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Corticosteroid effects on fetal metabolism and studies on steroid-receptor complexes in placenta of mice Wong, Ming Dak 1973

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CORTICOSTEROID EFFECTS ON FETAL vMETABOLISM AND STUDIES ON STEROID-RECEPTOR COMPLEXES IN PLACENTA OF MICE by MING DAK WONG B.Sc, Simon Fraser University, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biochemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Biochemistry The University of British Columbia Vancouver 8, Canada Date » August 2,1973 i ABSTRACT Teratogenic and lethal effects have been reported from treatment of pregnant animals with both synthetic and natural corticosteroids, and were also observed in this investigation. Injection of dexamethasone into pregnant mice on gestational day 18 resulted in decreasing net transfer of labeled glucose from mother to fetus with increasing steroid dosage. Inhibi-tion was also demonstrable when injections were made into the fetus in utero. It was concluded that the deleterious effects of dexamethasone on the survival of mouse fetuses might be attributable to this action. A l l steroid systems studied to date appear to involve a similar sequence of interactions that precede the different biochemical effects that are observed in target tissues. Bind ing of the steroid to a specific cytoplasmic macromolecule and translocation of this complex to the nucleus are required in the mechanism of steroid action. The placenta, being the site of maternal-fetal transmission of nutrients, was therefore examined for the presence of intracellular steroid receptors. Using Sephadex chromatography, the i n vitro binding of radioactive steroids to components in mouse placental nuclei and cytoplasm was investigated. Specificity was indicated in competition studies using excess unlabeled competing steroids. This spe c i f i c i t y was confirmed since only the active glucocor-ticoids formed complexes which demonstrated the a b i l i t y to translocate to the nucleus. The binding properties of the cytoplasmic steroid-receptor interaction were also studied. i i From the time course of binding the complex was shown to possess more s t a b i l i t y at 0°C than at 37°C, and the d i s t r i -bution of receptors in the cytosol appeared to be homogen-eous. The cytoplasmic complex showed l a b i l i t y when heat denaturation and proteolytic digestion were investigated, but did not appear to be affected by nucleases or the sulf-hydryl reagents. Kinetic analysis of the binding revealed the presence of high a f f i n i t y specific binding sites with a dissociation constant of 17.5 nM and a receptor site concen-tration of 0.26 pmoles/mg protein. Using sucrose density gradient centrifugation, the molecular weight of the cyto-plasmic corticosterone-receptor complex was estimated to be approximately 55•800. This investigation has revealed the existence of gluco-corticoid receptors in a target tissue in which the regulation of glucose a v a i l a b i l i t y to the fetus may be mediated by c o r t i -costeroids. In view of the c r i t i c a l role that glucose plays in the nutritional status of the fetus; and the preeminent influence that glucocorticoids have on glucose uptake in other target tissues, i t is then very plausible that the cortico-steroids have a regulatory function in fe t a l growth and via-b i l i t y . i i i TABLE OF CONTENTS Page ABSTRACT . . . . . . . . . . . i TABLE OF CONTENTS i i i LIST OF TABLES . v i LIST OF FIGURES v i i i LIST OF APPENDICES ix ACKNOWLEDGMENTS x ABBREVIATIONS USED x i INTRODUCTION . . . . . 1 Glucocorticoid Effects on Liver Metabolism . . . . 3 Glucocorticoid Effects on Peripheral Tissue . . . 8 Carbohydrate Metabolism in the Fetus and Placenta 11 The Role of Receptors in Glucocorticoid Action . , 15 The Present Problem . . . . . . . 21 MATERIALS AND METHODS 23 Chemicals 23 Solvents . . . . . . 23 Materials . . . . . . 2k Radiochemicals 2k Animals 2k Methods 2k 1. Steroid injections into pregnant mice . . . . 2k 2. Steroid injections into the fetus . . . . . . 25 3. Isolation of acid-soluble fraction of tissues . 25 iv TABLE OF CONTENTS (continued) Page k. Determination of radioactivity . . . . . . . 26 5. Semi-micro determination of blood glucose . . 26 6. Isolation of nuclear steroid-receptor complex 28 7. Cytosol exchange assay . . . 31 8. Other procedures 32 RESULTS 33 Steroid Effects on Fetal Glucose Metabolism . . . 33 A. Preliminary experiments on precursor incorporation 33 B. Dexamethasone effects on glucose incorporation into the acid-soluble fraction of f e t a l tissues 35 C. Effect of natural glucocorticoids . kJ D. Effect of steroid injected directly into the fetus k7 E. Glucose concentration and relative radioactivity of maternal blood . . . . . . . 51 F. Effect of dexamethasone on amino acid incorporation into the acid-soluble fraction of f e t a l tissues . . . . . 53 Steroid-receptor Complexes in Mouse Placental Tissue . 53 A. Preliminary isolation of nuclear complexes , 55 B. Specificity of nuclear receptor binding . . . 55 C. Characterization of the bound steroid . . . . 64 D. Isolation of cytoplasmic receptor 6k E. Binding properties of the cytoplasmic receptor . . . . . 69 V TABLE OF CONTENTS (continued) Page F. Kinetic examination of the binding . . . . . 72 G. Sucrose density gradient analysis . . . . . . 77 H. Relating the presence of placental receptors to the inhibition of glucose transfer 78 ^  DISCUSSION 8 4 BIBLIOGRAPHY 93 APPENDICES 100 v i LIST OF TABLES Page Table I. Table II. Representative values of radioactivity recovered from f e t a l extracts Effect of glucocorticoid treatment on fetal v i a b i l i t y Table III. Effect of dexamethasone upon the incor-poration of labeled acetate or glucose into a chloroform-methanol extract of SWV mouse fet a l tissues Table IV. Effect of various doses of dexamethasone upon the incorporation of labeled glucose into A/J mouse f e t a l tissues Table V. Strain differences in the effect of various doses of dexamethasone upon the incorporation of labeled glucose into mouse fetuses Table VI. Effects of corticosterone and Cortisol upon the incorporation of labeled glucose into SWV mouse f e t a l tissues Table VII. Table VIII. Effect of dexamethasone injected direct-l y into the fetus upon the incorporation of labeled glucose into SWV mouse fe t a l tissues Table IX. Glucose concentration and relative radio-activity of maternal blood from steroid-treated mice Table X. Effect of dexamethasone upon the incor-poration of labeled c i t r u l l i n e into SWV mouse fetuses Table XI. Effect of washing and addition of blood on the isolation of a labeled steroid complex from nuclei Table XII. Effect of cortexolone on the isolation of a labeled steroid complex from placental nuclei 27 3^ 36 37 44 *5 Relative activity of steroids on thymus involution 46 50 52 5* 59 62 v i i LIST OF TABLES (continued) Table XIII. Table XIV. Competitive interaction of steroids with -^H-corticosterone binding Chromatographic characterization of labeled steroid from isolated nuclear complexes Table XV. Identification by crystallization of labeled steroid from isolated nuclear complexes Table XVI. Table XVII. Table XVIII. Specific and nonspecific binding using the cytosol exchange assay Sensitivity of the cytosol steroid-receptor complex to heat, hydrolytic enzymes, and sulfhydryl reagents 1 Influence of actinomycin D on dexa-methasone -induced effects in pregnant mice Table XIX. Effect of cortexolone on dexamethasone inhibition of the incorporation of labeled glucose into SWV mouse fetuses Page 65 66 6? 70 74 82 83 • • • V l l l LIST OF FIGURES Page F i g . 1. Nuclei i s o l a t e d using hypotonic MgCl 2 29 F i g . 2. E f f e c t of various doses of dexamethasone upon the incorporation of labeled glucose into A/J mouse fetuses 39 F i g . 3. S t r a i n differences i n the e f f e c t of dexa-methasone upon the incorporation of labeled glucose into mouse fetuses 41 F i g . 4. E f f e c t of various doses of dexamethasone injected d i r e c t l y into the fetus upon the incorporation of labeled glucose into SWV mouse fetuses 48 F i g . 5- I s o l a t i o n of a -^H-corticosterone-receptor complex on Sephadex G-25 56 F i g . 6. Steroid s p e c i f i c i t y i n the i s o l a t i o n of a nuclear complex 58 F i g . 7. E f f e c t of cortexolone on the i s o l a t i o n of a ^ C - c o r t i s o l nuclear complex 61 F i g . 8. S p e c i f i c i t y of -^H-corticosterone binding i n placental homogenates 63 F i g . 9. F r a c t i o n a t i o n of placental tissue 68 F i g . 10. Time course of binding using the cytosol exchange assay 71 F i g . 11. Homogeniety of cytosol receptor d i s t r i -bution 73 F i g . 12. Scatchard p l o t of ^H-corticosterone binding i n SWV mouse placental cytosol 76 F i g . 13« Sucrose density c e n t r i f u g a t i o n of the cytosol steroid-receptor complex i s o l a t e d from Sephadex chromatography 79 LIST OF APPENDICES Appendix I. Preparation of Krebs-Eggleston Phosphate Buffer, pH 7.5 Appendix II. Structures of the Steroids Used Appendix III.Effect of Stress on Pregnancy in A/J and C57BL/6J Mice X ACKNOWLEDGMENTS I wish to express my gratitude by saying thank you to some of the many people with whom I have received helpful advice and discussion. I especially wish to thank Roger, Judy, Denise, Kash, Bruce, Brian, Jim, Bev, and Joyce. I wish to thank Dr. R.L. Noble, Director of the Cancer Research Centre, for the use of the f a c i l i t i e s at the Centre where most of this research was conducted. Many thanks also to Mr. B i l l Siep for his fine technical help in preparing chromatography columns. The considerate advice and encourage-ment offered by Dr. R.H. Pearce is also gratefully acknowled-ged. I especially wish to thank Dr. A.F. Burton for his patience and advice during the course of this research and the preparation of this thesis. Finally, most of a l l , I want to thank my family; Susan, Gregory, and Cheryl for their understanding and love. I wish to acknowledge the support of Medical Research Council of Canada Studentships for I969 to 1973. x i ABBREVIATIONS USED AMP adenosine-5'-monophosphate DEAE diethylaminoethyl DNA deoxyribonucleic acid DNAse deoxyribonuclease dpm disintegrations per minute EDTA (ethylenedinitrilo)-tetraacetic acid, tetrasodium salt i.p. intraperitoneal KEP Krebs-Eggleston phosphate buffer (see Appendix I) POPOP 1,4-Bis (2-(5-phenyloxazolyl))-benzene PPO 2,5-diphenyloxazole RNA ribonucleic acid RNAse ribonuclease s.c. subcutaneous TCA trichloroacetic acid TLC thin layer chromatography TRIS 2-amino-2-hydroxymethyl-l,3-propanediol uCi microcurie UDP uridine-5'-pyrophosphate ug microgram ul microliter uM micromolar 1 INTRODUCTION The metabolic effects of the glucocorticoid hormones have been examined actively over the past forty years. The pioneer work of Britton and Silvette in 1 9 3 2 arose from the observation that adrenocortical extracts had effects on carbo-hydrate metabolism and therefore a possible role in the regulation of glucose metabolism ( 1 ) . Even before this, in 1 9 2 7 . Cori and Cori had demonstrated that adrenalectomized rats and mice f a i l e d to maintain adequate glycogen stores ( 2 ) . How-ever, i t was not until the