 N O -4 CD (0 C) CD (3 O) C) C” CD Bound /Free (Liters/pg DNA xlO'9) n3 OJ J> CD CD CD '6 CD 55 I 700x 9 PRECIPITATE I , <— Kd~0.5x|0"°M 1 I l I .' j l - 4 _I 0 0.2 0.4 0.6 0.8 I.O Bound (Moles xpg DNA no“) I 1 Figure ZB CQOE NO NOMHQ I: HOMfiUHOU I OH X i‘l‘ifii UHUCCQum + AHTU >h€EEfiE Ca 02333 OHI ACmuuHCU MO Q$HJUGHOE N0 HOQEZZ AUCMUNCOU C0HU$fiUOWMflU~ UX n..:¢.¢ncU..vl~d {.51-L I.d...u¢-ad Adj-Inaqu shill-35!}: AUJ Ma.~1!u-fia.~ fiAJ-Ifidhfivhv N0 Idflufind nuhlv-00flouwu nhflv aha-.55..” om» Mvflfiflqa 56 .Uohm um conundocfl mooflam wanna» msflumuomH Eoum ooswmuno mooam> Eoum Amo.o.nmv ucouommwc maucmowmacmfim no: ohm momma voumsnodo ooom no oouonooae mooflam manna» mcwumuoma Eoum conflmuno mosam> coca Ho3OH maamoHDMHumum who memos woumsnpdn .Am saccmmmflv mGOmHummEoo oamfiuass now now» n.0mmonom means mmoam> voDMSmom so UoEHOMHom mmB mammamsm Hmowumflumum .HOMHuuoo UoHHoQMHss was Hemwuuoolmm mo coflumcwnsoo M mo 2 m OH x v on moo.o sufi3 Uov um woumnsoca ouoz moowam oommflam m.“ o om + mama o~.o n.ea ooumaflooao no + o and + mma oo~.o oo.oa ooumonoa mumuflmwooum usmumcuomsm opmufimwooum Damascuomdm o x ooh o x ooh come no uouuo oma Moo x unaccoum.fi HHoo >MMEEQE cw canon H .u z OHIOH Honeymoo mo moasooaos mo Monssz Ausmumsoo sowumwoommwvv QM n.0ov um msoo msfiumuomH scum moowam osmmfiu wumEEms on masocfln Homfluuoo mo muon oumnoumom mo mumsfism .m manna Scatcl'1 assue Slic single high i1: dexametl iii-E binding j. ecipi ta te Era: Scatcha isscciation :cse of cor‘ tissue slice: lssociation 13. 700 X 9 pr from lactatin 1"1541) those f0 SllCes bound :exanethasone 57 Scatchard plots of dexamethasone bound to mammary tissue slices from lactating cows at 37°C also revealed a single high affinity binding component, which was specific for dexamethasone and an unsaturable nonspecific dexametha— sone binding component in both 700 x g supernatant and precipitate tissue fractions. Table 4 summarizes the data from Scatchard plots of dexamethasone treated tissues. The dissociation constants were not significantly different from those of cortisol in 700 x g supernatant fractions of mammary tissue slices from lactating cows (Figures 1A and 2A). Dissociation constants for dexamethasone-receptor complexes in 700 x g precipitate fractions of mammary tissue slices from lactating cows were significantly greater (p< 0.01) than those for cortisol (Figures 1B and 23). Mammary tissue slices bound significantly more (p<:0.01) molecules of dexamethasone in mammary cells than cortisol. Mammary tissue slices bound approximately twice as many molecules of dexamethasone per mammary cell than cortisol (Table 4). Corticoid Binding in Mammary Tissue Slices from Virgin Heifers, 1-Month Prepartum, Lactating_andiDry Cows 1) Competition Experiments Virgin Heifers Unlabelled cortisol and dexamethasone reduced the binding of 3H-cortisol in 700 x g supernatant fractions by 11.7 (p<<0.05) and 15.6% (p<<0.05), respectively and by 43 CMTE uO mm H A400 >N$EE§E 2% DEUCE OCOWMSUOEMXQU 2 ON OH X GCOmczquGXOU NO GMHSUUHOE NO HQQEZZ AUCMumCOU COwumflUOmmflUC TM I. unU...k.Ml he and 3.300,). huv-idfaupdw4fi Esahh inched n ' Pun-lul- n a >.nl..:::'¢-: .uu, urn-n;— Fa-n can EO-bfidufllHP-UpUS-lwvflmvrv an: IUFVNAN Fe,u!.v-Ud-wr4uw. twee \nln fi-S-~..r.lu In! mfl.WA#fJ.N. S8 .HOmwuuoo nuw3 Oohm um woumndocw moowam momma» msflumuoma Eonm nocwmuno mosHm> Scum Amo.o.nmv usmuommwc hausmowmwcmfim no: mum mamas coumsnodo .Homfluuoo suds Oohm um woumndosw moowam mammflu mcwumuomH Eoum pocflmuno mosam> can» Aao.ouvmv uoumoum anamoflumaumum mum mamas cmumsnomn .Am chsommdv msomwumgfioo mamwuass How umou m.owmonom msflms modam> topmonom co oosuomuom mm3 mammamsm Hmowumwumum .oGOmmnuosmxoo voaaonMHsd can osommnuos Imxoonmm mo sowumswnsoo m «o z m OH x a on moo.o nuw3 Uobm um conundosw mums moowam osmmwam mm + New om .+. memm me 22 membranous: 1+. H . . woman a nma won nmm moon no v om NH 0 .o oumuwmfioonm ucmumsuomdm oumuwmwooum unnumcuomdm o x ooh o x ooh come no mm.H Haoo mumssds ca canon ocommnuosmxoo 2 0H OH x mnemmnuosmxoo mo moasooaos mo nonfisz Aucmumcoo COHUMHUOmmva QM 0.0onm um mzoo mswumuoma Scum moofiam osmmflu kHMEEME on mcwpcwn osommnuoemxmn mo muon numnoumom mo mumafidm .v manna 59 (p‘<0.01) and 62.2% (p<=0.01), respectively, in 700 x 9 pre- cipitates (Table 5). Progesterone, l7B-estradiol and testo- sterone did not significantly (p> 0.05) reduce binding of 3H-cortisol in either fraction. In the absence of unlabelled cortisol, uptake of 3H-cortisol (DPM/pg total DNA) was 21 times greater in 700 x g supernatants than precipitates (Table 5). In experiments where 3H-dexamethasone was used in place of 3H-cortisol, unlabelled cortisol and dexametha- sone reduced binding by 12.7 (p< 0.05) and 27% (p< 0.01), respectively, in 700 x g supernatants. Unlabelled dexametha— sone reduced 3H-dexamethasone binding by 75% (p<=0.01) in 700 x g precipitates (Table 5). Unlabelled progesterone, l78-estradiol and testosterone were without effect on 3H-dexamethasone binding. The uptake of 3H—dexamethazone, in the absence of unlabelled hormones, was only 5.7 times greater in 700 x g supernatants than precipitates (Table 5). l-Month Prepartum Cows Unlabelled cortisol and dexamethasone reduced the bind- ing of 3H-cortisol in 700 x g supernatant fractions 22.1% (p<<0.01) and 19.8% (p<:0.05), respectively, and by 55.2 (p<<0.01) and 65.5% (p‘<0.01), respectively, in 700 x 9 pre- cipitates (Table 6). In experiments where 3H-dexamethasone was used in place of 3H-cortisol, unlabelled cortisol and dexamethasone reduced the binding of 3H-dexamethasone in 700 x g supernatants by 17.4 (p<:0.05) and 19.0% (p<=0.05) Caz: _quE t:\z;: a2: Farce tzxzcc .o:0mo£uoEaxoUI:m u HOmquOOIIW 5.0.».0Nfinvz CHTHflxr truhuu o..- .¢ -‘ no..v.\¢ a a .) .v.......o.:- ..- \.-.¢-:-<.- 9e--¢:Is-. cc.-.n.xeo-.u'.-- w:~oce Howl-4L ~nJ..u.lu~. Ls... .savu Q ‘.~‘--‘ NP._~AJ.=-n.- u... rv~nr-..~. 60 .mcowuomum oumuwmflooum pom ucmumcnomom m x 005 as «non unanom on poms mmB use .mofloofluuoonmm mo sofluomuuxo Honooam dunno Houmm oumufimaowum m x con ca vousmmoa mmz £29 Hmuoev .Aao.ouvmv Houuooo Horooao Harem can» mmoqo .Amo.ouvmv Houusoo Honooam ahnuo can» mmoqn .Honooam amnuo GM 2 mnoa x h.~ mm3 ocommnuoemxovlmm use Homwuuooumm mo sowumuusoosoo on» oafin3 .Honooam dunno CH 2 m on x 5.0 was mowououm vmaaonmasa mo cowumuucoosoom H.HH mo n.m sh Houucoo Honooam Hanna m.ma an h.m up osououmoumoa «.3 S a. .m ma Honoafimoa S e.HH mm e.m om oaououmooohm ow.~ oov ov.a nmo ocommnuosmxoo . . and Ho m on nmm 0H m now A .u o mumuwmwooum usmumsuomdm oumufimflooum ucmumsuomim o x ooh o x ooh o x ooh o x ooh oaosuor ooHHooaHao oazo Houoa m:\2mo oazo Hobos mn\zmo osommnuosmxoclmm HOwwuuooumm m.muomwon smefl> Bonn moofiam oommflu xumsEdE on mcflocfin oCOmmnuomeooImm 0cm HomHuHOOImm mo sofiuflnwnsfl HMGOEnom .m manna III) Ill .HMEEME ou mcflosfln oGOmMSummeovlmm ocm HOmfluHOOImm mo :oHuflnwnsw Hmcofiuom .o manna respectively, an :espectively, in _:::;esterone, l7 faintly (p > 0,C :E-éexamethasone 20's (Table 6) . L: the absence c mater respectj 35:63 . Lactati, \ Unlabelled .3! 3: h-cortisol : ZP’0.05) reduce binding of either 3H-cortisol or 3H-dexamethasone in mammary slices from 1-month prepartum cows (Table 6). Uptake of 3H-cortisol and 3H-dexamethasone, in the absence of unlabelled hormones, was 23 and 29 times greater respectively in 700 x g supernatants than precipi- tates . Lactating Cows Unlabelled cortisol and dexamethasone reduced binding of 3H-cortisol in 700 x g supernatant fractions by 17.4 (p‘<0.05) and 20.4% (p<:0.01), respectively, and by 50.0 (p<20.01) and 55% (p<<0.01), respectively, in 700 x 9 pre— cipitates (Table 7). As previously shown, unlabelled progesterone, 17B—estradiol and testosterone did not signifi- cantly (p>'0.05) reduce the binding of 3H—cortisol in either fraction of lactating tissue slices. The uptake of 3H- cortisol in the absence of unlabelled hormones was 52 times greater in 700 x g supernatants than precipitates. Cortisol and dexamethasone reduced the binding of 3H- dexamethasone in 700 x g supernatants by 9.7% (p<:0.05) and by 44 (p‘<0.01) and 70% (p<<0.01), respectively, in 700 x g precipitates (Table 7). The other unlabelled steroids failed to significantly (p>'0.05) reduce 3H-dexamethasone w v V” *. . . M n m n» a 33443 nae: w d 9.4 .3. 9.4 =:v,d H 0.06... .v- ton-[ecu a * ~9.......o~:- an. a --¢w._~u.- ad-.e$—-b.a woo-.unv‘cb-SII-w rug-'3 H..v~‘ o I3.v.)-n .H... .nva a ~e~wr~a- ~13-Ad.-~..u~ u\ v..~..~u...~. - ‘alc an... Lrtr—L c:\w( rmC (ZQ HQUOEH. o~A\ZQQ I; 63 .mcofiuomum oumuwmfioonm use unnumcuomsm m x 005 an aunt umsnom on com: mmz tam .mofloofluuoonm mo cofiuomuuxo Honooam Hanuo uoumm oumuflmfiooum m x 005 CH cousmmos mm3 £20 Houoa M NO cowumuucoocoo on» oawnz .Honooam Hanuo ca 2 U .Aao.ouvmv Houucoo Honooam dunno can» mmoqo .Amo.ouvmv Houucoo Honooam Hmnuo son» moon n .Honooam ahnuo ca mica x h.m was ocowmnuoamxoc can Homwuuooumm OH x v.0 mmz mononoun voaaoancs mo cowumuucoocoum m v.m mva m.H mm Houusoo Honooam Hanan ~.o OMH o.~ om osououmoumoa 0.9 mma m.m mm Howomuumotmha o.m «NH m.H mo oaououmoooum om.m nHmH oom.o own ocommnuoamxoo oh.v nama omm.o nam acmwuuou oumuwmfiooum ucmumcuomdm oumuflmwooum ucmumsquSm o x ooa o x com o x ooh o x ooh oaosuor ooaaonoaao oazn Hobos mn\zmo ocomwnuoamxoOIm m azo Hobos on\zmo c Homwuuoolmm m.m300 msflumuUMH Scum moofiam mommau humsEmE.ou mqqpsan os0mmnuoamxovlm use Homauuoolm mo coaufinansw Hmsosuom .h wanna m m ;::.ding- The “F :Labelled hOmC starts than pre Drv Cows Competitive nary tissue 5 cortisol and de: 32') (p< 0.01) a1 fractions (Tablc also reduced tn. £2.22 (p< 0.01) Table 8). Whe Certisol, unlab re“lace the bind fractions. Unl binding by 17.9 331 ‘ Walled cort is KinethaSOne } ‘capectively I I 64 binding. The uptake of 3H-dexamethasone, in the absence of unlabelled hormones, was 17 times greater in 700 x 9 super- natants than precipitates. Dry Cows Competitive hormone binding experiments carried out on mammary tissue slices from dry cows showed that unlabelled cortisol and dexamethasone reduced the uptake of 3H-cortisol 22% (p< 0.01) and 13.6% (p<:0.05) in 700 x g supernatant fractions (Table 8). Unlabelled cortisol and dexamethasone also reduced the uptake of 3H-cortisol by 43 (p<:0.01) and 62.2% (p<:0.01), respectively, in 700 x g precipitates (Table 8). When 3H-dexamethasone was substituted for 3H- cortisol, unlabelled cortisol did not significantly (p> 0.05) reduce the binding of 3H-dexamethasone in 700 x g supernatant fractions. Unlabelled dexamethasone reduced 3H-dexamethasone binding by 17.9% (p< 0.05) in 700 x g supernatant fractions. Unlabelled cortisol and dexamethasone, however, reduced 3H- dexamethasone binding by 40.2 (p<=0.01) and 77% (p<=0.01), respectively, in 700 x g precipitate fractions. Unlabelled progesterone, l7B-estradiol and testosterone failed to reduce 3H-cortisol and 3H-dexamethasone binding in mammary tissue fractions from dry cows (Table 8). Uptake of 3H- cortisol and 3H-dexamethasone, in the absence of unlabelled hormones was 22 and 6 times greater, respectively, in 700 x g supernatants than precipitates. 65 .mcofluomum oumuwmwooum new ucmumcuomsm m x oon cw dump umsnom on cow: was 0cm .mofloofluuoonmm mo sowuomuuxo Honooam dunno nouum mumuwmfiooum m x con cw vouammos mm3 umEEME ou msflocwn 0:0mmnuosmxootmm new HomAUHOOImm uo cowuwnfinCfl accosuom .m manna 66 2) Scatchard Analysis: Virgin Heifers, 1-Month Prepartum, Lactating and Dry Cows (nonpregnant and nonlactating) A summary of the results obtained from adjusted and un— adjusted Scatchard plots using 3H-cortisol and 3H-dexametha— sone in mammary tissue slices from virgin heifers, l-month prepartum, lactating and dry cows are given in Tables 9-12. Scatchard plots of hormone bound in the various physiologi- cal states displayed two components which bound cortisol and dexamethasone in 700 x g supernatant and precipitate fractions. One component had high affinity for corticoid and was saturated at low hormone concentrations. This com- ponent was believed to represent the specific binding component for corticoids. The other component had low affin- ity for corticoid and was practically unsaturable. This component was thought to be nonspecific for corticoids. Mammary tissue slices from lactating cows, virgin heifers, 1-month prepartum and dry cows, incubated at 37°C with various concentrations of 3H—cortisol bound this hormone 1° to 20 x 10’10 M, with high affinity (Kd = 0.30 x 10' Table 9). The Kd for the cortisol-receptor complex in 700 x g supernatant fractions of virgin heifers, was signifi- cantly (p<<0.05) greater when compared with those for l-month prepartum, lactating and dry cows (Table 9). Furthermore, the Kd for the cortisol-receptor complex in 700 x g mammary supernatant fractions of l-month prepartum 67 .Amo.o vac snoaooeueaonm nonuno asonoo were or» an muorpo fine .Amo.o.nm ..o.nc economuho >Hucm0HMHsofim no: one mumwuomuomdm mean any mswumnm .sssHoo mean on» cw memos coumsflo¢ v.0.n .A: xfivsommnv msomflummsoo onHuHSE now umou m.ommonom manna mosam> nonmanvm co oosuowuom mm3 mamhamcm Hmoaumwuoumm .ocfiosfln owmaoommsoc Mom wouoouuoocd muon oumnoumom Bonn ooswmuno .moSHm> noumsnvmss Anvmsdv .oswpcfln owmaoommco: Mom wouooHuoo muoam cnmnoumom Scum nosflmuno .moaam> woumsnpm Amway m.a nm.~ m.o om.n m3oo Amcflumuomaco: .ucmsmoumsosv who ov.o 00m.o m.m ov.oa ozou msaumuomq H.H omm.o H.o om.m msoo Eduummoum nusozla m.~ n¢.m ha now muowfiom samufi> racers. Afloat Aflooaoc inane oumuflmflooum uswumsuomsm o x ooh S on x Aucmumcoo :oflumH00mmwov QM oumum Hmowmoaoflmwnm on- Homnuuoo m.m3oo Amswumuomasoc .usmsmoumdocv who one maaumuoma .Esuummoum nuCOEIH .mquHon camuw> Scum moowam manna» whmEEME ou msficsfin Homfiuuoo you Am.vxv musmumcoo coHuMAUOmmwo uon uumnoumom mo mumeesm .m manna 68 cows was significantly (p< 0.05) lower when compared with those for lactating and dry cows (Table 9). Dissociation constants for cortisol-receptor complexes in 700 x g mammary supernatant fractions of lactating and dry cows were not significantly (p> 0.05) different from one another (Table 9). The Kd's for cortisol-receptor complexes in 700 x g precipitate fractions of virgin heifers and dry cows were significantly (p< 0.05) greater than those for l-month pre- partum and lactating cows (Table 9). There was no signifi- cant (p> 0.05) difference between the cortisol Kd's of 700 x g precipitates of virgin and dry and between l—month pre- partum and lactating cows (Table 9). Statistical analyses of the number of cortisol mole— cules bound in mammary cells showed that virgin heifers and dry cows bound significantly (p< 0.05) fewer molecules of cortisol in 700 x g supernatant fractions when compared with l-month prepartum.cows and lactating cows (Table 10). But there was no significant difference (p> 0.05) between the number of molecules of cortisol bound in virgin heifers and dry cows (Table 10). Lactating cows bound significantly (p‘<0.05) more molecules of cortisol in 700 x g supernatant fractions than l-month prepartum cows (Table 10). There was no significant (p>’0.05) difference between animals of various physiological states with regard to the numbers of cortisol molecules bound in 700 x g precipitate fractions (Table 10). J.- n.— -.-.o..:- a....‘- o .a-ec u-u nd>I-I—.u'Iq-uunl in: Illa-aauu:an\ .‘1.I. I - . . - 1 69 .Amo.ouvmv hauamofiwwamHm Hommnc asnaoo 05mm onu aH muonuo Ada .Amo.o.nm ..o.HV uaoquMHc wauamoHMHamHm uoa mum mumHuomuomnm mean on» mawumnm .afinaoo mean on» aH mamoa coumnncan .Am chaommav maomHummEoo onHanE How umou m.ommonom maHmn monam> coumnncm ao cosuomuom mm3 mHmwamam Hmowumwumumm .mchaHn owmwoommaoa mom couoouuooan muoam cumnoumom scum coaHmuno .monam> coumnncman Ancmanv .mchaHn UHMHoommaoa How couoouuoo muon cumnoumow Scum coaHmuno .monam> coumnflcm Ancmv +| on on no H mm we H 2.3 o3 H Sn 260 3:38.035: Scheme—macs aha o H 2 non H am 2 H 8mm mo H 2.2 9.60 33303 m H mm n2 H mm on H $3 mm H omm 9:8 .538on 8.282% 3 H on no H om eo H 88 n2 H 8m 333: 5m»; 1823 393 33:3 783 . oumuwmwooum uamumauomnm o x o2. mm.“ HHoo >HMEEME aH canon Homwuuoo mo moHnooHoa mo Honfinz ououm Hwowmoaowmmnm a.m300 Amawumuomaaoa .uamamoumaoav mac can maHumuomH .Enunmmoum nuaoEIH .muomwmn aHmHH> Bonn mooHHm mnmmHu >HMEEME mo “acme mo mw.H Haoo mumsama aH canon HomHunoo mo moanooaos mo nonsnav mumoououawnx uoam cumnoumom mo humafinm .oa wanna 70 The Kd for the dexamethasone-receptor complex, in 700): g mammary supernatant fractions of virgin heifers was sig- nificantly (p<=0.05) lower when compared with that for lactating and dry cows (Table 11). There was no significant (p>'0.05) difference between dexamethasone—receptor Kd's in 700 x g mammary supernatant fractions of lactating and dry cows (Table 11). Dissociation constants for dexamethasone- receptor complexes in 700 x g precipitate fractions did not significantly differ with regard to physiological state (Table 11). Statistical comparisons of the number of dexamethasone molecules bound in mammary tissue slices showed a signifi— cant difference between animals of various physiological states (Table 12). For example, virgin heifers and dry cows bound significantly (p‘=0.05) fewer molecules of dexametha- sone in 700 x g supernatant fractions when compared with lactating cows (Table 12). There was no significant dif- ference between the number of molecules of dexamethasone bound in 700 x g supernatants of virgin heifers and dry cows (Table 12). There was no significant difference between the number of dexamethasone molecules bound in 700 x g precipitate fractions of mammary slices from virgin heifers and lactat- ing cows (Table 12). However, 700 x g precipitate fractions from virgin mammary slices bound significantly (p< 0.05) .Amo.ouvmv haucmOHMHcmHm HowwHo :Esaoo 08mm may cw muonuo Had .Amo.o.nm ..o.Hv ucmumMMHo xaucmoHuHcmHm poc mum mumwuomummdm meow may mCHumnm .casaoo meow may cH memos vmumsnodo.n .Am xwpcmmmtv mCOmHummEoo mHmHuHsE HOM umou m.mwmm£om msHmn mosam> omumsnom so UoBMOMHmm mm3 mwmaamcm HMUHumHumumm .msHoan UHMHommmco: How owuoouuoocd muon oucnoumom Eoum owcwmuno .mmsam> vmumsnomcs Ancmcsv .oCHocHn OHMHoQOcoc Mom omuomuuoo muon oumnoumom scum vocflmuno .mmsam> coumsflom Annoy 71 m.a ov.m v.m o~.m mzou Amcwumuomaco: .ucmcmoumcocv hum v.v ov.v m.ma oa.mH m3ou mcwumuomq o.m on.m m.m am.m mummwmm sHouH> Aflomcsv Amway Anomasv Anna. oumuHmHowum accumcuomdm mxoon 2 OH x Ausmumsoo coHumHUOmmHov UM mumum HMUHmoHOHmwnm can mGOmmcuomeoo m.m3oo Amcflumuoma Icon .ucmcmoumcocv who can mCHumuoma .muwmwmn chHH> Eouu mooHHm osmmHu aumesde ou mcwocwn mcommnumamxmo now Am.oxv mucmumsoo coHuMHoomeo uon oumnoumom mo humaasm .HH manna 72 .306 v3 Suamowmacmflm 33% 5530 «5m 93 fi 3930 H2 .305 Am :93 ucmumMMHp haucmoHMHcmHm you can mumHuomummsm meow may GGHHMSm :EDHOO menu on» GH memos voumsnocorn .Am xHUsommdv mQOmHHmmEoo mHmHuHsE Mom ummu m.ommm£om mcams mosHm> Umumsnom so vosuomuom mm: mwmaamcm HMUHumHumumM .mCHocHn UHMHoommcoc you omuomuuoocs muoam oumzoumom scum oocwmuno .mmsam> omumsflomss Anvmcsv .msHocwa UHMHommmco: Mom omuoouuoo muon vumnoumom Baum omcwmuno .mmsam> Umumsnom Annoy M300 3 H 3m 08 H m2 S H 2.: n2 H Be .mfiumuoflcoa Jamcmmumcoa sun 3 H 08 n3 H E «3 H 32.. 