extensive studies of Long, Katzin, and Fry in 1 9 ^ 0 , characterizing the f a l l in blood sugar and l i v e r glycogen of fasted, adrenalectomized rats and mice ( 3 ) that the adrenocortical hormones were generally recognized as having a profound effect on carbohydrate and protein metabolism as well as on salt and water metabolism. The separation of the extract from the adrenal gland into different components with glucocorticoid and mineralocorticoid a c t i v i t i e s arose with the identification and synthesis of the different steroids. This in turn has led to the elucidation of the basic aspects of steroid action. The glucocorticoids are generally considered to be cata-bolic hormones since their most pronounced metabolic effect in the animal as a whole results in wasting of muscle tissue with an accompanying negative nitrogen balance. On the whole, the actions of glucocorticoids in carbohydrate, protein, and l i p i d metabolism result in glucose 'sparing', as evident from the 2 increased catabolism by the peripheral tissues as well as de-creased glucose uptake. This i n turn leads to increased blood glucose and amino acid l e v e l s , thereby increasing gluconeo-genesis from the available amino acids mainly by the l i v e r ( 4 ) but also by the kidney ( 5 ) « Adipose tissue undergoes increased l i p o l y s i s and decreased lipogenesis ( 6 ) , thus increasing the blood concentrations of g l y c e r o l and f a t t y acids ( 7 ) . The gly c e r o l provides an additional substrate f o r hepatic gluconeo-genesis while the free f a t t y acids supply an alternate energy supply ( 8 ) and at the same time t h e i r high concentration may i n h i b i t the key g l y c o l y t i c enzymes of the l i v e r ( 9 ) , allowing gluconeogenesis to predominate i n that organ. In normal animals many of these e f f e c t s are not evident due to the compensatory actions of i n s u l i n which i s secreted i n response to the hyper-glycemia. Only by prolonged or excessive administration of the glu c o c o r t i c o i d s can many of these e f f e c t s be demonstrated i n vivo. Paradoxically, along with these catabolic e f f e c t s , the glucocorticoids also simultaneously exert an anabolic action. The l i v e r , i n contrast to the peripheral t i s s u e s , responds to the hormone with dramatic stimulation of several major hepatic metabolic processes. The anabolic e f f e c t s of glucocorticoids on the l i v e r r e s u l t i n glycogen deposition, increased gluconeo-genesis, increased uptake of amino acids, and increased syn-thesis of RNA and protein. Urea and ketone body production are also increased by the steroids but probably represent secondary changes i n the l i v e r (10) and are not d i r e c t l y affected by the 3 hormones. Glucocorticoids thus appear to have two distinct metabolic effects: (a) an anabolic action on the l i v e r leading to deposition of glycogen as well as formation of RNA and pro-tein, and (b) a decreased glucose and amino acid uptake by extrahepatic tissues, as well as a catabolic breakdown of pro-tein, l i p i d , RNA, and DNA by these tissues. Glucocorticoid Effects on Liver Metabolism The gross hepatic effects of glucocorticoid administration result in hypertrophy of the organ since total weight increases whereas the DNA levels remain essentially constant ( 1 0 ) . The general anabolic effect on the l i v e r is most readily manifested by the increase in glycogen deposition, although increased RNA and protein are also evident. These metabolic effects general-l y require 2 to 4 hours to be demonstrated in vivo with adren-alectomized, fasted animals ( 1 1 ) . Well before these effects, about 80 to 1 0 0 min after glucocorticoid administration, there is observed an increase in blood glucose ( 1 2 ) . This increase may result from a decrease in peripheral glucose u t i l i z a t i o n or an increase in hepatic glucose production. Although an increase in the rate of gluconeogenesis in l i v e r slices has been reported ( 1 3 ) , in vivo stimulation by glucocorticoids has been d i f f i c u l t to demonstrate using iso-lated perfused l i v e r (14, 1 5 ) . However, when 10 mM alanine was used as substrate ( 2 0 to 30 times the circulating physio-logical l e v e l ) , impaired incorporation of alanine into glucose was demonstrated using perfused livers from adrenalectomized rats ( 1 6 ) . This was restored to normal by the addition of a 4 glucocorticoid to the perfusion medium. Since no effect was seen using physiological concentrations of amino acids (15)» i t seems that hepatic gluconeogenesis is unaffected by gluco-corticoids i f the amino acid level is within the range that is normally handled by the l i v e r . However, the steroids w i l l in-crease the maximal capacity for hepatic gluconeogenesis under conditions where the amino acid load is high (13)-An early effect of the glucocorticoids is the rise in hepatic amino acid levels as a consequence of their release from the peripheral tissues, especially muscle (13, 17) . The uptake of these amino acids by the l i v e r may also be steroid-enhanced as suggested by the increased hepatic uptake of the nonmetabo-liz e d amino acid, 2-aminoisobutyric acid (18), after glucocor-ticoid administration. The high concentrations of amino acids in the l i v e r become ready substrates for steroid-induced gluco-neogenesis under these conditions. Since alanine comprises more than 80% of the total amino a c i d N 2 r e l e a s e d by muscle ( 1 3 ) , the rise in hepatic amino acid levels may play more.than just a precursor role. Alanine has been reported to inhibit l i v e r pyruvate kinase activity ( 19) . This inhibition would prevent the flow of substrate from phosphoenol-pyruvate to pyruvate, thus promoting gluconeogenesis. While the process of gluconeogenesis i t s e l f has been d i f -f i c u l t to demonstrate, the activity of any rate-limiting enzyme in the glucogenic sequence is increased in activity in response t to glucocorticoid treatment ( 1 7 ) . This effect'^is most l i k e l y a result of induction of the gluconeogenic enzymes, a fact borne 5 out by the observation that the stimulation of gluconeogenesis induced by steroids in l i v e r slices from adrenalectomized rats' is blocked by actinomycin D ( 2 0 ) . Control of phosphoenolpyru-vate carboxykinase synthesis ( 8 ) is probably the major mechanism in the longer-term adaptation of gluconeogenesis to glucocorti-coid administration. Cortisol has been shown to increase hepatic levels of phosphoenolpyruvate while leaving malate levels un-changed ( 2 0 ) . The ac t i v i t i e s of pyruvate carboxylase ( 8 ) , glucose-6-phosphatase ( 2 1 ) , and fructose - 1 , 6-diphosphatase ( 2 2 ) have a l l been reported to be increased after glucocorticoid ad-ministration. Both pyruvate carboxylase and glucose-6-phospha-tase may be involved in the steroid effect although the stimu-lation of the latter enzyme may not be due to stimulation of synthesis since the steroid effect is not blocked by actinomycin D ( 8 ) . The importance of the increase in fructose - 1 , 6-diphos-phatase to the steroid effect is dubious since the enzyme increase follows, not precedes, the increased rate of gluconeogenesis ( 2 3 ) . In addition, gluconeogenesis from fructose is not impair-ed in l i v e r slices from adrenalectomized rats nor are fructose - 1 , 6-diphosphate levels increased ( 8 ) . Along with the early rise in hepatic amino acid levels, there is also an early rise in the free fatty acid levels of the l i v e r ( 7 , 2 3 ) . This results from the permissive l i p o l y t i c action of the glucocorticoids on adipose tissue, causing the release of free fatty acid and glycerol ( 6 , 7 ) . The glycerol supplies additional substrate for gluconeogenesis while the e l -evated hepatic fatty acid level results in inhibition of glyco-6 l y t i c enzymes, chiefly pyruvate kinase ( 9 ) ' Inhibition of the hexose monophosphate pathway and the Krebs cycle enzymes, iso-citrate dehydrogenase and fumarase ( 2 3 ) by the high fatty acid level blocks other pathways of glucose oxidation in the l i v e r , allowing maximum efficiency for the conversion of lactate and pyruvate to glucose. The energy requirements for the l i v e r must now be obtained from other sources, namely, the partial oxidation of long-chain fatty acids which then result in the accumulation of ketone bodies. In order for gluconeogenesis from amino acids to occur rapidly the enzymes involved in transamination and deamination must also be increased, i f they are rate-limiting ( 1 ? ) . Glutamic-pyruvic transaminase (2k), tyrosine aminotransferase ( 2 5 ) , and tryptophan pyrrolase ( 2 6 ) a c t i v i t i e s are among the many trans-aminase enzymes increased and provide one of the earliest and most sensitive l i v e r responses to glucocorticoids. The enzymes involved in urea biosynthesis also increase ( 1 0 ) but this is most l i k e l y a delayed response to the increased mobilization of amino acids to the l i v e r since their increase occurs some time later. Studies using immunochemical determination of enzyme activity ( 2 7 ) , supported by experiments with puromycin (28), have demonstrated that hormonal induction of the transaminase enzymes also involve accelerated rates of synthesis and elevated levels of enzyme protein. The hepatic enzyme induction by the glucocorticoids does not occur in rodents pretreated with a c t i -nomycin D ( 1 0 ) , again indicating that this is a process also dependent upon continued RNA synthesis. 7 Even before i t was known that glucocorticoid-mediated enzyme induction was sensitive to actinomycin D, i t was found' that these steroids enhanced the incorporation of inorganic 12 J P into hepatic RNA ( 2 9 ) . This enhancement occurred in a l l subcellular organelles, and was reflected in an increased speci-f i c activity for transfer RNA, ribosomal RNA, and DNA-like RNA. 14 3 Using C-uridine and -guanine as precursors, Feigelson ( 1 0 ) found an increased rate of synthesis of a uracil-rich RNA species within 1 0 min after the administration of glucocorticoids in vivo, followed by an increased rate of synthesis of a guanine-rich species several hours later. The increased uracil-rich RNA synthesis precedes the induction of tryptophan pyrrolase and tyrosine aminotransferase ( 1 0 ) , and may be the template speci-fying the synthesis of the inducible enzymes. The guanine-rich RNA species synthesized later most l i k e l y represents an increased rate of synthesis of ribosomal RNA, The increase in hepatic RNA synthesis after glucocorticoid treatment may result from an i n -crease in RNA polymerase activity. Several groups ( 3 0 , 3 1 ) have reported a rapid rise in hepatic RNA polymerase activity after in vivo administration of glucocorticoids. Although the RNA and protein content of the l i v e r are elev-ated approximately 3 0 $ and 2 0 $ , respectively, within 1 2 hours of cortisone treatment ( 1 0 ) , the 3 0 $ increment in hepatic dry weight that occurs in this period is due mainly to the increased glycogen content. Steroid-stimulated l i v e r s have a decreased concentration of glucose-6-phosphate and uridine diphosphate glucose ( 1 7 ) , suggesting an effect on the activity of glycogen 8 synthetase by the glucocorticoids. Most Likely