03 H ES .38 33303 on H can nmm H mmm 8 H 33 n3 H m? 8533 59.; 3:23 A .88 x 333 x has oumuHmHooum unmumsuwmsm m x eon mm.H Hamo mMMEEME CH mason ocommsumadxwp mo mmasooaofi mo Monsdz mumum HMOHmoHOHmmnm m.m3oo Amcwumuomacoc .ucmcmmumcocv who can mcHumuowH .mumMHmn chuH> scum monHm osmmHu humeama no “come no mm.H Haoo mumaems cH canon mcommnumamxdo mo moasomaoa no Monascv mummoumucHtx uon oumnoumom mo humsasm .NH manna 73 more molecules of dexamethasone when compared with dry cows (Table 12). Lactating cows bound significantly (p<10.05) more molecules of dexamethasone in 700 x g precipitates than dry cows (Table 12). Tables 13 and 14 show the statistical comparisons be— tween cortisol and dexamethasone Scatchard plot slopes (Kd's) and x-intercepts (numbers of molecules of corticoid bound in mammary cell) in mammary slices from animals in various physiological states. The Kd for the cortisol-receptor complex in 700 x g supernatant fractions of virgin mammary tissue slices was significantly (p<<0.01) greater than that for dexamethasone (Table 13). However, the Kd for the cortisol-receptor complex in 700 x g precipitate fractions of virgin mammary slices was significantly (p<<0.05) less than that for dexamethasone (Table 13). There was no sig- nificant (p>’0.05) difference between the cortisol and dexa- methasone Kd's of 700 x g supernatant fractions of mammary tissue slices from lactating or dry cows (Table 13). In contrast, the Kd for cortisol in 700 x g precipitate frac- tions of mammary slices from lactating cows was significantly (p<:0.01) less than that for dexamethasone (Table 13). The cortisol—receptor complex Kd for cortisol in 700 x g precipi- tate fractions of dry cows was significantly (p< 0.05) less than that for dexamethasone (Table 13). There was no significant (p=>0.05) difference between the number of cortisol and dexamethasone molecules bound in 74 .Amo.o.nm ..0.Hv ms0mwnuwfioxmo can HOmHuHoo How mmaam> cowsuon mocmuwMMHv acmoHuHsmHm ozo .Amo.ouvmv mc0mmsumamxmo Mom 05Hm> sown acoummqu haucmowmwcmwmo .AHo.ouvmv mnemmguosmxmo you 05Hm> scum ucmHmMMHp kHHGMOHMHcmHmo .mcommnuoamxmo n xoa .HOmHuHoo u uuoun .Am chcommdv mCOmHHmmEoo onHuHsE How umou m.mmmonom mchs mosam> nonmanvm co oofiuomuom mm3 mwmaamcm HmowumHumumM v.0 om.m «.0 om.h mzoo Amcwumuochos .ucmcmoumcocv mun e.v oom.o H.ma m¢.oH msou mcaumuooq h.m vv.~ m.m oom muomwmm ckuH> x no x uo on u o n on Q» U mumuHmHomum usaumcummsm m x cos 2 0H oa x Aucmumaoo coaumfioommficv ax mumum HmoflmoHoamanm m.moumum HMUHmoHOHmacm mnoHHm> sH mamsasm Baum mmowam mumaama cH mnemonuwemxmc can HOmHuuoo Mom Aw.oxv momoam uon oumnououm mo cowauumaoo HMUHumHumum .ma manna 7S .Amo.o.nm ..o.HV mGOmmzuomemv 0cm HOmHuuoo Mom moaam> cmm3uwn monouommwo unmoHMHcmHm ozm .Amo.ouvmv ocOmmnuoemxoo How msHm> Soum ucouwmwwo maucmowmwcowmo .Aao.ouvmv mcommnumamxoo How 05Hm> Eoum ucmuwmuwo aaucmowmwcmwmo .GCOmMSHwfiflunmU " nag ~H0mHUHOO fl #HOUQ Am xaocmmmcv mcomfiummEoo mHmHuHsE you umou m.mumw:om ochs mosam> noumsnom so omsuowuom mmz mwmhamcc HMUHumHumumM msoo mN.H mNH oo.H mm NN.H hov mmH.H hon Amcwumuomaco: .ucmcmoumcosv aha mH.H 55H ooa.H Hm Ho.H mesa umv.H mvma mzoo mcaumuomq mm H mom om H mm 2 H m3 «3 H man 383% £3; x00 uuoo x00 uuoo oumuHmHomum n ucmumcummdmn m x 005 mm.H Hamo hquEoE GH @2503 oHoowuuoo mo moaaomHOE mo Honfisz wumum HmowmoHonanm ca named: «.mmumum HmonoHonanm muoHum> oaooauumoomwomu mmowam hunsane cw «cemmnuoamxmv can Homfluuoo Mow «ammo mumssms 2H wcson . . m so ' _ . H mHOE Mo Hun—.55 mummououcw x UOHQ vumnouwom Mo COmHHmQEOU HmUHumHHMHm .VH MHQMB 76 the 700 x g supernatant fractions of virgin heifers and dry cows (Table 14). Mammary tissue from lactating cows, however, bound significantly (p < 0.05) more molecules of dexamethasone than cortisol in 700 x g supernatant frac- tions (Table 14). Tissue slices from cattle in all physio- logical states studied, bound significantly (p< 0.01) more molecules of dexamethasone in 700 x g precipitate fractions than cortisol (Table 14). Tissue slices from all animals studied bound approximately twice as many total molecules of dexamethasone (number of molecules bound in 700 x g supernatant plus the number of molecules bound in 700 x g precipitates) than cortisol (Table 14). m_9_t-éll:>olism of 3H-cortisol The majority of radioactivity in the bound and free fractions of 700 x g supernatants, isolated by Sephadex G"200 chromatography, migrated in a spot on thin-layer Ch’5‘<>Inatograms which superimposed on standard cortisol (Figure 3) . No metabolites of 3H-cortisol were found in the 700 x g supernatant fractions. 77 .mzoo mcwumuomH Eoum mwowam msmmHu wumfieme mo musmumc lquSm m x con mo msowuomum =mmuw= can acadonnsHmuoum= Eoum cwuomuuxo HOmHuuoolm mo hamwumoumeouno MommHIane m .m musmHm 78 m magmas “88 £35 Eat 3535 m. _. o. m m s o n e 5:23 oomuo Bot .63 .358: \ c638 oomuo 60.: x03 .38.“... T236250 -xn.2m--l A... Loou noon ndoc L 000 1 00m lNdO 79 Physicochemical Characterization of Corticoid Receptors in Mammary Tissue From Lactating Cows Gel Filtration Chromatography l) Manunary gytosol Fractions 700 x g Supernatant Two radioactive peaks were present in the Sephadex (}—25 elution profiles of 700 x g supernatant samples obtained fh:om.mammary tissue slices treated with either 3H-cortisol pfiLus ethyl alcohol or 3H-cortisol plus unlabelled cortisol (I?igure 4). The peak of 3H-cortisol radioactivity in frac- ‘tgion number 5 was associated with a major protein peak from 'tlae 700 x g supernatant fractions. The peak of radioactiv- :ii;y found in fraction 12 had no corresponding peak of assayable protein. Addition of unlabelled cortisol reduced the quantity of radioactivity present in the protein-rich fraction (number 5) by approximately 66%, when compared with 3H—cortisol-ethyl alcohol controls. 15,000 and 100,000 x g Supernatants The profiles of radioactivity (not shown) for the 700, :L5:¢300 or 100,000 x g supernatants after Sephadex G-200 chromatography were similar to those shown in Figure 4 for 3H”cortisol plus ethyl alcohol. The amount of 3H--cortisol associated with the proteins of 700, 15,000 and 100,000 x g suPet-natants were comparable and averaged 3,400 i 134, ¥ Figure 4. 80 Gel filtration (Sephadex G—25) elution profile of 700 x g mammary supernatant from tissue slices of lactating cows treated with 3H-cortisol plus ethyl alcohol or 3H-cortisol plus unlabelled cortisol. cpm cpm 800 600 400 200 IOOO 800 600 400 200 I; 81 G- 25 3,;- Cortisol 3" -Cortlsol I T - p b I’m-Cortisol + Cortisol 3.1- Cortisol [‘WProtein I; \.\o-o-o ’.-\\_’ l l l A 1 I 1 I 3 5 7 9 ll l3 l5 l7 l9 2| Fraction Number Figure 4 3.0 2.0 LO 3.0 2.0 ’0 mg Protein mg Protein 82 4,900 i 146 and 3,900 i 138 cpm/mg protein, respectively. These mean values were not significantly different (p<<0.05) from one another which suggested that experimental use of either of these tissue supernatants would be representative of 3H-cortisol binding within mammary cytosol. 2) Mammary Nuclear Fractions 700 x g_Precipitate The Sephadex G-25 elution profiles of KCl extracts of 700 x g precipitate fractions of mammary tissue slices from lactating cows labelled with 3H-cortisol plus ethyl alcohol and 3H-cortisol plus unlabelled cortisol are shown in Figure 5. Tritiated cortisol was associated with proteins which eluted at fraction 7 (Figure 5). Radioactive corti- sol in fraction 7 was reduced by 67%, with the addition of unlabelled cortisol (Figure 5). A large quantity of 3H- cortisol remained unbound in the nuclear extract (fractions 10 and 21). Unbound cortisol had no associated protein peak. Bgtential Binding Inhibitors l) Enzymes Trypsin markedly reduced the 3H-cortisol bound in the Eraction associated with protein after chromatography of 700 x g supernatants on Sephadex G-200 (Table 15). The reduction in binding was three times greater than that Figure 5. 83 Gel filtration (Sephadex G-25) elution profile of 0.3 M KCl extract of 700 x g mammary pre- cipitate, isolated from tissues incubated with 3H-cortisol plus ethyl alcohol or 3H-cortisol plus unlabelled cortisol. 30.000 . 20,000 I 0.0 OOJ 8.000 7.000 6,000 5,000 4,000 3.000 2.000 I 7 l I I moo}. 4 400 350 300 .250 200 ISO IOOI- 50 I 84 6-25 o——o 3l-l - Cortisol + Ethanol 0—0 3H- Cortisol + Cortisol H Protein 3H - Cortisol + Ethanol T A—--A Protein 3H - Cortisol + Cortisol A--AA-- ‘A’ O A’ A‘ ‘ - ‘ :;:--:. 4 1 1 l J L 1 l l I I 3 7 I3 I5 I7 I9 2| FRACTION NUMBER Figure 5 3.0 2.5 2.0 E w '— 8 LS a U' E I.0 0.5 Table 15. Enzymatic inhibition of 3H-cortisol binding to 700 x 9 super- natant protein fractions of mammary lactating cows.a tissue slices from Treatment CPM/mlb 3H-cortisol ethyl alcohol 450 3H-cortisol trypsin 50 3H-cortisol RNase 550 3H-cortisol DNase 440 3N-cortisol lipase 400 3H-cortisol hyaluronidase 425 3H-cortisol cortisol 150 k a ' - Chancentration of 3H-cortisol was 1.3x10 7 M. a concentration of 2.4x10"6 M. Unlabelled cortisol had The respective enzyme concentrations were 0.5 mg/ml of Tris-EDTA buffer, pH 7.4. Data expressed in CPM/ml, are mean values for peaks of 3I-l-cortisol tXD‘und to macromolecular components from.Sephadex G-200 columns. 86 obtained with unlabelled cortisol. Lipase, RNase, DNase or hyaluronidase did not affect binding of 3H-cortisol in the protein-rich fraction of the 700 x g supernatant. A large quantity of 3H-cortisol remained unbound (not associated with protein). The 3H-cortisol associated with these frac- tions was not altered by enzymes. 2) p-Chloromercuribenzoate (PCMB) Unlabelled cortisol significantly reduced binding of 3H-cortisol in 700 x g supernatant and precipitate fractions (as expected in control tissue slices (Table 16). However, PCMB-treated slices unexpectedly bound more 3H-cortisol in the presence of unlabelled cortisol. 3) Oubain (G-Strophanthin) 5 Oubain (10- mM) had no significant effect on the up- ‘take or binding of 3H—cortisol in either 700 x g supernatant can mcHauoum mchsHo HomHuuoo Eamon .ncHououm mcwocwn HOmHuuoo humEEma .ucmuocumnsm m x com «0 maesao> coausam loomuo xmomnmmmv coflumuuaao Hoe .m mHSmHm 90 m ousmwm :E 235 .6 oeao> O¢ mm on ON ON m. o. m o ----.--—---u-u-—udqu-——-—--_u-.q_i < omofloscoem 5.32020on £63230 coca zze 520$ 9.550 .3500 E23 toucooum, ooom :0:on coin 136 529;. 9:25 .9530 b oEEoE o x 00h 520.5 052.5 82.30 Eaton EoEta 0.. 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Ha anamflm comes _ sod 3 2nd . sec _ an. _ 2:0 I 5352 20.551... an we we cc we 08 on on em mm on on on em mm cu m. w. c. w. o. o m v a .....|.1I41...x1.. x 00. 000 r 000.. x .000. am :- 8.x 3 m I om x ' _ .ooou : x \\\\\u. .oonu zao . 1000» 8* 1000c looon umoaajmo mHuoomnou can» moons .hao>Huoommou .Honooao Hanna CH 2 m 0H K v can S OH x o.H who: HonHuHoo COHHonHss can HomHuuoolm mo msoHumuucoocoom m m m.o m.o Human ocH>om no.H m.a swasnam aspen ocH>on «a aH.H m.H usouocuomsn u a con unease: am . H o . a 8.33 «Bonnie QH . H u . H 294 mad HOmHuuoo + HamHuuoolm Honooao Hanna + HamHuuoolm oHooE coHuonsocH m m 65,5 138. mason o.msoo msHumuooH scum noowam osmnHu humafioe no mmHMUHmHomum m x com «o mouoaomw moumladou on HomHuuoolmm mo wsHUsHm .hH magma 107 Table 18. Binding of 3H-cortisol to cell-free isolates of 100,000 x g precipitates (microsomes) of mammary tissue slices from lactating cows. Hormone CPM + SE 3H-cortisola + ethyl alcohol 36,650 :_202 3H-cortisol + cortisolb 34,482 i 121 3H-cortisol + dexamethasoneb 36,329 :_210 a 3H-cortisol concentration was 1.6 x 10 9 M in ethyl alcohol. Unlabelled cortisol and dexamethasone concentrations were 4 x 10“8 M in ethyl alcohol. 108 .mcoHuomum oumuHmHoon com ucmuocummsm m x 005 :H sumo pounce on cons mm3 can moHooHuHooimm mo cowuomuuxo Honooam Hanna Houmm ouMUHmHooum m x con CH oousmmoe m83 42o Hmuoao .xao.o vac Honucoo Hogooao dunno con» mooqo .xmo.o vac Houuooo Hoaooao Assam can» woman .Hosooao Hanna GH 2 mica x h.~ mos osommnuofioxoclmm 6cm HomHuHOOImM mo coHuouucmocoo any mHHs3 .Honooam Hanna GH 2 m OH x h.@ mo3 noHououm ooaaonoaas mo coHuoHucoocoom m.m omH m.H moa Houuooo Hoaooao Hanan m.m ems m.H moa Hoaeouumo.mea o.o mod o.H ooa ocououmomoum om.H coma 05.0 new oconmsuoeoxoo om.m some om.o 0mm HOnHuuou om.m nova oo.H omn osoHaxmuuoo oo.m omma om.o omh ocoaocwoaowus oumuwmwooum ucoumcuomdm ououHmHooum ucmumsummsm ocoahon voadoanca o x can m x com o x 005 m x can oazo Hence mn\zmo ooze deuce mn\zmo acomocumonooimm HomHuHOOImm .msoo a mcwuouooa Bonn moowan asmnHu muoasoa mo mcoHuoouu ouauwmwooum can ucoumsuom5n m x 005 cu msHoan anamosuosoxapimm can HomHuHoolmm mo coHananH osoaoxouuoo was osoaocwoaowue .ma Canoe 109 However, progesterone and 178-estradiol did not signifi- cantly (p>'0.05) reduce binding of 3H-cortisol in either fraction (Table 19. When 3H-dexamethasone was used in place of 3H-cortisol and the various unlabelled hormones were added to mammary slices as described above, triamcinolone, cortexelone, 3 cortisol and dexamethasone reduced the binding of 3H-dexa- f methasone in 700 x g supernatants by 8.0, 6-7, 9.3 and 14.0% I respectively; and by 42.3, 36.5, 51.9 and 76.9% respectively, B in 700 x g precipitates (Table 19). Progesterone and 178- E estradiol did not significantly (p>’0.05) effect 3H-dexa- methasone binding in 700 x g supernatant or precipitate fractions. Cl4-Glucose Uptake Unlabelled cortexelone, cortisol, triamcinolone and dexamethasone inhibited C14-g1ucose incorporation into mammary tissue slices from lactating cows (Figure 13). Inhibition of C14-g1ucose incorporation was apparent with all corticoids at hormone concentrations as low as 10"8 M when compared with ethyl alcohol controls (Figure 13). Progesterone served as a second control. No inhibition of C14-glucose was observed with progesterone. As the concen- tration of corticoids increased, inhibition of Cl4-glucose uptake increased in a dose response relationship (Figure 13). Inhibition of Cl4-glucose incorporation was greatest at assess-id Starfltuglzl 4.. S . Figure 13. 110 Effects of various unlabelled corticoids on C14-glucose uptake into mammary tissue slices from lactating cows at 37°C. The percent inhi- bition of Cl4—glucose uptake was calculated from controls (100% uptake, 0% inhibition). O——O cortexelone (ll—deoxycortisol) cortisol triamcinolone dexamethasone progesterone ethyl alcohol controls % Inhibition of C'4-GIucose Uptake 111 so 1 50 - 40. so. .01 I 0- I0"8 I0"7 l0'6 I0"5 1 , IC'4 Steroid Concentration (MI Figure 13 112 hormone concentrations of 10"4 M, when compared with ethyl alcohol controls. At concentrations of 10.4 M, cortisol and dexamethasone reduced the uptake of Cl4-glucose into mammary tissue by approximately 55% when compared with ethyl alcohol controls. Triamcinolone and cortexelone reduced glucose uptake 48 and 30%, respectively, at concentrations : of 10..4 M, when compared with ethyl alcohol controls g (Figure 13). f The inhibition of 3H-cortisol and 3H-dexamethasone : I uptake by unlabelled corticoids served as a semi-quantitative index of corticoid binding in 700 x g supernatant and pre- cipitate fractions of mammary tissue slices. Product-moment correlation analysis showed that corticoid binding in 700 x g supernatant fractions of mammary tissue slices from lactating cows was significantly (p‘<0.01) correlated (r = 0.91, for 3H-cortisol and Cld-glucose; r = 0.93, for 3H-dexamethasone and Cl4-glucose) with the inhibition of C14-glucose uptake. 6 M cortisol significantly In other experiments, 1 x 10- (p‘<0.05) reduced the uptake of C14-glucose into mammary tissue 42%, while 1 x 10.4 M cortexelone significantly (p‘<0.05) reduced C14-glucose uptake by 30% (Table 20). When cortisol and cortexelone were simultaneously added to mammary tissue slices from lactating cows, glucose uptake was reduced by only 4.0% (Table 20). Cortexelone, therefore, interacted with cortisol to increase C14-glucose uptake into mammary tissue slices. 113 Table 20. Cl4-glucose uptake into mammary tissue slices from lactating COWS . Treatment CPM/pg Total DNA :_Std Error [E§E.x 100]f Ethyl alcohol control Cortisol (l x 10_6 M) Cortexelone (l x 10.4 M) Cortisol (1 x 10.6 M) plus cortexelone (l x 10'4 M) 36.0 i 0.25 20.8 25.1 34.5 a a b + + + 0.50 0.43 0.36 42. 30. aLess than control (p< 0.05). bGreater than uptake seen for cortisol or cortexelone alone (p< 0.05) but not significantly different from control (p>’0.05). c==C14-glucose uptake of control without corticoid. e==C14-glucose uptake with corticoid. fTotal DNA was measured in 700 x g precipitates and was used to adjust data. 114 In Vivo Experiments Mammary Uptake of Corticoids Table 21 shows the external pudic artery and mammary vein concentration (ng/ml) of serum corticoids before and after milking. This table also illustrates arterial-venous concentration differences (ng/ml) for corticoids before and after milking. Serum corticoids, obtained from external pudic artery samples, averaged 6.72 i 1.33 ng/ml (base line : S.E. of mean) prior to the initiation of milking, at time zero (Table 21). At milking, arterial corticoids increased to 13.13 i 1.14 ng/ml and continued to increase until twelve minutes after the initiation of milking to 17.34 i 1.02 ng/ml. After this interval, corticoids decreased to a final level of 3.61 i 0.95 ng/ml sixty minutes after the initiation of milking. Serum corticoids, obtained from mammary vein samples, averaged 6.65 i 1.19 ng/ml (base line) prior to the initia- tion of milking (Table 21). At milking, venous corticoids increased to 14.82 i_l.40 ng/ml, dropped slightly at six minutes, then increased again at twelve minutes after the initiation of milking to 14.45 i 1.11 ng/ml. After this interval, venous corticoids decreased to a final level of 3.55 i 1.0 ng/ml sixty minutes after the initiation of milking. 115 some mucoHoMMHo >I¢ Mom msowum>uomno *\oum2\, u mucoummmwv >I4 and mo .m.m .Aoa.osvm0 ones can» uoumoum haucoowuwcmHmn .moussHa m fiaouoswxoummo How ooscHucoo can capoHUHcH nm3 mCHxHHzo wo.o+ om.on mH.OI 00.HI mm.m+ nam.m+ m0.HI Ah.o+ mm.OI mv.o+ mucoummmwv unoco>ldmfluouu< mm.m mm.m mH.OH mo.mH mv.vH no.0H N0.¢H v0.0 Vh.0 mm.v mzocm> H0.m NH.m vo.oa No.NH vm.bd vm.ma MH.MH mm.m on.m mo.m Hmfiumuud iasxoco meaooauuoo Hoops oo+ cm+ o~+ 03+ ~H+ 0+ 0 on man on- moaxaaa 1+0 “memo one .IV on Howum nouscwa cw oEHB .9528 nouns can ououon moHooHuuoo Eamon mo cowuouusoocoo sHo> muoEEoE use xuouuo owosm Hmsuouxm .HN wands 116 Arterial-venous differences in corticoids were positive at 6 and 12 minutes after the initiation of milking. The mean A-V differences at these time intervals were 5.81 ng/ml :_2.40 (mean A-V difference :,SE of mean) and 2.89 ng/ml : 2.40, respectively (Table 21). These positive A-V differ- ences in corticoids were not statistically significant from Ii zero at the pi<0.05 level (Table 21). ‘4’...’...‘€{o~ DISCUSSION Unlabelled cortisol, cortexelone, triamcinolone, and dexamethasone significantly reduced the binding of tritiated cortisol and dexamethasone in 700 x g supernatant and precip- itate fractions subsequently isolated from mammary tissue slices of lactating cows (Tables 1, 2 and 19). Unlabelled progesterone, testosterone and l7B-estradiol did not signifi- cantly reduce corticoid binding to mammary tissue from lactating cows (Tables 2, 7 and 19). These results provided qualitative evidence that mammary tissue slices from lactat- ing cows were capable of specifically binding cortisol and dexamethasone in 700 x g supernatant and precipitate frac- tions. Unlabelled corticoids also reduced uptake of labelled triamcinolone acetonide and dexamethasone in mam- mary tissues from lactating rats (Gardner and Witliff, 1973a), mice (Shyamala, 1973a) and voles (Turnell et al., 1974b). However, other noncorticoid hormones reduced uptake of various corticoids in these systems. For example, progesterone reduced binding of various corticoids in rat (Gardner and Witliff, 1973a), mouse (Shyamala, 1973a) and vole (Turnell et al., 1974b) mammary tissues and progester- one and 170 hydroxyprogesterone reduced binding of cortisol 117 118 in cultured bovine mammary cells (Tucker et al., 1971). Furthermore, Gardner and Witliff (1973a) reduced tritiated triamcinolone binding in mammary tissue from lactating cows with high doses (5 x 10'6 M) of unlabelled 17B-estradiol, and Turnell et al. (1974b) reduced tritiated triamcinolone binding in mammary tissue from lactating voles with high doses (5 x 10.6 M) of 17B-estradiol and 0.5 to 5 x 10-6 M testosterone. we conclude that the bovine mammary tissue slice system may be more specific for binding corticoids than those reported by other workers. Unlabelled cortisol and dexamethasone were also capable of reducing tritiated cortisol and dexamethasone binding in mammary tissue slices from virgin heifers, one-month prepar- tum and dry cows (nonpregnant, nonlactating) (Tables 5-8). Only subtle differences were apparent, however, between the results previously described for mammary tissue slices from lactating cows. For example, unlabelled dexamethasone reduced tritiated dexamethasone binding in 700 x g precipi- tate fractions of virgin mammary tissue, while unlabelled cortisol did not (Table 5). Unlabelled cortisol did not reduce tritiated dexamethasone binding in 700 x 9 super- natant fractions from dry cows (Table 8). Furthermore, the total uptake of tritiated corticoids, in the absence of unlabelled corticoids, was greater in mammary tissue slices from lactating cows when compared with mammary tissue from "6‘3 9.7] ‘1... “an . 