there i s an activation due to increased precursor supplies as well as a direct stimulation of the conversion of glycogen synthetase D (inactive) to I (active). This activation, which may result in a 200% increase in l i v e r glycogen content within 4 hours (10) is apparently due to an activation of existing enzyme since actinomycin D generally has no effect on the process (17). The stimulatory effect on glycogen synthesis may not be a direct effect of glucocorticoid but may be mediated by insulin which is stimulated by the high blood glucose levels. The glucocor-ticoid-induced hyperglycemia could also result in glucose a c t i -vation of glycogen synthetase (32) and inactivation of phospho-rylase a. Glucocorticoid Effects on Peripheral Tissue Interest in the peripheral action of glucocorticoids arose with the discovery that C o r t i s o l injection into fasted, adrenal-ectomized rats produced an increase in blood glucose well before demonstrable effects on l i v e r glycogen (12) or gluconeogenesis. Decreased peripheral glucose uptake would result in this effect and experiments to locate the sites of this decrease were met with success using adipose tissue (33i 3*0 i skin (35)» and thy-mus (36, 37). Along with the decreased glucose u t i l i z a t i o n by these tissues there is also increased catabolism of protein from muscle and l i p i d from adipose tissue, leading to elevated hepatic levels of amino acids and fatty acids. In general, nucleic acid metabolism is also depressed by the effect of the glucocorticoids on the peripheral tissues. Q Experiments to demonstrate decreased glucose uptake by muscle have been contradictory (38). This stems from the low normal u t i l i z a t i o n of glucose by muscle (17), e s p e c i a l l y i f free f a t t y acid i s available as f u e l . Several hours a f t e r s t e r o i d administration i n vivo i t i s possible to show decreased glucose uptake by r a t hemidiaphragm or heart i n v i t r o (39). with a s p e c i f i c e f f e c t on the i n i t i a l phosphorylation. This may not be a d i r e c t e f f e c t of the g l u c o c o r t i c o i d since the r i s e i n free f a t t y acid concentration could serve to i n h i b i t the u t i l -i z a t i o n of glucose by muscle (17). The increased egress of amino acids into the c i r c u l a t i o n from muscle, even i n eviscer-ated preparations ( 3 2 ) » i s one of the f i r s t e f f e c t s of glucocor-t i c o i d administration i n vivo. Also demonstrable i s decreased incorporation of labeled amino acids into muscle i s o l a t e d from animals pretreated with g l u c o c o r t i c o i d s . Thus, the e f f e c t s of glucocorticoids on muscle are quite l i k e l y both catabolic and anti-anabolic, r e s u l t i n g mainly i n an elevated supply of amino acids to the l i v e r . The glucocorticoid-induced i n h i b i t i o n of glucose uptake by adipose tissue r e s u l t s i n a decrease i n g l y c e r o l phosphate and f a t t y acid production and may p a r t l y explain the anti - 1ipogenic e f f e c t since there i s no convincing evidence that glucocorticoids d i r e c t l y influence lipogenesis. What has been c l e a r l y e s t a b l i s h -ed i s that the glucocorticoids have a permissive role i n f a t t y acid mobilization by l i p o l y t i c agents (6) which, i f unopposed by i n s u l i n , r e s u l t i n release of free f a t t y acids and g l y c e r o l from adipose t i s s u e . Fat mobilization by the catecholamines 1 0 and l i p o l y t i c peptide hormones i s believed to involve c y c l i c AMP a c t i v a t i o n of a c e l l u l a r hormone-sensitive l i p a s e ( 6 ) . One a t t r a c t i v e p o s s i b i l i t y f o r the involvement of glucocorticoids i s that the steroids i n h i b i t c y c l i c AMP phosphodiesterase ( 3 2 ) , thus allowing the l i p o l y t i c e f f e c t to be manifested. The work of Munck and h i s collaborators showed that thymus tissue exhibited the same behavior as other peripheral tissues when exposed to glucocorticoids. After s t e r o i d i n j e c t i o n there i s decreased incorporation of glucose into thymuses of rats and decreased glucose uptake by incubated thymus c e l l s ( 3 8 ) . An i n v i t r o decrease i n both protein synthesis (37) and nucleic acid metabolism (40) was also observed. The i n h i b i t o r y e f f e c t s on glucose uptake were thought by Munck to be responsible f o r the catabolic e f f e c t s of the glucocorticoids ( 3 8 ) . This was i n d i c -ated by the observation that e f f e c t s on protein synthesis and nucleic acid metabolism occur much more slowly than the decrease i n glucose uptake (37)» and c y t o l o g i c a l indications of catabol-ism take even longer to appear ( 4 l ) . Experimental support f o r t h i s hypothesis has come from the work of Young, which showed an absolute requirement f o r glucose before the e f f e c t s of C o r -t i s o l on protein synthesis and nucleic acid metabolism take place (42). Further work u t i l i z i n g lymphosarcoma c e l l s has i n -dicated that the ultimate catabolic action of the g l u c o c o r t i -coids leads to i n h i b i t i o n of growth and f i n a l l y c y t o l y s i s . The general conclusion that emerges i s that f o r lymphoid t i s -sue, and also f o r adipose tissue and skin, i n h i b i t i o n of g l u -cose uptake i s probably an e s s e n t i a l f i r s t step i n the catabol-i c actions of the glucocorticoids. ] 1 Carbohydrate Metabolism in the Fetus and Placenta The fetus during intrauterine l i f e is dependent upon mater-nal sources for a l l of the nutrients and energy supply neces-sary for growth and differentiation. Measurements of the rate of glucose uptake in fet a l lambs suggest that i t is fast enough to account for the fet a l oxygen consumption (4-3) and may thus be the principal metabolic fuel of the fetus. Since carbohy-drates are also transported, stored, and synthesized by the pla-centa, each of these functions w i l l have an effect on the avail-a b i l i t y of carbohydrate to the fetus. Placental transport of glucose has been characterized as occurring by 'facilitated diffusion' (44) since travel is in the same direction as for a diffusion gradient but the rate exceeds the calculated rate predicted on physicochemical grounds. This transport system is mediated by a carrier probably a protein component of the c e l l membrane which can reversibly bind the glucose and carry i t across the membrane. The transport is usually down the concentration gradient and can proceed from mat-ernal to fetal or in reverse, depending on the direction of the gradient (45). At very high glucose concentrations i t has been possible to saturate the carrier system. Other sugars may also act as competitive inhibitors of the transport although the car-r i e r demonstrates a strong specificity for glucose. This is evident from the observation that fructose, a hexose of similar molecular weight to glucose, crosses the placenta at a tenth of the rate (44). A stereospecificity also exists in the system since the biologically-significant D-sugars are transported much faster than the corresponding L-isomers. 12 A l l mammalian p l a c e n t a s s t u d i e d to date have been shown to take up and u t i l i z e g l u c o s e . Since the uptake of glu c o s e by other t i s s u e s i s under hormonal r e g u l a t i o n , the r a t e - l i m i t i n g passage of glucose through the p l a c e n t a l c e l l membrane may a l s o have s i m i l a r c o n t r o l s . Changes i n the uptake and u t i l i z a t i o n of g l u c o s e by the p l a c e n t a would allow i t to i n f l u e n c e the quan-t i t y of carbohydrate made a v a i l a b l e to the f e t u s . There have been c o n f l i c t i n g o b s e r v a t i o n s c o n c e r n i n g the e f f e c t of i n s u l i n on p l a c e n t a l glucose uptake. I n s u l i n f a c i l -i t a t i o n of glucose uptake by human p l a c e n t a l s l i c e s has been r e p o r t e d but has not been confirmed. T h i s may r e s u l t from d i f -f e r e n c e s i n t i s s u e p r e p a r a t i o n , l e n g t h of g e s t a t i o n , or v a r i a t i o n i n the amount of i n s u l i n a s e (45), known to be p r e s e n t i n p l a c e n t a l t i s s u e i n d i f f e r e n t experimental systems. A n a e r o b i o s i s has been shown to a c c e l e r a t e p l a c e n t a l g l u c o s e uptake (46) although i t i s not known i f t h i s i s a d i r e c t s t i m u l a t i o n of the r a t e -l i m i t i n g membrane t r a n s p o r t step as has been demonstrated f o r muscle t i s s u e (47). A Pasteur e f f e c t has been invoked to ex-p l a i n the i n c r e a s e d uptake caused by anoxia (48) s i n c e d e p l e t i o n of glucose-6-phosphate w i l l r e s u l t i n i n c r e a s i n g the r a t e of p h o s p h o r y l a t i o n of g l u c o s e , thus a l l o w i n g the t r a n s p o r t of more glucose a c r o s s the membrane. In s i d e the p l a c e n t a l c e l l p h o s p h o r y l a t i o n to g l u c o s e s -phosphate i s the f i r s t step i n glucose metabolism. T h i s i s a p p a r e n t l y accomplished by a n o n s p e c i f i c hexokinase s i n c e g l u -cokinase has not been found i n human or guinea p i g p l a c e n t a (49). The glucose-6-phosphate then e n t e r s g l y c o g e n s y n t h e s i s or glucose 11 u t i l i z a t i o n via the hexose monophosphate shunt and glycolysis. The hexose monophosphate pathway has significant activity early in gestation (49, 50), presumably to provide pentoses for nucleic acid production, after which the enzymes of the shunt decrease in activity towards term (49). The glycolytic pathway, which is quantitatively the most significant route of glucose u t i l i z -ation by the placenta, is accelerated by both anoxia (46) and maternal diabetes (45). The placental glycogen, which varies with gestational age, has no clearly established function to date. It has been suggested that the glycogen could serve as a reservoir of carbohydrate for the fetus during the period of rapid growth preceding the termination of gestation (51). The fetus, although i t receives maternal amino acids, pre-sumably conserves them for protein synthesis since trans-or de-amination does not occur during fe t a l l i f e (52) and gluconeo-genesis from amino acids commences only after birth (53). Thus the fetus uses glucose as virtually i t s only energy source and to some extent i t s metabolism is analogous to an adult animal receiving a high-carbohydrate diet. However, not a l l the glu-cose which crosses the placenta is oxidized. Some is used for lipogenesis and a large proportion is stored as l i v e r glycogen. It is well established that at a particular developmental stage on and until birth, the fetal l i v e r accumulates large amounts of glycogen which are rapidly exhausted at birth. This evidently w i l l provide the newborn with a supply of glucose after the severance of the maternal supply and before an endo-genous source is available. In a l l species studied (52), 14 the glycogen concentration in the fe t a l liver and skeletal muscle begins to rise during the last third of gestation. Near term l i v e r glycogen is at least twice the normal adult concen-tration for most species whereas the skeletal muscle glycogen concentration is 3 to ^ times the corresponding adult level (54). This elevated level drops considerably and l i v e r glycogen reaches IQfo or less of i t s i n i t i a l value within 2 to 3 hours of birth. Skeletal glycogen also f a l l s to, or below, the adult level with-in 1 to 3 days after birth. There is very l i t t l e known about the factors responsible for the f i r s t appearance of glycogen in fetal tissues and the subsequent increase in content. Phosphoglucomutase, UDP-glucose pyrophosphorylase, and glycogen synthetase a l l appear and become increasingly active in fe t a l l i v e r tissue just prior to glycogen storage (55). Burton, Greenall, and Turnell (56) have shown a correlation between the increase in l i v e r glycogen levels of fetal mice and the appearance of steroid reductase activity (which increases active corticosteroids) in fe t a l l i v e r . Stim-ulation of fetal l i v e r glycogen deposition by glucocorticoids probably results from the rise in blood glucose concentration, both in maternal and fetal plasma. Injection of corticosteroids early in gestation w i l l induce l i v e r glycogen storage to begin earlier in both rats (55) and mice (56).