119 cattle in other physiological states (Table 7). The greater uptake of corticoids observed in mammary tissue slices from lactating cows was believed to be associated with lactation- al events occurring within mammary cells. The total uptake of tritiated dexamethasone was greater than tritiated corti- 'l‘ sol in tissue slices from one month prepartum and lactating cows when compared with virgin heifers and dry cows (Tables 5-8). The enhanced uptake seen for dexamethasone, when com- pared with cortisol, might have been attributable to lacta- ‘p-‘:-..-.\_l . tional events or to the greater biological activity of dexamethasone (Westphal, 1971 and Yoshitaka et al., 1966). Gardner and Witliff (1973a) demonstrated that unlabelled corticoids reduced triamcinolone acetonide binding in mammary tissue from pregnant rats. However, these workers failed to reduce tritiated triamcinolone binding in virgin rat mammary tissue with the addition of unlabelled corticoids. They suggested that this response was due to the paucity of re- ceptor molecules for triamcinolone in virgin rat mammary tissue. Competitive hormone binding experiments are not quanti— tative. Scatchard plot analysis, however, allows one to calculate several constants which are directly related to the kinetics of hormone-receptor interactions. For example, association constants (Ka) and dissociation constants (Kd = i3) for hormone receptor complexes, can easily be calculated 120 from Scatchard plots (Scatchard, 1949). The total number of hormone molecules bound per cell can also be determined. This in turn gives an estimate of the number of hormone receptor sites per cell, if one assumes that the receptor site binds one molecule of hormone per molecule of receptor. Scatchard analyses of mammary tissue slices from lac- n tating cows revealed a single high affinity binding component I 10 (Kdtcflo- M) for cortisol and dexamethasone. This constant was in close agreement with those found in mammary tissues %. i 9 to 10'7 M ‘ of other species, which ranged from 6 x 10- (Gardner and Witliff, 1973a; Shyamala, 1973a and Turnell et al., 1974b). The 1299 to 1957 (adjusted) molecules of corticoids bound with high affinity to fresh mammary tissue slices from lactating cows, were less than the 7500 mole- cules of cortisol bound with high affinity to bovine mammary cells cultured in_gi£59_(Tucker et al., 1971) and less than the number of molecules of corticoid bound to mammary tissue from other species (Shyamala, 1973a; Gardner and Witliff, 1973a and Turnell et al., 1974b). The low number of corti- coid molecules bound in mammary tissue slices from lactating cows may have been attributable to stress, induced at slaughter, in cattle used in this study. This stress may cause elevated serum corticoids which would then bind to specific corticoid receptor sites. The number of receptor sites detectable by Scatchard assay would therefore be underestimated. 121 Mammary tissue from lactating cows bound significantly more total molecules of dexamethasone than cortisol. The greater number of apparent binding sites for dexamethasone may have been a result of the synthetic corticoid's greater biological activity (Westphal, 1971 and Yoshitaka et al., 1966). A single homogeneous population of specific corticoid receptor molecules was observed in these studies and has been described for a number of other mammary tissue systems (Gardner and Witliff, 1973a; Shyamala, 1973a and Turnell et al., 1974b). However, the fact that only one high affin- ity component for corticoid binding was observed for corti- sol and dexamethasone in our system was contrary to results observed in cultured bovine mammary cells where two high affinity components bound cortisol (Tucker et al., 1971). The discrepancies between these two systems may have been a result of cellular dedifferentiation in cultured cells, with a resultant change in receptor site affinity or synthesis of a mixed population of receptors. In addition to the high affinity binding component, anOther component that had a low affinity, but larger capa- city for corticoid was apparent in mammary tissue slices from lactating cows (Figure 1A and 18). This component was thought to be analogous to the nonspecific component found in cultured bovine mammary cells (Tucker et al., 1971). we?“ 1' .a. 7:. #- I-r-"W- "I. m" . n, "a . .. .. be“ L“ r 122 The component was present in all preparations studied and could not be eliminated by washing the tissue. Attempts were made to eliminate this component from 700 x 9 super- natant fractions by charcoal and florisil treatment, but this resulted in absorption of bound cortisol from specific receptor sites. Mathematical transformation of Scatchard :7 M data was therefore necessary to subtract nonspecific bind- ing in tissue fraCtions which might have been associated with the Specific high affinity binding component (Appendix I D). This correction allowed for a more precise estimation of receptor-hormone dissociation constants and numbers of corticoid molecules bound per mammary cell. Scatchard plots carried out on tissue slices from lactating cows at 37°C and 4°C indicated that, similar to many steroid hormone binding systems (Sandberg et al., 1966) the mechanism for corticoid binding was temperature dependent. Reducing the incubation temperature did not markedly alter the affinity of mammary tissue for cortisol, but the total number of molecules bound with high affinity was reduced by approximately 82% (Table 3). Fresh mammary tissue slices from lactating cows did not metabolize cortisol prior to its becoming bound in the 700 x g supernatant (Figure 3). This was unlike the results found for liver cells (Baxter and Tomkins, 1971a) and mam— mary cells cultured in_vitro (Tucker et al., 1971) where a 123 substantial portion of cortisol recovered from the cytosol was in a form other than authentic cortisol. The absence of bound metabolites of cortisol in cytosol fractions of fresh mammary tissue from lactating cows was analogous to that observed for rat thymus (Munck and Wira, 1971) and mouse mammary tissue (Shyamala, 1973a). Further experiments 3 are needed in order to determine if cortisol is metabolized g prior to becoming bound in 700 x g mammary nuclear fractions. I Experiments designed to determine if the uptake of I. corticoids into mammary tissue slices from lactating cows 35 was dependent on a facilitated active transport system were carried out using 10.5 mM Ouabain (cardiac glycoside). This concentration of Ouabain was chosen, since it blocks the Na+/K+ pump in cardiac and smooth muscles (Repke, 1963). Ouabain had no effect on uptake of cortisol. Therefore, it was assumed that corticoid uptake into mammary slices from lactating cows was either passive or that uptake could possibly be blocked by other transport inhibitors or other concentrations of Ouabain. Scatchard analyses of cortisol and dexamethasone bind— ing in virgin heifers, one-month prepartum and dry cows revealed a two component corticoid binding system which showed some similarities with those previously described for mammary tissue from lactating cows. For example, one com- ponent had high affinity for corticoid and was saturable. 124 The other component had low affinity for cortisol and proved unsaturable. In general, no specific trends among reproductive states were observed for Kd's of corticoids bound to either 700 x g supernatant or precipitate fractions, even though statistical analyses revealed significant differences (Tables L 9 and 11). The number of corticoid molecules bound in 700 x g supernatant and precipitate fractions also varied sig— nificantly among reproductive states (Tables 10 and 12). But, specific trends were present when the number of corti— coid molecules bound per mammary cell were compared among reproductive states. For example, mammary slices from virgin heifers and dry cows bound fewer molecules of corti- sol when compared with prepartum and lactating cows (Table 10). There was no significant difference between the number of molecules of cortisol bound in tissue fractions of virgin heifers and dry cows (Table 10). Furthermore, lactating cows bound more molecules of dexamethasone than virgin heifers and dry cows (Table 12). As a general rule, mammary tissue slices from all cattle bound approximately 2.0 times more molecules of dexamethasone than cortisol. This might be explained by the well-known greater biological activity of dexamethasone (Westphal, 1971 and Yoshitaka et al., 1966).