-If decapitated fetuses of adrenalectomized rats are given Cortisol the l i v e r glycogen content is increased (55)- In rab-bits, however, corticosteroids were found to have no effect on glycogen storage even when amounts up to 3 mg were administered. 15 Only when p i t u i t a r y p r o l a c t i n was g i v e n together w i t h the c o r t i -c o i d was glycogen d e p o s i t i o n e v i d e n t i n the d e c a p i t a t e d r a b b i t f e t u s e s (55). The concept t h a t arose from these o b s e r v a t i o n s i s t h a t glycogen d e p o s i t i o n i n the f e t a l l i v e r i s under a dual hormonal c o n t r o l , both by a d r e n a l s t e r o i d s and a p i t u i t a r y hor-mone. With'some animals such as the r a t a p r o l a c t i n - l i k e f a c t o r i s produced by the p l a c e n t a and t h e r e f o r e i n these s p e c i e s c o r -t i c o s t e r o i d s become the l i m i t i n g f a c t o r f o r g l y c o g e n d e p o s i t i o n i n f e t a l l i v e r . The p h o s p h o r y l a t i o n of glucose i n the f e t a l l i v e r i s accom-p l i s h e d by hexokinase s i n c e g l u c o k i n a s e , the s p e c i f i c enzyme of a d u l t l i v e r , i s absent (53) and f i r s t appears a few days a f t e r b i r t h . The glucose-6-phosphate i s then c h a n n e l l e d i n t o g l y c o g e n f o r m a t i o n , g l y c o l y s i s , and the hexose monophosphate shunt. To-wards the end of term an i n c r e a s e i n the enzymes concerned with g l y c o g e n o l y s i s and gluconeogenesis a l s o occurs. Thus phosphory-l a s e a c t i v i t y shows an i n c r e a s e some time a f t e r g l y c o g e n depo-s i t i o n has commenced (55) w h i l e glucose-6-phosphatase a c t i v i t y r i s e s v e r y s h a r p l y s h o r t l y before b i r t h . Pyruvate c a r b o x y l a s e and f r u c t o s e diphosphatase are a l s o r e a d i l y d e t e c t a b l e s h o r t l y before b i r t h and a t t a i n a d u l t l e v e l s a t term. However, g l u c o -neogenesis may not be o p e r a t i v e u n t i l a f t e r b i r t h s i n c e phos-phoenolpyruvate carboxykinase has no d e t e c t a b l e a c t i v i t y before b i r t h (55). The Role of Receptors i n G l u c o c o r t i c o i d A c t i o n I t i s c l e a r t h a t there are a m u l t i t u d e of e f f e c t s a t t r i b u t e d to g l u c o c o r t i c o i d s . As w e l l as i n i t i a l d i r e c t e f f e c t s upon 16 s p e c i f i c p r o c e s s e s , there are the c e l l u l a r m o d i f i c a t i o n s t h a t occur as a p h y s i o l o g i c a l response to l o n g term e f f e c t s of the hormone. T h i s m u l t i p l i c i t y of b i o c h e m i c a l responses to g l u c o -c o r t i c o i d s has f r u s t r a t e d the search f o r the f i r s t e f f e c t of the hormone w i t h i n the c e l l . N e v e r t h e l e s s the b a s i c a c t i o n of the g l u c o c o r t i c o i d s on most me t a b o l i c processes has been thought to i n v o l v e m o d i f i c a t i o n of the t r a n s c r i p t i o n a l or t r a n s l a t i o n a l aspects of g e n e t i c e x p r e s s i o n . K a r l s o n ( 5 7 ) e l a b o r a t e d the h y p o t h e s i s t h a t the hormones, by i n t e r a c t i o n s w i t h the genome, would c o n t r o l the s y n t h e s i s of p r o t e i n s i n the r e c e p t o r organ by r e g u l a t i n g messenger RNA syn-t h e s i s . D i r e c t i n t e r a c t i o n of the s t e r o i d s w i t h DNA d i d not seem to be a f e a s i b l e method f o r c o n t r o l s i n c e i t i s d i f f i c u l t to see how the s t e r o i d s themselves c o u l d c o n t a i n enough s p e c i f -i c i t y f o r b i n d i n g to s p e c i f i c r e g i o n s of DNA. H i s t o n e s have "been shown to b i n d C o r t i s o l ( 5 8 , 5 9 ) , although the a f f i n i t y of s t e r o i d s f o r t h i s f r a c t i o n g e n e r a l l y appear to be r a t h e r low ( 6 0 ) . There i s a l s o a r e l a t i v e l a c k of s p e c i f i c i t y i n the s t r u c t u r e of the h i s t o n e s , l e a d i n g to the i d e a t h a t they i n t e r -a c t mainly by n o n - s p e c i f i c a l l y masking DNA ( 6 1 ) . The non-histone or a c i d i c p r o t e i n s have been thought to be r e s p o n s i b l e f o r un-masking s p e c i f i c DNA sequences by c o u n t e r a c t i n g the i n h i b i t o r y e f f e c t s of h i s t o n e s ( 6 2 ) . U s i n g adrenalectomized r a t s , S h e l t o n and A l l f r e y made the o b s e r v a t i o n t h a t both RNA and a c i d i c p r o -t e i n s y n t h e s i s i n l i v e r c e l l s were enhanced w i t h i n 2 to 3 hours a f t e r a s i n g l e injection of Cortisol ( 6 3 ) . Whichever component of the nucleus is affected, g l u c o c o r t i c o i d treatment has been shown to increase DNA-dependent RNA polymerase a c t i v i t y ( 3 0 , 3 1 ) . 17 These experiments, along with the o b s e r v a t i o n by F e i g e l s o n and h i s c o l l a b o r a t o r s ( 1 0 , 2 9 ) of an enhanced, r a t e of s y n t h e s i s of DNA-like RNA as w e l l as ribosomal RNA a f t e r s t e r o i d treatment, i n d i c a t e t h a t messenger RNA s y n t h e s i s and w i t h i t p r o t e i n syn-t h e s i s i s a c c e l e r a t e d by s t e r o i d a d m i n i s t r a t i o n . By f o l l o w i n g the s u b c e l l u l a r d i s t r i b u t i o n of g l u c o c o r t i c o i d s i n t a r g e t t i s s u e s an understanding of t h e i r a c t i o n s might be o b t a i n e d . In v i v o the l i v e r i s the main organ t h a t c o n c e n t r a t e s g l u c o c o r t i c o i d s above the blood l e v e l s i n c e i t i s an important s i t e of s t e r o i d metabolism as w e l l as b e i n g a t a r g e t t i s s u e . A f t e r an i . p . i n j e c t i o n of l a b e l e d g l u c o c o r t i c o i d , there i s r a p i d accumulation i n the l i v e r w i t h most of the r a d i o a c t i v i t y p r e s e n t i n the c y t o s o l and l e s s e r amounts i n the other s u b c e l l u l a r f r a c -t i o n s . The c y t o s o l r a d i o a c t i v i t y , of which 6 to ljfo i s bound to macromolecules, i s e l u t e d from columns of DEAE Sephadex A - 5 0 a s s o c i a t e d with f o u r p r o t e i n peaks which L i t w a c k ( 6 0 ) has l a b e l e d b i n d e r s I, I I , I I I and IV. Peaks I and I I I have been found to b i n d mostly to p o l a r m e t a b o l i t e s of C o r t i s o l w h i l e IV binds most e f f e c t i v e l y to m e t a b o l i t e s of t e s t o s t e r o n e and p r o g e s t e r -one. B i n d e r I I i s a s s o c i a t e d w i t h a much h i g h e r p o r t i o n of the unchanged s t e r o i d immediately a f t e r hormone a d m i n i s t r a t i o n and may r e p r e s e n t the c y t o s o l g l u c o c o r t i c o i d r e c e p t o r i n the l i v e r . I t s b i n d i n g p r o p e r t i e s s a t i s f y the b a s i c requirements of a p h y s i o l o g i c a l hormone r e c e p t o r ; i t i s s a t u r a t e d i n v i v o very r a p i d l y ( 5 to 10 min) and w i t h i n a p h y s i o l o g i c a l c o n c e n t r a t i o n of s t e r o i d ( 0 . 1 nM). Due to i t s r e l a t i v e l y poor blood supply the thymus does not c o n c e n t r a t e s t e r o i d s e f f e c t i v e l y in v i v o . However, u s i n g i n v i t r o in i n c u b a t i o n s to circumvent the problems of blood c i r c u l a t i o n and c o m p e t i t i o n f o r hormone uptake, thymus c e l l s have been found to a l s o accumulate r a d i o a c t i v e g l u c o c o r t i c o i d s i n a l l s u b c e l l u l a r f r a c t i o n s ( 6 0 , 6 4 ) . Munck has shown t h a t a d d i t i o n of l a b e l e d g l u c o c o r t i c o i d s of h i g h s p e c i f i c a c t i v i t y to a thymus c e l l sus-pension r e s u l t s i n i t i a l l y i n two types of i n t e r a c t i o n s ; non-s p e c i f i c and s p e c i f i c b i n d i n g ( 6 5 ) . The n o n s p e c i f i c b i n d i n g i s v i r t u a l l y i nstantaneous ( 3 8 )» and r e s u l t s i n d i s t r i b u t i o n of the s t e r o i d throughout the c e l l mainly by a l o o s e a d s o r p t i o n onto v a r i o u s macromolecules and s u b c e l l u l a r o r g a n e l l e s . T h i s form of b i n d i n g does not s a t u r a t e even a t high s t e r o i d concen-t r a t i o n s ( 1 0 uM and up) and has no c o r r e l a t i o n w i t h i n v i v o g l u -c o c o r t i c o i d a c t i v i t y . The p h y s i o l o g i c a l l y important b i n d i n g accounts f o r o n l y a s m a l l f r a c t i o n of the t o t a l amount of bound s t e r o i d and becomes s a t u r a t e d a t about 1 uM ( 6 5 ) . I f c e l l s t r e a t e d w i t h r a d i o a c t i v e g l u c o c o r t i c o i d are d i l u t e d i n t o a medium with no s t e r o i d , the bound s t e r o i d w i l l d i s s o c i a t e a t a r a t e determined by i t s b i n d -i n g a f f i n i t y . At 3?°C most of the s t e r o i d d i s s o c i a t e s w i t h i n 1 min and t h i s r a p i d l y d i s s o c i a t i n g f r a c t i o n r e p r e s e n t s non-s p e c i f i c a l l y bound s t e r o i d . A c o r t i c o s t e r o i d such as c o r t i s o n e which i s not b i o l o g i c a l l y a c t i v e per se, i s bound almost e n t i r e l y i n t h i s manner and e x h i b i t s no s p e c i f i c b i n d i n g ( 3 8 ) . F o r the a c t i v e g l u c o c o r t i c o i d s a l a r g e p o r t i o n a l s o d i s s o c i a t e s r a p i d l y , but t h e r e remains a f r a c t i o n of h i g h e r b i n d i n g a f f i n i t y which d i s s o c i a t e s much more s l o w l y . The p r o p e r t i e s of t h i s f r a c t i o n ( 6 6 ) i n d i c a t e t h a t i t c o n s i s t s of hormone molecules s p e c i f i c a l l y bound to the g l u c o c o r t i c o i d r e c e p t o r s . 