- Interpretation of competitive hormone binding experi- ments and Scatchard analyses of corticoid binding to whole 125 mammary tissue slices is somewhat restricted because the tissue slices represent a mixed pOpulation of cell types. The experimenter can not be certain of the binding in a particular cell type. Results of binding experiments would, therefore, vary from one experiment to the next depending upon the number of specific cell types present which are capable of specifically binding corticoid. However, charac- terization of corticoid receptors in mammary tissue from lactating rats (Gardner and Witliff, 1973a), mice (Shyamala, 1973a) and voles (Turnell et al., 1974) has recently been reported in the literature. These receptors were character- ized as proteins, since binding was altered by proteolytic enzymes and mercurials. Evidence presented in this disser— tation suggested that the corticoid receptors in mammary tissue from lactating cattle were proteins, because trypsin (Table 15) and p-chloromercuribenzoate (Table 16) signifi- cantly reduced specific cortisol binding in 700 x 9 super- natant fractions of mammary tissue slices from lactating cows. Results of hormone binding experiments carried out on cattle in various reproductive states may be brought into focus if it is assumed that corticoid binding is specific for mammary alveolar epithelial cells (Tucker et al., 1971) or proteins closely associated with these cells (Gardner and Witliff, 1973a; Shyamala, 1973a and Turnell et al., 126 1974b) and that the physiological function of corticoids, and other mammotropic hormones, is involved with growth and maintenance of metabolically active alveolar cells. The mammary gland of virgin heifers consists primarily of ducts and connective tissue. The degree of development is primarily dependent upon the number of estrous cycles the animal has after puberty. In general, estrogen causes proliferation of ductular epithelial tissue, whereas pro- gesterone causes proliferation of alveolar epithelium. The few alveolar epithelial cells present in the virgin mammary gland are not representative of the metabolically active milk secreting epithelial cells of the pregnant and lactat- ing animal and have no dependence on lactogenic hormones. The paucity of receptor sites for corticoid (Tables 10 and 12) in mammary tissue from virgin heifers could be attribu- table to the lack of alveolar epithelial cells. Gardner and Witliff (1973a) have also reported a low number of cor- ticoid receptor sites in mammary cytosol fractions from virgin rats when compared with pregnant or lactating rats. During pregnancy, the mammary gland, with its rudimen- tary ductular structure is transformed into fully secreting tissue. Ovarian, pituitary, cortical and pancreatic hor- mones (mammotropic hormones) are believed to act synergis- tically throughout this transformation to stimulate the production of metabolically active alveolar cells. 127 Cellular differentiation progresses throughout pregnancy and remains dependent upon corticoids and other mammotropic hor- mones. Lumina of terminal ducts and alveoli are swollen with proteins and other secretory products. At this stage of mammary development, the gland is similar to the lactat- {’4 ing gland, with the exception that "milk" is not being pro— IT‘-. I! I ' duced. The large numbers of metabolically active alveolar cells present in one-month prepartum mammary tissue could .~ . . ..... wfiuum._‘n w.n-_-u‘- ’ . e" . ‘ M“... conceivably contribute to the greater number of corticoid V57. receptor sites when compared with virgin and dry cows (Tables 10 and 12). Shortly before parturition, there is an increase in serum concentrations of corticoids and other lactogenic hormones, as well as a decrease in progesterone. These responses are thought to affect the initiation of lactation. The fully lactating mammary gland represents the summit of cellular differentiation. Alveolar cells are completely efficient metabolic factories for the production of casein, milk, fat, B-lactoglobulin, lactose and other milk compon- ents. It is conceivable that the rise in corticoids and other lactogenic hormones seen shortly before parturition, may stimulate alveolar cells to produce milk components and specific species of protein hormone receptors. The abundance of metabolically active (protein synthesizing) alveolar cells could contribute to the greater number of corticoid 128 receptor sites when compared with virgin heifers, one-month prepartum and dry cows (Table 10 and 12). Gardner and Witliff (1973a) reported more specific corticoid binding in mammary glands of lactating rats as opposed to the glands of virgin and pregnant rats. Furthermore, Emery (1969) showed a sixfold increase in corticosterone binding in lac- tating rats when compared with animals in their sixteenth day of pregnancy. At the end of lactation, regression of the mammary gland takes place. This response is believed to be a result of changes in lactogenic and mammotropic hormone concentra- tions which may be rate-limiting to milk secretion and mam» mary development. Alveoli lose their structural integrity and become engorged with lipid droplets and protein granules. The overall anatomy of mammary glands from dry cows (non— pregnant, nonlactating) resembles that for virgin heifers. There are very few, if any, functional epithelial cells and the gland is primarily composed of connective tissue and ductular epithelium. Some nonsecretory alveolar cells associated with terminal ducts remain intact. The reduction in metabolic activity and associated regression of alveolar epithelial tissue caused by the reduction of rate limiting lactogenic and mammotropic hormones could account for the paucity Of receptor sites seen in mammary tissue slices from dry cows (Tables 10 and 12). This could also account for 129 the close agreement between the number of binding sites observed in dry cows and virgin heifers. Several attempts were made in this study to relate specific corticoid uptake and binding to physiological responses occurring within mammary tissue. Cameron (1973) and Turnell et al. (1974a) demonstrated that glucocorticoids inhibited uptake of Cl4-g1ucose into mouse mammary tissue slices and rat thymocytes, respectively. In this disserta- tion, cortisol, cortexelone, triamcinolone and dexamethasone were effective in inhibiting C14-glucose incorporation into mammary tissue slices from lactating cows (Figure 12). The concentrations of corticoids necessary to inhibit glucose incorporation were slightly greater than those used to achieve saturation of the high affinity binding component in Scatchard analyses. The high positive correlation between corticoid binding and inhibition of Cl4-glucose incorpora- tion by corticoids suggested that these responses were closely related. Further confirmation of these data was provided in experiments using cortexelone and cortisol. Turnell et al. (1974a) and Munck and Wira (1971) showed that cortexelone does not have the biological activity that most glucocorti— coids have on glucose uptake into thymus tissue, but that it is an avid competitor for tritiated—triamcinolone and tritiated-cortisol binding. When cortexelone plus 130 triamcinolone or cortisol were simultaneously added to thy- mus preparations, cortexelone acted as an antiglucocorticoid and reduced the ability of corticoids to inhibit glucose incorporation. These workers proposed that cortexelone occupied specific binding sites which were normally occupied by more biologically active corticoids. Cortexelone was I thought to affect the stereochemistry of the hormone receptor . complex, so that it no longer exerted its normal physiologi- cal action on the cell. When cortisol and cortexelone were mm4‘fii'- . ' '4 simultaneously added to mammary slices from lactating cows in this study, glucose uptake was increased. This suggested that cortexelone interacted with cortisol to increase glu- cose incorporation into mammary tissue slices. Since cortex- elone reduced corticoid binding in mammary tissue slices from lactating cows and had reduced biological activity with regard to inhibiting C14-glucose incorporation, when compared with cortisol, it was further suggested that corticoid bind- ing to specific receptor molecules in mammary tissue slices from lactating cows was definitively correlated with glucose uptake. 13 3112 experiments designed to measure the arterial- venous concentration differences of corticoids across the mammary gland suggested that corticoids were taken up by the mammary gland shortly after the onset of milking. The mean A-V differences at 6 and 12 minutes after the start of 131 milking (5.81 ng/ml and 2.89 ng/ml) were comparable to A-V differences in corticoids observed by Patterson and Linzell (1974) in lactating cows. Whether or not the uptake was specific to the mammary gland and represented utilization of corticoids for milk secretion or was merely nonspecific and a result of increased arterial corticoid concentrations remains to be elucidated. The rise in arterial and venous corticoids noted at 30 and 15 minutes before milking (Table 21) was believed to be a result of exteroceptive stimuli induced by the presence of the milker or milking unit. Similar results have been reported by Smith et al. (1972) who showed that the milking stimulus per se and exterocep— tive stimuli caused increased serum corticoids in cows. Hormone binding experiments, described in this disser- tation, suggested that the specific corticoid binding component of mammary tissue from lactating cows was not a result of tissue contamination with the corticoid binding globulin (CBG) of blood. For example, CBG has a higher affinity for progesterone at 37°C than for corticoids (Rosenthal et al., 1969; Sandberg et al., 1966; and Seal and Doe, 1966) but, tritiated cortisol or tritiated dexa- methasone binding to mammary tissue could not be competi- tively inhibited with unlabelled progesterone at 37°C. Furthermore, dexamethasone is only a moderate to poor com- petitive inhibitor of