19 C o r texolone, a s t e r o i d without i_n v i v o g l u c o c o r t i c o i d a c t -i v i t y s i n c e i t l a c k s an oxygen f u n c t i o n a t carhon - 1 1, does com-pete e f f e c t i v e l y _in v i t r o w i t h C o r t i s o l f o r the s p e c i f i c b i n d i n g s i t e s . Munck found t h a t 10 uM co r t e x o l o n e would completely a b o l i s h the m e t a b o l i c e f f e c t s of 1 uM C o r t i s o l on thymus c e l l s ( 6 5 , 6 7 ) , thus a c t i n g as an a n t i - g l u c o c o r t i c o i d . These experim-ents w i t h c o r t e x o l o n e , which have been confirmed by other work-ers ( 6 8 , 6 9 , 7 0 ) , i n d i c a t e t h a t the s p e c i f i c b i n d i n g s i t e s have a r o l e i n s t e r o i d a c t i o n . F u r t h e r evidence t h a t the g l u c o c o r t i -c o i d r e c e p t o r s mediate the hormonal e f f e c t i s p r o v i d e d by s t u d i e s on c u l t u r e d hepatoma c e l l s ( 7 1 , 7 2 ) , lymphoid c e l l s ( 7 3 ) , and f i b r o b l a s t s (7*0 which showed t h a t absent or decreased s e n s i t -i v i t y to s t e r o i d s i s accompanied by a d i m i n i s h e d c o n c e n t r a t i o n of s p e c i f i c g l u c o c o r t i c o i d r e c e p t o r s i t e s . The o v e r a l l m olecular mechanism of g l u c o c o r t i c o i d a c t i o n on t a r g e t c e l l s appears q u i t e s i m i l a r to the the o r y e l u c i d a t e d f o r the other s t e r o i d hormones ( 7 5 i 7 6 , 7 7 ) . A f t e r p e n e t r a t i o n of the c e i l membrane, the s t e r o i d i s f i r s t l o o s e l y bound by the r e v e r s i b l e n o n s p e c i f i c b i n d i n g d e s c r i b e d e a r l i e r . I n t e r a c t i o n then occurs with a l i m i t e d number of s p e c i f i c b i n d i n g r e c e p t o r s p r e s e n t i n the c y t o s o l . I t i s not known a t p r e s e n t i f these are f r e e l y d i s p e r s e d throughout the cytoplasm or a t t a c h e d to membrane s t r u c t u r e s . A f t e r i t s f o r m a t i o n , the s t e r o i d - r e c e p t o r complex i s able to p e n e t r a t e the n u c l e a r membrane and bin d to s p e c i f i c s i t e s on the chromatin of the c e l l n u c l e u s . In t h i s manner the g l u c o c o r t i c o i d - r e c e p t o r complexes are thought to add a r e g u l a t o r y element which can then modify RNA t r a n s c r i p t i o n or some p o s t - t r a n s c r i p t i o n a l event ( 7 8 ) . A c u r r e n t model concerning the i n i t i a l i n t e r a c t i o n between g l u c o c o r t i c o i d s and c y t o p l a s m i c r e c e p t o r s p o s t u l a t e s t h a t the r e c e p t o r i s an a l l o s t e r i c p r o t e i n with a c t i v e and i n a c t i v e con-f o r m a t i o n s . In the absence of s t e r o i d s the r e c e p t o r i s p r e s e n t mainly i n the i n a c t i v e form. A b i o l o g i c a l l y a c t i v e g l u c o c o r t i -c o i d w i l l b i n d s p e c i f i c a l l y to the a c t i v e conformation of the r e c e p t o r , thereby f a v o r i n g a s h i f t of the a l l o s t e r i c e q u i l i b r i u m to the a c t i v e form ( 7 1 ) . In t h i s way more complex i s formed u n t i l e i t h e r the s t e r o i d or the r e c e p t o r has been d e p l e t e d . I n -t e r a c t i o n of these complexes with the n u c l e a r chromatin w i l l r e s u l t i n a maximal g l u c o c o r t i c o i d response by the t a r g e t c e l l . The i n a c t i v e c o r t i c o s t e r o i d s d i s p l a y no a f f i n i t y f o r e i t h e r con-f o r m a t i o n of the p r o t e i n and t h e r e f o r e have no e f f e c t on t h i s system. In c o n t r a s t , g l u c o c o r t i c o i d a n t a g o n i s t s w i l l bind p r e -f e r e n t i a l l y to the i n a c t i v e c o n formation of the r e c e p t o r and form a n o n - f u n c t i o n i n g complex. T h i s w i l l s h i f t the e q u i l i b r i u m ( 7 2 ) , r e s u l t i n g i n d e p l e t i o n of a c t i v e r e c e p t o r molecules and p r e v e n t i o n of the b i o l o g i c response even i n the presence of a c t i v e g l u c o c o r t i c o i d s . Some experimental evidence to support t h i s model has been o b t a i n e d u s i n g the i n d u c t i o n of t y r o s i n e a m i n o t r a n s f e r a s e by g l u c o c o r t i c o i d s i n r a t hepatoma t i s s u e c u l t u r e c e l l s ( 7 2 ) . The a c t i v e g l u c o c o r t i c o i d s were observed to bind to the r e c e p t o r s w i t h an a f f i n i t y which i s d i r e c t l y r e l a t e d to t h e i r a b i l i t y to induce t y r o s i n e a m i n o t r a n s f e r a s e . A n t i - i n d u c e r s such as p r o -gesterone and 17 Oi -methyl t e s t o s t e r o n e bind to the r e c e p t o r s w i t h an a f f i n i t y p r e d i c t e d from t h e i r c a p a c i t y to i n h i b i t en-zyme i n d u c t i o n . The k i n e t i c s of s t e r o i d b i n d i n g and d i s s o c i a t i o n 21 a t 37 C a r e a l s o r a p i d enough to a c c o u n t f o r the k i n e t i c s of enzyme i n d u c t i o n and d e i n d u c t i o n . The P r e s e n t Problem The f o c u s of t h i s s t u d y c o n c e r n s an a t t empt to e l u c i d a t e the b i o c h e m i c a l mechanisms i n v o l v e d i n g l u c o c o r t i c o i d hormone a c t i o n . The i n f l u e n c e of t h e s e hormones i n the e n d o c r i n o l o g y of pregnancy was i n v e s t i g a t e d u s i n g s e v e r a l s t r a i n s of i n b r e d p r e g n a n t mice. D i f f e r e n c e s i n the r e s p o n s e to c o r t i c o s t e r o i d s of s e v e r a l s t r a i n s of mice have been a t t r i b u t e d t o d i f f e r e n c e s i n m e t a b o l i s m o f the s t e r o i d s . The A / j s t r a i n has b e e r , shown to be p a r t i c u l a r l y s u s c e p t i b l e to i n d u c t i o n o f c l e f t p a l a t e and death of a h i g h p r o p o r t i o n of f e t u s e s when p r e g n a n t mice were t r e a t e d w i t h g l u c o c o r t i c o i d s ( 7 9 ) - T h i s was a t t r i b u t e d to the g r e a t e r uptake of i n j e c t e d s t e r o i d by f e t u s e s o f the A / j s t r a i n as compared w i t h l e s s s e n s i t i v e s t r a i n s (80, 8 1 ) . C o r r e c t i o n of a c o n g e n i t a l eye anomaly i n mutant mice by a d m i n i s t r a t i o n of c o r t i s o n e (82) a l s o c o r r e l a t e d w i t h h i g h e r uptake of l a b e l e d s t e r o i d ( 8 3 ) by one of the mutant l i n e s . W h i l e the amount of s t e r o i d r e a c h i n g f e t a l t i s s u e s can v a r y w i t h the s t r a i n , f a c t o r s o t h e r t h a n the d i s p o s i t i o n of the s t e r o i d must i n f l u e n c e the r e s p o n s e of the f e t u s . The w e l l-documented e f f e c t s of g l u c o c o r t i c o i d s on g l u c o s e m e t a b o l i s m l e d to the con-s i d e r a t i o n t h a t the a v a i l a b i l i t y of g l u c o s e t o the f e t u s might p o s s i b l y be m o d i f i e d by c o r t i c o s t e r o i d t r e a t m e n t . S i n c e g l u c o s e i s the major n u t r i e n t w h i c h the f e t u s r e c e i v e s f r o m the mother, t h i s i n f l u e n c e o f the s t e r o i d s would have i m p o r t a n t consequences i n both f e t a l development and s u r v i v a l . 22 A corticoid effect on glucose transport to the fetus would indicate a steroid influence on placental function, since i t is mainly responsible for the transmission of nutrients from mother to fetus ( 4 4 ) . The placenta might then be a good target tissue for investigation of the site(s) of specific corticosteroid action. MATERIALS AND METHODS Chemicals The f o l l o w i n g chemicals were purchased from the Sigma Chemical Company: C o r t i s o l , c o r t i s o n e , progesterone, c o r t i -c o s terone, d i t h i o t h r e i t o l , and N-ethyl maleimide. Dexamethasone (Decadron powder) was the g i f t of Merck and Co. to Dr. M. Dar-r a c h i n t h i s department. Cortexolone was o b t a i n e d from N u t r i -t i o n a l B i o c h e m i c a l s Corp; 1 1 - e p i c o r t i s o l , bovine serum albumin, and human 7 g l o b u l i n were a l l from Mann Research Labs; and 11-e p i c o r t i c o s t e r o n e was a g i f t from Dr. J . Babcock of The Upjohn Company, Kalamazoo, Michigan. Pronase (45,000 PUK units/mg), deoxyribonuclease (42 , 3 0 0 Dornase units/mg, RNAse content = 0.102$), r i b o n u c l e a s e ( 4 7 K u n i t z units/mg), a c t i n o m y c i n D, and p-c h l o r o m e r c u r i b e n z o a t e were a l l o b t a i n e d from Calbiochem. PP0 and P0P0P ( s c i n t i l l a t i o n grade) and B i o - S o l v s o l u b i l i z e r f o r m u l a BBS - 3 were a l l from Beckman Instruments, The G l u c o s t a t reagent was o b t a i n e d from Worthington B i o c h e m i c a l Corp. and Nembutal (sodium p e n t o b a r b i t a l ) was purchased from Abbott L a b o r a t o r i e s L t d . , M ontreal. The sesame o i l used (U.S.P. grade) was from Bush Boake A l l e n . A l l other chemicals used were of reagent grade and were purchased from F i s h e r Chemical Co., Vancouver. S o l v e n t s A l l s o l v e n t s used were of reagent grade and were p u r i f i e d by d i s t i l l a t i o n before use. Materials S c i n t i l l a t i o n v i a l s (low potassium glass) and p l a s t i c caps were from Fraser Medical Supplies, Vancouver. Pharmacia sup-p l i e d the Sephadex G-25 (coarse grade). Radiochemicals The following radiochemicals were purchased from New Eng-land Nuclear Corporation, with the indicated s p e c i f i c a c t i v i t i e s : 4- C - c o r t i s o l (52 mCi/mmole), 1,2--^H-cortisone (51.6 Ci/mmole) i r-^C-progesterone (52.8 mCi/mmole) , and u r e i d o - ^ C - c i t r u l l i n e (4.28 mCi/mmole). Amersham/Searle Corporation supplied the re-3 14 mainder: 1, 2- H-corticosterone (36 Ci/mmole), U- C-D-glucose (288 mCi/mmole), and l - ^ C - a c e t i c acid (52.9 mCi/mmole). A l l radiochemicals were used upon r e c e i p t and were examined or p u r i -f i e d r o u t i n e l y by TLC or paper chromatography. Animals The A/j and C57BL/6J inbred st r a i n s of mouse were purchased from Jackson Memorial Laboratories, Bar Harbor, Me. Pregnant females usually arrived between day 10-13 of pregnancy. The SWV mice were randomly bred using the f a c i l i t i e s of the Cancer Research Centre at t h i s university. A l l animals were fed a di e t of Purina Breeder Chow and water ad l i b . For experiments requ i r i n g timed pregnancies the date of f i n d i n g the vaginal plug was designated as day zero of pregnancy and a l l experiments were performed on the morning of the 18th day. Methods 1. Steroid i n j e c t i o n s into pregnant mice An intra p e r i t o n e a l i n j e c t i o n of the st e r o i d , suspended i n 25 sesame o i l , was made i n t o pregnant mice while c o n t r o l s received the same volume of sesame o i l only. One hour l a t e r 5 uCi of l a b e l e d precursor d i s s o l v e d i n 0.1 ml p h y s i o l o g i c a l s a l i n e ( 0 . 1 5 M ) was i n j e c t e d subcutaneously i n t o the upper abdomen. A f t e r an-other 15 min each mouse was s a c r i f i c e d and the f e t u s e s were q u i c k l y e x c i s e d , weighed, and placed on i c e . 2. S t e r o i d i n j e c t i o n s i n t o the f e t u s Pregnant SWV mice ( g e s t a t i o n a l day 18, weight 4 5 - 5 0 gm) were ane s t h e t i z e d by i n j e c t i o n of 0.1 ml sodium p e n t o b a r b i t a l s o l u t i o n ( 1 5 mg/ml i n p h y s i o l o g i c a l s a l i n e ) subcutaneously and maintained w i t h a d d i t i o n a l doses when r e q u i r e d . The peritoneum was c a r e f u l l y opened to expose the fet u s e s which were v i s i b l e through the ute-r i n e membrane. I n j e c t i o n s of v a r y i n g doses of dexamethasone sus-pended i n 0 . 0 5 ml sesame o i l were made i n t o the f e t u s e s i n utero. The f e t u s e s were then r e s t o r e d i n t o the abdominal c a v i t y and both the p e r i t o n e a l membrane and s k i n were sutured. One-half hour 14 l a t e r 5 uCi of U- C-D-glucose d i s s o l v e d i n 0.1 ml p h y s i o l o g i c a l s a l i n e was i n j e c t e d subcutaneously i n t o the upper abdomen of the mother. A f t e r another 15 min each mouse was k i l l e d by c e r v i c a l d i s l o c a t i o n and the fet u s e s were again removed, weighed and placed on i c e . 3. I s o l a t i o n of a c i d - s o l u b l e f r a c t i o n of t i s s u e s E i t h e r whole fe t u s e s or i n d i v i d u a l t i s s u e s were f i n e l y minced and homogenization was accomplished i n p h y s i o l o g i c a l s a l i n e ( 5 ml/gm wet wt t i s s u e ) using a motor-driven T e f l o n P o t t e r - E l v e h -jem t i s s u e g r i n d e r . The heavy residue was removed by c e n t r i f u -g a t i o n at 1200xg f o r 5 min and 1 ml of 60% (w/v) TCA was added 26 to every 5 mi of saline supernatant. Small molecules such as monosaccharides, amino acids, and nucleotides remain soluble iri the d i l u t e TCA solu t i o n but oligopeptides and other components of s i m i l a r size are completely p r e c i p i t a t e d (84). After c e n t r i -fugation at 1200xg f o r 10 min, the acid-soluble supernatant was decanted into a test tube and the r a d i o a c t i v i t y determined. The r a t i o of acid-insoluble to acid-soluble counts recovered did not d i f f e r from one experiment to another (Table I ) . 4. Determination of r a d i o a c t i v i t y An aliquot of the sample to be counted was placed into a s c i n t i l l a t i o n v i a l and 10 ml of s c i n t i l l a t i o n f l u i d consisting of 4 gm PPO and 100 mg P0P0P per l i t e r of toluene ( 8 5 ) was added. Bio-Solv s o l u b i l i z e r formula BBS-3 was also added to the samples u n t i l the s o l u t i o n appeared clear. The samples were then assay-ed f o r r a d i o a c t i v i t y using a Packard Tri-Carb l i q u i d s c i n t i l -l a t i o n spectrometer (Model 3 0 0 3 ) . Sample quenching was monitored by the channels-ratio method ( 8 6 ) using a commercially available quench set (Amersham/Searle). The counting e f f i c i e n c y f o r "^C was approximately 78$ while the JK counting e f f i c i e n c y was 3 2 $ . 5 . Semi-micro determination of blood glucose Blood samples were obtained from the throat, 0 . 4 ml blood added to 0.1 ml sodium c i t r a t e (28 mg/ml). A 1:40 Somogyi f i l -t rate ( 8 7 ) was then prepared by adding 0.1 ml of the whole blood to 1 . 9 ml d i s t i l l e d water, mixing, and adding 1.0 ml 1.8$ Ba (OH)2' 8H" 20. After thorough mixing, 1.0 ml 2$ ZnSO^. 7H"20 was added and the solu t i o n was mixed and then centrifuged to remove the p r e c i p i t a t e . 2? TABLE I. Repre from sentative values of f e t a l extracts r a d i o a c t i v i t y recovered Dexamethasone dose (ug) Total counts recovered (dpm) Acid-insoluble ^ Acid-soluble Ratio b (a./b) Control 1 0 , 3 5 0 3 5,400 0. 293 1 2 , 2 1 0 38 ,280 0 . 3 1 9 50 14 , 1 0 0 4 5 , 8 0 0 0 . 3 0 8 1 2 , 3 2 0 42 , 0 0 0 0 . 2 9 4 2 0 0 1 2 , 1 5 0 38,800 0 . 3 1 4 1 1 , 2 0 0 36,750 0 . 3 0 5 Pregnant mice were injected with various doses of dexameth-asone and labeled glucose as described i n Methods. The fetuses were removed, and the acid-soluble f r a c t i o n f o r each was isola t e d using the procedure indicated i n Methods. The acid-insoluble f r a c t i o n f o r each was also retained and s o l u b i l i z e d by heating at 1 0 0 ° f o r 60 min i n 1 ml 5N KOH. The alk a l i n e solutions were then neutralized by addition of 5N p e r c h l o r i c acid and radioact-i v i t y was determined on aliquots of both f r a c t i o n s . G l u c o s t a t i s a p r e p a r e d r e a g e n t f o r the q u a n t i t a t i v e , c o l -o r i m e t r i c d e t e r m i n a t i o n of g l u c o s e which makes use of two c o u p l e d r e a c t i o n s c a t a l y z e d by g l u c o s e o x i d a s e and a p e r o x i d a s e enzyme ( 8 8 ) . The G l u c o s t a t r e a g e n t c o n s i s t i n g of enzymes and chromogen was d i s s o l v e d i n 50 ml d i s t i l l e d w a t e r and 2 ml of t h i s was added to 2 ml of each b l o o d f i l t r a t e sample o r g l u c o s e s t a n d a r d . A f t e r a 10 min i n c u b a t i o n a t room t e m p e r a t u r e the r e a c t i o n was s t o p p e d by a d d i t i o n of 1 drop of 4 N H C l . A f t e r a f u r t h e r 5 min the ab-s o r b a n c e was d e t e r m i n e d a t 420 nm. 6. I s o l a t i o n of n u c l e a r s t e r o i d - r e c e p t o r complex P r e g n a n t SWV mice ( g e s t a t i o n a l day 13 t o 18) were k i l l e d by c e r v i c a l d i s l o c a t i o n and the p l a c e n t a s removed and p l a c e d on i c e . E x t r a n e o u s t i s s u e was trimmed away and two p l a c e n t a s p e r sample were t h e n l i g h t l y homogenized w i t h 2 t o 3 s t r o k e s of a l o o s e - f i t -t i n g t i s s u e g r i n d e r i n 2 ml. KEP b u f f e r , pH 7.4, c o n t a i n i n g 2.8 mM g l u c o s e . R a d i o a c t i v e s t e r o i d d i s s o l v e d i n e t h a n o l , a l o n e o r w i t h n o n - r a d i o a c t i v e s t e r o i d s ( a l s o d i s s o l v e d i n e t h a n o l ) , was added and the samples l e f t on i c e f o r 15 min w i t h o c c a s i o n a l a g i t a t i o n . The samples were t h e n i n c u b a t e d a t 37°C f o r 10 min i n a s h a k i n g w a t e r b a t h , p u t back on i c e and d i l u t e d by a d d i t i o n of 50 ml i c e c o l d 1.5 mM MgClg. T h i s p r o c e d u r e has been used by W i r a and Munck (64) to s h a t t e r c e l l membranes, d i s r u p t i n g c y t o -p l a s m i c m a t e r i a l but l e a v i n g n u c l e i i n t a c t ( F i g . 1). A f t e r s i t -t i n g on i c e f o r 10 to 15 min, the e x c e s s s u p e r n a t a n t f l u i d was d e c a n t e d o f f and each sample was c e n t r i f u g e d a t 1200xg f o r 10 min a t 6°C. The r e s i d u e of n u c l e i was washed t w i c e w i t h 10 ml i c e c o l d 1.5 mM M g C l 9 and r e s u s p e n d e d i n 0.7 ml of c o l d b u f f e r 29 Fig. 1. Nuclei isolated using hypotonic MgCl 2 Placental tissue was homogenized with a loose-f i t t i n g tissue grinder in 2 ml KEP buffer (pH 7.2*. with 2.8 mM glucose), then diluted with 50 ml ice cold 1.5 mM MgCl2» The prepared nuclei were harvested by centrifu-gation at 1200xg and resuspended in freshly prepared 5$ glutaraldehyde in 1.5 mM MgCl2« Smears were prepared and stained with toluidine blue. X 640 31 composed of 0 . 6 M K C l , 10 mM TRIS, and 1 . 5 mM EDTA, pH 8.0. The n u c l e i were t h e n homogenized w i t h 8 t o 10 s t r o k e s of a t i g h t -f i t t i n g Ten-Broeck ground g l a s s homogenizer and c e n t r i f u g e d a t 1 0 5 , 0 0 0 x g f o r 10 min a t 0 t o 4°C i n a r e f r i g e r a t e d u l t r a c e n t r i -f u g e . An a l i q u o t ( u s u a l l y 0 . 5 ml) o f t h i s n u c l e a r f r a c t i o n was the n removed and p l a c e d on 11 x 180 mm columns of Sephadex G - 2 5 u s i n g 0 . 6 M K C l b u f f e r w i t h 10 mM TRIS and. 1 . 5 mM EDTA, pH 8.0. The e l u t i o n s were c a r r i e d o u t a t 6°C and the peak of bound ma-t e r i a l c o u l d be c o l l e c t e d w i t h i n 10 min a f t e r a p p l i c a t i o n of the sample. F r a c t i o n s of a p p r o x i m a t e l y 1 ml were c o l l e c t e d and the m a c r o m o l e c u l a r peak, was i d e n t i f i e d by p r o t e i n d e t e r m i n a t i o n s u s i n g the method of Lowry, Rosebrough, F a r r , and R a n d a l l ( 8 9 ) . R a d i o a c t i v e d e t e r m i n a t i o n s were made on 0 . 1 ml a l i q u o t s i n the same manner as p r e v i o u s l y s t a t e d . 7. C y t o s o l exchange a s s a y P r e g n a n t S W V mice ( g e s t a t i o n a l day 13 to 18) were k i l l e d and the p l a c e n t a s removed and trimmed as b e f o r e . The p o o l e d t i s s u e ( 7 t o 11 p l a c e n t a s ) was f i n e l y minced and washed t h r e e t i m e s w i t h 10 ml i c e c o l d KEP b u f f e r , pH 7 . 4 , c o n t a i n i n g 2.8 mM g l u c o s e . The t i s s u e was t h e n r e s u s p e n d e d i n the KEP b u f f e r (5 ml/gm wet wt t i s s u e ) and homogenized u s i n g a T e f l o n P o t t e r - E l v e h j e m homoge-n i z e r (8 t o 10 s t r o k e s ) . The p r e p a r a t i o n was c e n t r i f u g e d a t 1 2 0 0 x g f o r 10 min a t 6°C and the s u p e r n a t a n t was r e c e n t r i f u g e d a t 1 0 5 , 0 0 0 x g f o r 60 min a t 0 to 4°C i n a r e f r i g e r a t e d u l t r a c e n t -r i f u g e t o o b t a i n the c y t o s o l f r a c t i o n . The c y t o s o l exchange a s -say c o n s i s t e d of the i n c u b a t i o n of a l i q u o t s of the c y t o s o l f r a c -t i o n w i t h - ^ H - c o r t i c o s t e r o n e ( 1 3 « 8 nM) d i s s o l v e d i n e t h a n o l i n 32 two series. The f i r s t series had 200 uM unlabeled corticoste-rone dissolved in ethanol added while the second set, contained 2 0 0 uM unlabeled epicorticosterone (also dissolved in ethanol). After incubation, 0 . 5 ml aliquots of the samples were placed on columns of Sephadex G-25 at 6°C using KEP buffer, pH 7 . 4 , with 2 . 8 mM glucose. The protein and radioactivity determinations on 1 ml fractions were assayed as before. Specific binding in this case represented the difference between the binding in the presence of epicorticosterone and corticosterone ( 9 0 ) . 8. Other procedures Other procedures, used less frequently, w i l l be described as they are required in the following experiments. 