cortisol binding to CBG (Pizarro, 132 1969; Peets et al., 1969 and Baxter and Tomkins, 1971a). But, dexamethasone proved to be an excellent competitive inhibitor of cortisol binding in the bovine mammary slice system. Also, the binding affinity of CBG to cortisol is reduced by decreasing the incubation temperature (Sandberg et al., 1966). As described earlier, reducing the tempera— ture from 37° to 4°C did not effect the affinity of cortisol binding to mammary tissue slices from lactating cows, but did reduce the total number of molecules specifically bound with high affinity. In experiments where the binding of tritiated cortisol, dexamethasone and progesterone was examined in sera, sera bound more cortisol and progesterone at 37°C when compared with dexamethasone. This gave further confirmation that the corticoid receptor found in mammary tissue was unique. Additional attempts were made to distinguish corticoid binding in mammary tissue from that of blood using several physicochemical techniques. The results of these attempts confirmed the hormone binding experiments previously described. Namely, the corticoid binding component of mam— mary tissue was unique and not a contaminate from blood (CBG). For example, gel filtration chromatography and sucrose gradient analyses of mammary supernatant fractions suggested that the protein(s) binding cortisol was a macro— molecule with a molecular weight of approximately 2.5 x 105 133 to 3 x 106 (Figures 6 and 7); whereas, the major protein which bound cortisol in blood had an approximate molecular weight of 6 to 8 x 104 (Figures 6 and 7). Disc gel electro- phoresis and DEAE cellulose chromatography confirmed that the corticoid binding protein(s) of mammary tissue had dif- ferent electrochemical characteristics than serum binding components. The physicochemical techniques employed to characterize the mammary corticoid receptor in this study did not com- pletely resolve the biochemical nature of the binding pro- tein. Future attempts are necessary to determine the structure of this macromolecule. The size of the cytosol binding protein suggested that it may be composed of sub- units. These subunits may be nucleic acid(s) carbohydrate(s) or other proteins which do not participate in the binding reaction. Intact tissue was not a necessary determinant for specific binding of cortisol to mammary tissue in isolated 700 x g supernatant or precipitate (containing microsomes and other cellular debris) fractions, because unlabelled corti— sol reduced tritiated cortisol binding in these fractions (Figure 12, Table 17). In contrast, isolated 100,000 x g precipitate (microsomes) devoid of other cellular debris did not specifically bind cortisol (Table 18). 134 Evidence presented in this dissertation suggested that mammary tissue slices possessed protein(s) receptor mole- cules which were capable of specifically binding corticoids. The fact that mammary tissue slices from lactating cows bound more molecules of corticoids than tissue from cattle in other reproductive states and that binding and uptake of corticoids appeared related to events associated with milk secretion, may form the basis for further studies to deter- mine the specific role which corticoids play in lactation. Based on the data reported in these studies, one could specu- late that specific corticoid binding is involved in the growth and maintenance of metabolically active alveolar epithelial cells. SUMMARY Evidence presented in this dissertation suggested that bovine mammary tissue slices possessed specific receptor molecules for corticoids. Specificity for corticoids was based on the observations of hormone binding experiments and Scatchard analyses. Unlabelled cortisol, cortexelone, triamchinolone and dexamethasone reduced binding of tritiated cortisol and dexamethasone in mammary tissue slices from lactating cows. Unlabelled progesterone, testosterone and 17B-estradiol had no effect on corticoid binding in fresh mammary tissue slices from lactating cows. Scatchard analysis revealed two components which bound cortisol and dexamethasone in mammary slices from lactating cows. One component had high affinity (Kd :10‘10 M) for both corticoids and was saturated at low hormone concentration. The other component had low affinity for both corticoids and was practically unsaturable. It is believed that the high affinity component represented specifically bound corticoid. The unsaturable component was thought to represent nonspecifically bound hormone. Mammary tissue from lactating cows bound approximately 1263 and 1855 135 136 total molecules of cortisol and dexamethasone, respectively, per mammary cell. Attempts were made to compare corticoid binding in mam— mary tissue slices from lactating cows with binding in mammary tissue slices from cattle in other physiological states. These physiological states included virgin heifers, Auto-.1- l-month prepartum and dry (nonpregnant, nonlactating) cows. 1.“ .h. 4 J ‘ .I Results of competitive hormone binding experiments, carried out using tritiated cortisol and dexamethasone and the other I unlabelled steroids mentioned above, were similar to those described for mammary tissue slices of lactating cows. Scatchard analyses of corticoid binding in mammary tissue slices from virgin heifers, l-month prepartum and dry cows showed similarities to those for mammary tissue slices from lactating cows. For example, a two component Scatchard plot was resolved for each physiological state. Dissociation constants (Kd) for cortisol and dexamethasone ranged from 0.3 to 20 x 10"10 M. Mammary slices from lactating cows, bound more molecules of corticoid per mammary cell when com- pared to the other physiological states examined. Virgin heifers and dry cows bound the fewest number of corticoid molecules per mammary cell. Mammary tissue slices from all physiological states examined bound more molecules of dexa- methasone than cortisol. Thin-layer chromatography of specifically bound tritiated cortisol, in 700 x g supernatants 137 of mammary tissue slices from lactating cows, indicated that the majority of bound radioactivity was authentic cortisol. Gel filtration chromatography experiments showed that the specific corticoid receptor in 700 x 9 15,000 x g and 100,000 x g supernatants and 700 x g precipitate fractions of mammary tissue slices from lactating cows was a macro- molecular protein(s). This protein(s) had an approximate molecular weight of from 2.5 x 105 to 3 x 106. Binding of tritiated cortisol to this protein(s) was reduced by tryp- sin and mercurials. Further biochemical analyses of the corticoid binding protein(s) in mammary tissue from lactat- ing cows, using the techniques of disc gel electrophoresis, DEAE cellulose chromatography and gel filtration chromatog- raphy, showed that the protein(s) was unique to mammary tissue and not a contaminate from blood (CBG). Cell-free preparations of 700 x g supernatants and precipitate fractions specifically bound cortisol. Cell- free preparations of 100,000 x g precipitates bound large quantities of tritiated cortisol, however, this binding was not specific since unlabelled corticoids failed to reduce binding of tritiated cortisol. Glucose uptake into mammary tissue slices from lactat- ing cows was reduced by various unlabelled corticoids. The ability of cortisol to inhibit glucose incorporation was 138 reduced by the addition of cortexelone (ll-deoxycortisol). Cortexelone reduced tritiated cortisol binding in fresh lactating tissue slices and proved to have an antiglucorti- coid action on glucose uptake. Correlation analyses sug- gested that specific corticoid binding in mammary tissue slices from lactating cows was correlated with glucose incorporation into the mammary cell. Furthermore, corticoids were taken up from the blood by the mammary gland shortly after initiation of the milking response. In summary, data presented in this dissertation sug- gested that mammary tissue slices possessed protein(s) receptor molecules which were capable of specifically bind- ing corticoids. The fact that mammary tissue slices from lactating cows bound more molecules of corticoids than tis- sue from cattle in other physiological states and that binding and uptake of corticoids appeared related to events associated with milk secretion, may form the basis for further studies to determine the specific role which corti- coids play in lactation. 5.7-E‘s 7" ‘t’u— stw.->.. .. BIBLIOGRAPHY BIBLIOGRAPHY Adams, W. M. and W. C. Wagner. 1970. The role of corticoids in parturition. Biol. Reprod. 3:223. :1 Ahren, K. and D. Jacobsohn. 1957. The action of cortisone on the mammary glands of rats under various states of hormonal imbalance. Acta. Physiol. Scand. 40:254. _..____.1 -—}..,.f-. .Txmarn‘d . . I" Anderson, R. R. and C. W. Turner. 1962a. Effect of adrena- lectomy and corticoid replacement on mammary gland I growth in rats. Proc. Soc. Exp. Biol. Med. 109:85. E . 1962b. Effect of adrenalectomy and corticoid replacement on lactational performance in rats. Proc. Soc. Exp. Biol. 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Count tritium standards with external standardization. Print out will give: Sample No. Time Channel A Channel B 4123 1000 247 290 Sample 4123 40 116 117 Sample 4123 40 273 157 Sample + Ext. Standard 133 Ba 2. for Channel A 273 - 116 57 for Channel B 157 — 117 = 40 . _ Channel A (57) ESR. A/B - CHEHHEI—B'TTOY 3. plot standard curve: X axis = ESR A/B (standards) Y axis = Percent Efficiency (standards) Linear regression of standard curve was carried out on an Olivette programable desk computer. 153 Act-11.1.7711”. '7," '- I; ,J. vw . . '7' 154 Quench Correction and Determination of DPM for Unknowns 1. Follow steps 1 and 2, knowing the A/B for unknown, read percent efficiency from standard curve. 