3 3 RESULTS S t e r o i d E f f e c t s on F e t a l Glucose Metabolism The g l u c o c o r t i c o s t e r o i d s have been r e p o r t e d to i n f l u e n c e f e t a l v i a b i l i t y , so both n a t u r a l and s y n t h e t i c c o r t i c o s t e r o i d s were employed to determine t h e i r e f f e c t s on the f e t u s . In p r e -l i m i n a r y experiments the f i n d i n g s of P i n s k y and DiGeorge ( 7 9 ) were confirmed. I n j e c t i o n of a sesame o i l suspension of dexa-methasone (see Appendix I I f o r s t r u c t u r a l formula) i n t o a p r e g -nant mouse r e s u l t e d i n death of a l l the f e t u s e s . With the A / j s t r a i n t h i s was accomplished u s i n g a t o t a l dose of s t e r o i d as low as 6 0 0 ug over three days (Table I I ) . In many cases where a h i g h dosage of dexamethasone had been maintained over a number of days, r e s o r p t i o n of the f e t u s e s was e v i d e n t ; they were d i s -c o l o r e d and emaciated. Only i n one i n s t a n c e was there no apparent e f f e c t of dexamethasone on f e t a l v i a b i l i t y . In t h a t animal the i n j e c t i o n s c o n t a i n e d a moderate dose ( 5 0 0 ug) but were onl y g i v e n f o r one day a t a very l a t e stage of pregnancy. Using C o r t i s o l t h e r e was no apparent e f f e c t even with a t o t a l dose as high as 8 mg which i s s i m i l a r to the d a t a of P i n s k y and DiGeorge which r e p o r t e d a much lower i n c i d e n c e of c l e f t p a l a t e u s i n g C o r t i s o l . A. P r e l i m i n a r y experiments on p r e c u r s o r i n c o r p o r a t i o n The i n f l u e n c e of dexamethasone treatment upon the f e t a l meta-b o l i s m of two r a d i o a c t i v e p r e c u r s o r s , a c e t a t e and g l u c o s e , was examined i n order to i n v e s t i g a t e the l e t h a l e f f e c t s of the s t e -r o i d . U s i n g a c e t a t e there appeared to be no d i f f e r e n c e i n the counts r e c o v e r e d i n a chloroform-methanol e x t r a c t of the s t e r o i d -34 TABLE I I . E f f e c t of g l u c o c o r t i c o i d t r e a t m e n t on f e t a l v i a b i l i t y Mouse Dosage per No. of G e s t a t i o n a l F e t u s e s s t r a i n i n j e c t i o n d a i l y p e r i o d •# dead i n j e c t i o n s (day) t o t a l 500 ug D 2 12 t o 15 11/11 500 ug D 2 14 t o 17 8/8 2 mg D 1 17 to 18 9/9 100 ug D 2 12 to 15 8/8 100 ug D 2 14 t o 17 7/7 500 ug D 2 18 to 19 0/9 2 mg D 1 12 t o 15 11/11 2 mg D 1 14 t o 18 7/7 1 mg D 1 16 to 19 10/10 2 mg D 1 16 t o 19 9/9 (0.5 ml o i l ) 2 15 t o 18 0/10 4 mg F 2 16 t o 17 0/12 50 ug F 2 15 t o 17 0/9 100 ug F 2 14 t o 17 1/9 P r e g n a n t SWV and A / J mice were t r e a t e d w i t h i . p . i n j e c t i o n ( s ) of s t e r o i d (D=dexamethasone, P = c o r t i s o l ) d i s s o l v e d i n 0.5 ml sesame o i l d u r i n g the i n d i c a t e d g e s t a t i o n a l p e r i o d . On the morning of the l a s t day i n d i c a t e d the mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and the f e t u s e s were removed and exam-i n e d . 35 t r e a t e d f e t u s e s when compared w i t h sesame o i l - t r e a t e d c o n t r o l s s i n c e r e c o v e r i e s f r om the i n d i v i d u a l t i s s u e s , a l t h o u g h d i f f e r e n t , d i d not c o r r e l a t e w i t h t r e a t m e n t ( T a b l e I I I ) . When l a b e l e d g l u c o s e was used, a d e c r e a s e i n i n c o r p o r a t i o n of l a b e l i n t o a l l the t i s s u e s examined was e v i d e n t , r e s u l t i n g i n a 53% drop i n the t o t a l c o u n t s r e c o v e r e d from the f e t a l t i s s u e s of the d e x a m e t h a s o n e - t r e a t e d a n i m a l compared to the c o n t r o l s . A l t h o u g h the a c t u a l c o u n t s f o r the f e t a l b r a i n t i s s u e were a l s o d e c r e a s e d by dexamethasone i n j e c t i o n , the p e r c e n t a g e drop v/as l e s s t h a n f o r the o t h e r t i s s u e s . I n f a c t , the f e t a l b r a i n a c t u a l -l y i n c r e a s e d i n the p r o p o r t i o n o f r e c o v e r e d t o t a l c o u n t s f r o m 14% f o r c o n t r o l s to 23% a f t e r dexamethasone a d m i n i s t r a t i o n . The d e c r e a s e i n t o t a l r a d i o a c t i v i t y r e c o v e r e d as w e l l as the change i n d i s t r i b u t i o n of the c o u n t s , s u g g e s t e d t h a t the s t e r o i d was a f -f e c t i n g b o t h the c a r b o h y d r a t e m e t a b o l i s m of the f e t u s i t s e l f as w e l l as t r a n s p o r t o f the l a b e l e d g l u c o s e f r o m the m a t e r n a l c i r -c u l a t i o n t o the f e t u s . B. Dexamethasone e f f e c t s on g l u c o s e i n c o r p o r a t i o n i n t o the a c i d - s o l u b l e f r a c t i o n of f e t a l t i s s u e s  I t was d e c i d e d t h a t the a c i d - s o l u b l e f r a c t i o n of f e t a l t i s -sues would most c l o s e l y r e f l e c t f r e e g l u c o s e t r a n s p o r t e d t o the f e t u s (84). The e f f e c t of dexamethasone t r e a t m e n t on the c o u n t s r e c o v e r e d i n t h i s f r a c t i o n were d e t e r m i n e d u s i n g p r e g n a n t A / j mice. Dexamethasone a t t h r e e c o n c e n t r a t i o n s was used ( T a b l e I V ) . The c o u n t s i n c o r p o r a t e d i n t o the f e t u s e s o f the s t e r o i d - t r e a t e d a n i m a l s i n a l l c ases were i n c r e a s e d over t h o s e o f the c o n t r o l f e t u s e s s i n c e the c o u n t s r e c o v e r e d i n the i n d i v i d u a l t i s s u e s were g e n e r a l l y a l l h i g h e r f o r the s t e r o i d - t r e a t e d a n i m a l s . However, 36 TABLE I I I . E f f e c t of dexamethasone upon the i n c o r p o r a t i o n of l a b e l e d a c e t a t e o r g l u c o s e i n t o a c h l o r o f o r m -methanol e x t r a c t of SWV mouse f e t a l t i s s u e s A c e t a t e G l u c o s e T i s s u e C o n t r o l Dexamethasone C o n t r o l Dexamethasone B r a i n 2.6 3.1 14 . 1 10.9 L i v e r 9.5 7.2 11.0 4 . 5 Gut 6.0 5.2 6.7 3.1 Lung 3-5 6.0 11.1 3.0 C a r c a s s 2.1 2.0 6.8 2.9 # F e t u s e s 9 5 13 9 # A n i m a l s T I " 2 I P r e g n a n t SWV mice ( g e s t a t i o n a l day 18) were t r e a t e d w i t h an i . p . i n j e c t i o n of 1 mg dexamethasone suspended i n 0.1 ml sesame o i l ( c o n t r o l s r e c e i v e d 0.1 ml sesame o i l ) 24 hour s "before-hand. A s.c. i n j e c t i o n of e i t h e r l a b e l e d a c e t a t e or g l u c o s e was made and 1 hour l a t e r the a n i m a l s were k i l l e d and the f e t u s e s were removed, weighed, and p l a c e d on i c e . The f e t a l b r a i n , l i v e r , g u t , and l u n g were e x c i s e d f r o m the c a r c a s s and each t i s s u e was p o o l e d and weighed. The t i s s u e s were homogenized i n p h y s i o l o g i c -a l s a l i n e (5 ml/gm wet wt t i s s u e ) , t h e n e x t r a c t e d t w i c e w i t h an e q u a l volume of c h l o r o f o r m - m e t h a n o l (2:1) and the r a d i o a c t i v i t y of the e x t r a c t was d e t e r m i n e d . The f i g u r e s r e p r e s e n t c o u n t s c o r -r e c t e d f o r the t i s s u e w e i g h t (dpm/mg wet wt t i s s u e ) . 37 TABLE IV. E f f e c t of v a r i o u s doses of dexamethasone upon the i n c o r p o r a t i o n of l a b e l e d g l u c o s e i n t o A / j mouse f e t a l t i s s u e s D o s e (ug) Tissue 0 5 0 100 200 B r a i n 1 6 . 0 2 3 - 3 18. 3 1 7 . 3 L i v e r 2 3 - 3 3 3 - 6 24.2 2 6 . 8 Gut 2 2 . 3 22.4 2 5 . 3 1 9 . 3 Lung 2 2 . 3 3 3 . 2 3 6 . 7 2 7 . 0 C a r c a s s 20. 3 3 8 . 3 2 9 . 6 24.0 # F e t u s e s 14 8 1 11 # A n i m a l s 2 1 1 1 P r e g n a n t A/.J mice were i n j e c t e d w i t h v a r i o u s doses of dexamethasone and w i t h l a b e l e d g l u c o s e as d e s c r i b e d i n Methods. The f e t a l b r a i n , l i v e r , g u t , and l u n g were e x c i s e d f r o m the c a r -c a s s and each t i s s u e was p o o l e d and weighed. A c i d - s o l u b l e r a d i o -a c t i v i t y was d e t e r m i n e d f o r each t i s s u e and the f i g u r e s r e p r e s e n t c o u n t s c o r r e c t e d f o r the t i s s u e w e i g h t (dpm/mg wet wt t i s s u e ) . 38 t h e r e was a n e g a t i v e c o r r e l a t i o n w i t h dosage o f s t e r o i d . The r e c o v e r e d c o u n t s were h i g h e s t i n f e t u s e s of mice t r e a t e d w i t h o n l y 50 ug dexamethasone whereas the 200 ug s t e r o i d - t r e a t e d samples had r e c o v e r e d r a d i o a c t i v i t i e s o n l y s l i g h t l y above the c o n t r o l v a l u e s . V a r i o u s doses of dexamethasone were used to d e t e r m i n e the dose - r e s p o n s e r e l a t i o n s h i p between the s t e r o i d and the i n c o r p o r -a t i o n of l a b e l e d g l u c o s e i n t o mouse f e t u s e s . F i g . 2 shows t h a t the two e f f e c t s a re e v i d e n t o ver the c o n c e n t r a t i o n range i n v e s -t i g a t e d when the A / j s t r a i n was used. A t the l o w e r s t e r o i d doses ( 5 0 and 1 0 0 ug) t h e r e were h i g h e r c o u n t s r e c o v e r e d i n t r e a t e d f e -t u s e s compared t o c o n t r o l s . However, the s l o p e of the cur v e i s n e g a t i v e , i m p l y i n g d e c r e a s i n g i n c o r p o r a t i o n w i t h i n c r e a s i n g s t e -r o i d dose, so t h a t a t the 3 0 0 ug dosage the r e c o v e r e d c o u n t s a re s i g n i f i c a n t l y below c o n t r o l v a l u e s ( P < 0 , 0 0 1 u s i n g t t e s t ) . Thus, t h e r e i s e v i d e n c e f o r i n h i b i t i o n o f i n c o r p o r a t i o n o f l a b e l e d g l u c o s e i n t o A / j f e t u s e s a t e v e r y c o n c e n t r a t i o n of s t e r o i d used. However, a t the l o w e r doses the i n h i b i t i o n appears t o be masked by an o p p o s i n g e f f e c t which promotes the t r a n s f e r of the l a b e l e d g l u c o s e . T h i s o p p o s i n g e f f e c t i s most l i k e l y due to the h y p e r -g l y c e m i a w h i c h r e s u l t s when g l u c o c o r t i c o i d s a r e a d m i n i s t e r e d ( 1 2 ) . The e l e v a t e d b l o o d g l u c o s e l e v e l s would i n c r e a s e the t r a n s f e r of g l u c o s e f r o m mother t o f e t u s s i n c e the g r a d i e n t d i f f e r e n c e would be i n c r e a s e d ( 4 5 ) . The t h r e e mouse s t r a i n s used were th e n compared a t the 2 0 0 ug dexamethasone dose and F i g . 3 shows the r e c o v e r i e s i n f e t u s e s r e l a t i v e t o c o n t r o l s of the same s t r a i n . A t t h i s dose the A / j 39 Fig. 2 . Effect of various doses of dexamethasone upon the incorporation of labeled glucose into A/j mouse fetuses Pregnant A/j mice were injected with various doses of dexamethasone and with labeled glucose as described i n Methods. Acid-soluble radioactivity was determined for each fetus to compute the corrected counts (dpm/mg wet wt fetus) which were then compared to controls ex-pressed as 100$. Each point represents the mean of at least 10 fetuses (and at least 3 animals) with the SEM indicated by the vertical lines. 40 Dexamethasone dose (jag) 41 Fig. 3 . Strain differences in the effect of dexamethasone upon the incorporation of labeled glucose into mouse fetuses Pregnant mice were injected with 200 ug dexa-methasone and with labeled glucose as described in Methods. Acid-soluble radioactivity was determined for each fetus to compute the corrected counts (dpm/mg wet wt fetus) which were then compared to controls of the same strain expressed as 100%. The vertical lines indicate SEM and the figures in brackets indicate the number of determinations. Acid-soluble counts in fetus relative to control (%) VJ1 CD O \J1 o c : C/i CD l/> «—«» O . * - « J v D TO — CO ^3 f e t u s e s e x h i b i t e d an i n c o r p o r a t i o n o f l a b e l which was e q u i v a l e n t t o the c o n t r o l v a l u e s (as shown i n F i g . 