100% 2' Percent Efficiency (unknown) = Factor Q 3. DPM for unknown = Multiply Factor Q by 10 minute unknown sample count for Channel. I . . .vw. «. 4*; ”mu..- A‘K‘nh‘L‘Qm' ear-Lina I Who ”I: .- A. .. I » . . r .1 APPENDIX C DNA ASSAY Reagents: Ether (anhydrous) 5% TCA (trichloroacetic acid) 95% ethyl alcohol saturated with sodium acetate (C2H3Na02) 1 N sodium hydroxide.(KOH) 6 N hydrochloric acid (HCl) 10% PCA (perchloric acid) 5% PCA Procedure: Part I 1. Sample contains 700 x g precipitate (nuclear frac- tion) plus 3 m1 of ethyl alcohol (ETOH) 2. Centrifuge 15 min at 18,000 RPM, pour off ETOH, add 3 m1 ether. 3. Centrifuge 15 min at 18,000 RPM, decant ether super- natant and discard. 4. Evaporate pellet, 20-30 min under air, add 5 ml 5% TCA (ice cold), centrifuge at 18,000 RPM for 15 min. Carefully decant supernate, discard. 5. To pellet add 5 m1 5% TCA, centrifuge at 18,000 RPM for 15 min. Carefully decant supernate, discard. 6. Add 5.0 ml of 95% ETOH sodium acetate, vortex, centrifuge at 18,000 RPM for 15 min. 7. Discard supernate, save pellet, pellet is used immediately or can be frozen. 155 156 Part II 1. To pellet obtained from Part I, step number 7, add 4.0 ml of 1N KOH, vortex, incubate at 37°C for 15 hrs. Part III 1. 2. Add 4 ml ice cold 6N HCl to pellet in Part II, step 1. Then add 5 ml of ice cold 10% PCA. Centrifuge at 18,000 RPM for 15 minutes, decant supernate (RNA) and discard. Wash pellet with 5 ml of ice cold 5% PCA, vortex, centrifuge at 18,000 RPM for 15 min, decant and discard supernate. To pellet, add 5 ml of 5% PCA, vortex and incubate at 70°C for 15 min, centrifuge at 18,000 RPM for 15 min, decant off supernate into 25 ml volumetric flask. To pellet, wash with 5 ml additional 5% PCA, vortex and centrifuge at 18,000 RPM for 15 min, add super- nate to volumetric flask. To pellet, repeat step 5, volumetric contains 15 ml of 5% PCA extract. 'Bring.volume up to 25 ml with additional 5% PCA. Read percent transmission or spectrophometer set at 268°A, convert to optical density (O.D.) from table. pg DNA = O.D. x 47 ug/ml x 25 mls. APPENDIX D SCATCHARD PLOT TRANSFORMATION CORRECTION FOR NONSPECIFIC BINDING Scatchard plots were corrected for nonspecific binding by the following mathematical and statistical transformations: ‘2‘ o ma 0 0 Q. . Line 1 (spec1fic component) m o ‘ HF4 L‘ a «fi x 1'» Line 2 (nonspecific component) :1 2'. 4(/’ “’0 ‘ , “ 1 5 a 3 8 8h 4 5 6 7 9 Dilution Bound (moles/pg DNA x 10"17 Figure A1. Scatchard plot unadjusted for nonspecific binding. Two Component Curve Analysis 1. Two separate least squares analyses were carried out for points on lines one and two of Figure A1. Each point (x's) represents a mean value from quadrupli- cate samples from a particular animal, obtained in four separate experiments, at one of a total of nine hormone dilutions. 2. Linear regression analysis was used to calculate the equation for line 2 (Figure A1) for each physio- logical state Scatchard plot. The regression equa— tion is represented by equation 1. 157 nib-‘h- -\-::1 n—u:a.:.¢-\-m _‘ .. < ' film F.:La‘aw~ {m ‘1'... :- .. . «:> X 158 = bo + b X (equation 1) 1 y intercept for line 2 slope for line 2 bound ree bound Transformation to produce "best fitting'I linear regression line representing high affinity—specific corticoid binding component. a. Substitute bound values (X) corresponding to Y experimental points on line 1 into linear regression equation 1. Obtain Y for each point on all four dilutions of line 1. Y (corrected) = Y (experimental) - Y Plot Y (corrected) values against bound (experimen- tal) values for dilutions of line 1 (Figure A1). Least squares analysis was performed on the means for each dilution to give a single line representing high affinity binding (line 3, Figure A2, on the following page). 1161““ n’ M} 9‘). 9‘“ O 4 I {Fflfliufl’ ‘ cm-fl ugly i. L “bu...— ‘ 159 ‘z‘ a.~ Us? A Line 3 (corrected specific $2 + A—A— component) H X or! :1 Zl___4§.. '2 . A—A— g a) H 8th Bound (moles/pg DNA x 10'17) Figure A2. Scatchard plot adjusted for nonspecific binding. A represents agggg (corrected) vs bound (experimental) values for each animal at each of four hormone dilutions. APPENDIX E LOWRY METHOD FOR PROTEIN DETERMINATION Reagents: A. Lowry A 1. Sodium carbonate (anhydrous) 60.0 g 2. Sodium hydroxide (pellets) 12.0 g 3. Sodium or potassium tartrate 0.6 g 4. Distilled H20 to make 3,000.0 ml B. Lowry B 1. Copper sulfate solution 0.5 g% (CuSO .SH 0) ' 4 2 C. Lowry C (prepared fresh daily) 1. Lowry A 50 parts 2 Lowry B 1 part Phenol reagent according to Folin Ciocalteu l. Phenol Reagent-concentrate 1 part (Central Scientific Co.) 2. Distilled H20 1 part Protein Standard 8.0 g% (Dade Reagents Inc., Miami, Fla. Lot No. PRS-406) l. Dilute with 100 ml distilled H20 to give 800 ug/ml Concentrations of protein standards used for determina- tion of standard curve: 0, 20, 40, 60, 80 and 160 ug/ml. 160 161 Procedure: 1. 1 m1 of protein solution (standard or unknown) added to 5 ml of Lowry C. 2. Incubate 20 min at room temperature. 3. 0.5 ml phenol reagent jetted in for rapid mixing. 4. Incubate 1/2 hr at room temperature (20-22°C), mix occasionally. 5. Read at 600 mu. APPENDIX F DISC GEL ELECTROPHORESISa Buffers: A. 6.0 gm TRIS 0.8 ml concentrated HC 100 ml distilled H20 B. 0.75 gm TRIS 0.40 ml Concentrated HCl 100 ml distilled H20 C. 0.6 gm TRIS 2.0 liters distilled H20 3.0 gm glycine Running Gel: 1.4 gm cyanogum -41 for 7% gel 20 ml buffer A 0.02 ml TEMED 0.02 gm Ammonium Persulfate Sample Gel: 4.0 gm acrylamide 5.0 gm sucrose 100 ml Buffer B 0.1 gm Ammonium Persulfate, 0.1 ml TEMED Spacer Gel: 0.4 gm Cyanogum -41 in 10 ml buffer B 0.1 m1 TEMED 0.01 gm Ammonium Persulfate aSamples were electrophoresed 4-5 hrs, 2 ma/disc at 4°C. 162 APPENDIX G COMPETITIVE PROTEIN BINDING ASSAY FOR SERUM CORTICOIDS Duplicate aliquots of serum (0.2 mls) are placed in l6 x 100 mm test tubes. In order to account for procedural losses, approximately 3,000 dpm of l, 2, 6, 7 3H-cortisol (Specific activity 91 ci/mmole) is added to a third aliquot from a series of unknowns (10 to 20 within each assay). Samples are vortexed vigorously for 1 minute with 2.0 ml nanograde 2, 2, 4 trimethyl pentane to remove progester- one. After extraction, samples are stored at -20°C for 1 hour. The solvent layer is decanted and discarded before the serum is thawed. Serum is allowed to warm to room temperature. After the samples reach room temperature, 2.0 ml of reagent grade methylene chloride is added in order to extract the corticoids. Samples are vortexed vigorously for 1 minute. After separation of solvent and serum, the lower methylene chloride layer, containing corticoid is carefully aspirated off with a pasteur pipette and saved in a 12 x 75 mm test tube for assay. Cortisol (Sigma Chemical Co.) is pipetted from a stock solution of 10 ng/ml in ethyl alcohol (100%) for use as standards. Three sets of standards (0.0, 0.1, 0.25, 0.5, 163 164 1.0, 1.5, 2.0, 2.5, 5.0, long) are included in each assay. Solvent in standard and unknown tubes is evaporated under air, and 1.00 ml of 1.25% dog plasma with approximately 40,000 dpm/m1 l, 2, 6, 7 3H-cortisol, is added to each tube.a Assay tubes are then stored at 4°C for 12 to 18 hours. After incubation at 4°C, assay tubes are placed in an ice bath and allowed to equilibrate for at least 15 minutes. In order to separate bound from free corticoids, 0.5 m1 of 1% dextran T70 (Pharmacia, Uppsula, Sweden) and 0.5% carbon decolarizing, neutral norit (Fisher Scientific Co.) in glass distilled water is added to each tube. Samples are then rapidly mixed and allowed to incubate in an ice bath for 5 minutes, then centrifuged at 2,000 x g for 15 minutes at 4°C. A 0.5 ml aliquot of the supernatant is diluted with 5.0 ml of liquid scintillation cocktail (3a 703, Research Products International Corp., Elk Grove Village, Ill.) for quantifi- cation of radioactivity. Standard sera with high and low corticoids are assayed with each set of unknown serum samples. ‘7 aDog plasma (Colorado Serum Co.) is diluted to 2.5% in 500 ml distilled water and mixed with 60 g Florisil (80 mesh; Matheson, Coleman and Bell) for 3 hours to eliminate endogenous steroids. The suspension is then centrifuged at 2,800 rpm for 15 minutes. The supernatant fluid volume is doubled.with glass distilled water to give 1.25% plasma. One, 2, 6, 7 H-cortisol is added to the 1.25% plasma to give approximately 40,000 dpm/ml and can be stored at 4°C for up to one month. ‘n "3* ,- .1 H. ._.J lwat. A. murW“ ,1." . O , I APPENDIX H STATISTICAL ANALYSIS OF SCATCHARD PLOT SLOPES AND X INTERCEPTS Multiple comparison of Scatchard plot slopes (Kd's) 1. Y = bo + bl X (linear regression equation for line 3, Figure A2) x = (—39) = x intercept; b = y intercept; 1 bl = slope 2. Determine SSx. and sz (pooled error variance)* for lines. 1 3. Test statistic: for (b. - b.') Scheffe, minimum significant difference between % 2 ' _ bi } bi i/ (t l)fo.os' t4, VEISp (1 + 1 )]*. SSxi SSxi t number treatments being compared VE degrees of freedom for error . 2 * pooled error variance, Sp = SSE1 + SSE df1 + df2 2 ** V (b1 ’ bi.) = [Sp2(SSx. + SSx.)] 1 1. Multiple comparison of Scatchard plot x - intercepts (mole- cules of corticoid bound in mammary cell) 2 = molecules of corticoid bound in mammary cell = -C b l o — (—) bi 165 l. 166 ha, from line 3 Figure A2. b from line 3 Figure A2. 1' 2 .c V(Z).=(—§—> v “ho/b1” C 2 .C «I 1 2 = (-——)[b 2 _ C22 0 V (b1) + b1 V (be) 2 bob1 COV 4 (bobi)]/bl 3| ‘x 11" V(bl) = (MSE/SSX), h0 = y - bl x CoV [(37- b1 3?), bl] = - 52v (b1) V(bo) = MSE nil?) + {52/3st approximate standard error of Z = S.E. = V V(Z) Test differences in x-intercepts (molecules bound) using the following Scheffé confidence interval: (zl - 22) i [(t-l) f W[V(zl) + V(zzn a, t-l, Ve(p) a = 0.05 a = 0.01 t = number treatments being compared ** pooled degrees of freedom for error ,‘ r Ihu—u‘u—l S "1 "I II n 3