2 ) . However, the C 5 7 B L / 6 j f e t u s e s showed a c o n s i d e r a b l e i n c r e a s e i n l a b e l i n c o r p o r a t e d r e l a t i v e t o c o n t r o l s w h i l e c o n v e r s e l y , the SWV showed a d e c r e a s e . C o n t r o l v a l u e s among the s t r a i n s a l s o d i f f e r e d s i n c e the SWV f e -t u s e s y i e l d e d much l o w e r r e c o v e r i e s t h a n e i t h e r the A./j or C57BL-/ 6 j f e t u s e s ( T a b l e V ) . However, the same g e n e r a l dose-response seems t o h o l d f o r the SWV and C57BL/6J s t r a i n s as f o r the A / j s i n c e the l o w e r s t e r o i d doses r e s u l t e d i n i n c r e a s e d i n c o r p o r a t i o n whereas h i g h e r doses g e n e r a l l y caused a de c r e a s e i n the amount of l a b e l r e c o v e r e d . C. E f f e c t of n a t u r a l g l u c o c o r t i c o i d s The n a t u r a l l y - o c c u r r i n g g l u c o c o r t i c o i d s , C o r t i s o l and c o r t i -c o s t e r o n e , were used t o determine i f th e y would have the same e f -f e c t on i n c o r p o r a t i o n of l a b e l e d g l u c o s e as the s y n t h e t i c s t e r o i d . T a b l e VI shows t h a t a l t h o u g h an i n c r e a s e d i n c o r p o r a t i o n of l a b e l i s a p p a r e n t , t h e r e i s no e v i d e n c e of i n h i b i t i o n s i m i l a r t o t h a t caused by dexamethasone even when t h e n a t u r a l s t e r o i d s were used a t doses as h i g h as 5 mg.. Both the n a t u r a l and s y n t h e t i c c o r t i -c o s t e r o i d s w i l l cause h y p e r g l y c e m i a and i n the absence o f o t h e r f a c t o r s , t h i s can e x p l a i n the i n c r e a s e d c o u n t s r e c o v e r e d i n the f e t u s . I n e l i c i t i n g o t h e r e f f e c t s , however, the n a t u r a l and s y n -t h e t i c c o r t i c o s t e r o i d s do n o t have e q u a l a c t i v i t i e s . Dexametha-sone has been r e p o r t e d t o produce twenty t i m e s as much l i v e r g l y -cogen d e p o s i t i o n as does C o r t i s o l i n the mouse ( 7 9 ) . T h i s i s s i m i l a r t o the d i f f e r e n c e i n a c t i v i t y e n c o u n t e r e d when the thymus i n v o l u t i o n a s s a y i s used ( T a b l e V I I ) . However, i f granuloma i n -h i b i t i o n or a n t i - i n f l a m m a t o r y r e s p o n s e i s used as the i n d e x of 44 TABLE V. S t r a i n differences i n the e f f e c t of various doses of dexamethasone upon the incorporation of labeled glucose into mouse fetuses Steroid A/j SWV C 5 7 P L / 6 J dose (ug) 0 ( 3 ^ . 6 + 1 . 2 ( 1 2 , 3 ) 2 5 . 8 + 0 . 7 ( 8 , 2 ) 3 9 . 7 + 1 . 3 ( 8 , 2 ) 50 4 8 . 7 + 1 . 5 ( 1 5 , 4 ) 2 8 . 8 + 2 . 3 ( 3 , 1 ) 6 8 . 4 . . ( 2 , 1 ) 1 0 0 4 2 . 3 + 1 . 1 ( 1 1 , 2 ) 3 3 . ^ + 2 . 1 ( 5 . 1 ) 2 0 0 3 2 . 8 + 0 . 8 ( 8 , 2 ) 1 0 . 5 + 0 . 3 ( 9 , 2 ) 6 1 . 0 + 6 . 4 ( 9 , 3 ) 3 0 0 2 8 . 8 + 1 . 2 ( 1 0 , 2 ) — 2 6 . 0 + 0 . 6 ( 4 , 1 ) Pregnant mice were injected with various doses of dexamethasone and labeled glucose as described i n Methods. Acid-soluble r a d i o a c t i v i t y was determined f o r each fetus and fig u r e s represent mean of corrected counts (dpm/mg wet wt fetus) + SEM. The f i g u r e s i n brackets represent the number of fetuses and anim-a l s , r e s p e c t i v e l y . \ 45 TABLE V I . E f f e c t s of c o r t i c o s t e r o n e and C o r t i s o l u p o n the i n c o r p o r a t i o n o f l a b e l e d g l u c o s e i n t o SWV mouse f e t a l t i s s u e s T i s s u e C o n t r o l C o r t i - C o r t i - C o r t i s o l c o s t e r o n e c o s t e r o n e (5 mg) ( 2 0 0 ug) (1 mg) B r a i n 3160 3 0 2 0 5140 4 0 8 0 L i v e r 5 4 6 0 6 3 5 0 8 5 6 0 7200 Gut 3570 3310 3520 3 8 3 0 Lung 2 ? 2 0 2 7 6 0 3340 3470 C a r c a s s 1 2 , 6 5 0 13,820 1 5 , 7 5 0 1 9 , 5 0 0 # F e t u s e s 18 1 0 7 9 # A n i m a l s 2 1 1 1 P r e g n a n t SWV mice were i n j e c t e d w i t h c o r t i c o s t e r o n e or C o r t i s o l and l a b e l e d g l u c o s e as d e s c r i b e d i n Methods. The f e t a l b r a i n , l i v e r , g u t , and l u n g were e x c i s e d f r o m t h e c a r c a s s and each t i s s u e was p o o l e d . A c i d - s o l u b l e r a d i o a c t i v i t y was de-t e r m i n e d f o r each t i s s u e and the f i g u r e s r e p r e s e n t t o t a l c o u n t s c o r r e c t e d f o r the number of f e t u s e s . The f e t a l w e i g h t s i n t h i s e x p e r i m e n t were a l l 950 + 85;<mg. 46 T A B L E V I I . R e l a t i v e a c t i v i t y of s t e r o i d s on thymus i n v o l u t i o n 3 S t e r o i d Mean thymus R a t i o x 10-w e i g h t (mg) thymu.s/body wt C o n t r o l 63 4 . 1 6 + 0.16 400 ug c o r t i c o s t e r o n e 44 2 . 2 9 + 0 . 1 0 400 ug C o r t i s o l 37 . 2.18 + 0.08 20 ug dexamethasone 28 1 . 5 9 + 0 . 1 2 The r e l a t i v e a c t i v i t y of s t e r o i d s was compared u s i n g a thymus i n v o l u t i o n a s s a y d e s c r i b e d by Dorfman and Dorfman ( 9 1 ) . Young f e m a l e SWV mice, 15 to 20-gm each, were i n j e c t e d s.c. w i t h s t e r o i d i n o l i v e o i l . Two i n j e c t i o n s of 0 . 2 ml o i l were made 5 h r s a p a r t . Thymuses were removed and weighed 23 h r s a f t e r the f i r s t i n j e c t i o n . Each f i g u r e r e p r e s e n t s a mean of 6 t o 8 d e t e r m i n a t i o n s . 4 ? potency, the difference in activity is closer to two hundredfold (92). Similarly, the difference ir. activity of C o r t i s o l and dexamethasone in inducing cleft palate in the mouse was reported to be several hundredfold (79 ) . Therefore, the lack of effect of the natural, glucocorticoids on glucose transfer, which correlates with their lack of lethal effects (Table II), may result from a large difference in their relative activity using this parameter. This is brought out by the similarity of the effect caused by the high doses of natural steroid and the lower doses of dexamethasone. (Tables V, VI, VII). There i s , therefore, justification from these results in using dexamethasone rather'than the natural glucocorticoids, since acute effects expected to be demonstrated by these experi-ments might be more easily achieved. D. Effect of steroid injected directly into the fetus Since the dose-response for dexamethasone treatment showed two distinct and opposing effects, an experiment was designed to separate these responses, Injections of steroid were perform-ed into the fetuses in utero before treatment of the mouse with labeled glucose, as was previously done. In this manner i t was hoped that only the local effects of the steroid on the feto-placental unit would be manifested, without provoking hypergly-cemia in the mother. As seen in Pig. 4 , the counts recovered in the fetuses were below control values at the lower steroid doses, while at the higher doses ( 5 0 and 100 ug) recoveries were similar to control values. Table VIII shows that the effect occurred in a l l of the fetal tissues examined although again 48 F i g . 4 . E f f e c t of v a r i o u s doses of dexamethasone i n j e c t e d d i r e c t l y i n t o the f e t u s upon the i n c o r p o r a t i o n of l a b e l e d g l u c o s e i n t o SWV mouse f e t u s e s Pregnant SWV mice were a n e s t h e t i z e d and the f e t u s e s i n j e c t e d w i t h the i n d i c a t e d dose of dexamethasone as d e s c r i b e d i n Methods. A f t e r a s.c. i n j e c t i o n of the l a b e l e d glucose i n t o the mother, f e t u s e s were removed and weighed. A c i d - s o l u b l e r a d i o a c t i v i t y was determined f o r each f e t u s to compute the c o r r e c t e d counts (dpm/mg wet wt f e t u s ) which were then compared to sesame o i l -t r e a t e d c o n t r o l s expressed as 100%. The v e r t i c a l l i n e s i n d i c a t e SEM f o r 3 to 4 d e t e r m i n a t i o n s . Comparison of the combined h i g h doses (50 and 100 ug) v e r s u s the l o w e r doses combined (5» 12.5* and 25 ug) u s i n g the t t e s t i n d i c a t e s t h a t the d i f f e r e n c e i s s i g n i f i c a n t , (P<0.01), 4 9 ^ IOO o o CD > > 90 -03 CD oo 1 3 O o 80 CD J O O O O I • a o < 70 -control 100 50 25 12.5 Dexamethasone dose (jag) 50 T A B L E V I I I . E f f e c t o f d e x a m e t h a s o n e i n j e c t e d d i r e c t l y i n t o t h e f e t u s u p o n t h e i n c o r p o r a t i o n o f l a b e l e d g l u c o s e i n t o SWV m o u s e f e t a l t i s s u e s T i s s u e D o s e ( u g ) 0 25 50 B r a i n 8.6 10.2 9.1 L i v e r 18 .0 i o . 5 17.7 G u t 9-9 5.8 7.6 L u n g 10.8 6 .7 9.1 C a r c a s s 13.4 9.3 13.6 A p r e g n a n t SWV m o u s e w a s a n e s t h e t i z e d a n d t h e f e t u s e s i n j e c t e d w i t h t h e i n d i c a t e d d o s e o f d e x a m e t h a s o n e a s d e s c r i b e d i n M e t h o d s . A f t e r a s . c . i n j e c t i o n o f t h e l a b e l e d g l u c o s e i n t o t h e m o t h e r ; t h e f e t a l b r a i n , l i v e r , g u t , a n d l u n g w e r e e x c i s e d f r o m t h e c a r c a s s a n d e a c h t i s s u e w a s t h e n w e i g h e d . A c i d - s o l u b l e r a d i o a c t i v i t y w a s d e t e r m i n e d f o r e a c h s a m p l e o f t i s s u e a n d f i g u r e s r e p r e s e n t t o t a l c o u n t s c o r r e c t e d f o r t h e t i s s u e w e i g h t ( d p m / m g w e t w t t i s s u e ) . 51 t h e d e c r e a s e w a s m u c h l e s s p r o m i n e n t i n t h e f e t a l b r a i n . T h i s i s c o n s i s t e n t , w i t h t h e p r o p o s a l t h a t l o w e r d o s e s e x e r t e d t h e i r e f f e c t o n l y o n t h e f e t o - p l a c e n t a l c o m p a r t m e n t , v / h e r e a s h i g h e r d o s e s w e r e a b l e t o i n f l u e n c e t h e m a t e r n a l c o m p a r t m e n t , a s i f i n ^ j e c t e d i n t o t h e m o t h e r s a n d w e r e t h u s a b l e t o e x e r t a n o p p o s i n g e f f e c t b y r a i s i n g t h e m a t e r n a l b l o o d g l u c o s e l e v e l . E . G l u c o s e c o n c e n t r a t i o n a n d r e l a t i v e r a d i o a c t i v i t y o f m a t e r n a l b l o o d ' •  T o d e t e r m i n e w h e t h e r o r n o t t h e s t e r o i d e f f e c t s c o u l d b e e x -p l a i n e d b y d i f f e r e n c e s i n m a t e r n a l h y p e r g l y c e m i a , t h e c o n c e n -t r a t i o n o f g l u c o s e a n d t h e r a d i o a c t i v i t y i n m a t e r n a l b l o o d w e r e m e a s u r e d . U s i n g A / j m i c e , t h e b l o o d g l u c o s e c o n c e n t r a t i o n f o r a l l t h e s t e r o i d - t r e a t e d a n i m a l s w a s f o u n d t o b e a t l e a s t t w i c e t h e c o n t r o l l e v e l ( T a b l e I X ) . H o w e v e r , t h e r e w a s n o c o r r e l a t i o n b e t w e e n t h e b l o o d g l u c o s e c o n c e n t r a t i o n a n d t h e d o s e o f s t e r o i d u s e d i n t h e i n j e c t i o n . T h e r e l a t i v e c o n c e n t r a t i o n o f g l u c o s e t o r a d i o a c t i v i t y i n m a t e r n a l b l o o d w a s f o u n d t o b e l o w e r f o r a l l t h e s t e r o i d - t r e a t e d a n i m a l s c o m p a r e d t o t h e A / j c o n t r o l s a l t h o u g h t h e r e w a s a g a i n n o c o r r e l a t i o n w i t h s t e r o i d d o s e . T h e SWV a n d C5 ? B L/6j m i c e t r e a t e d w i t h 200 u g d e x a m e t h a s o n e e x h i b i t e d t h e s a m e b l o o d g l u c o s e c o n -c e n t r a t i o n a s t h e A / j t r e a t e d a t t h e s a m e d o s e o f s t e r o i d a l -t h o u g h t h e r e l a t i v e r a d i o a c t i v i t y o f m a t e r n a l b l o o d f o r t h e f o r -m e r s t r a i n s a p p e a r e d h i g h e r t h a n f o r t h e A / j . F r o m t h e s e d a t a , t h e r e w a s n o i n d i c a t i o n t h a t t h e s t e r o i d - i n d u c e d d e c r e a s e i n t r a n s f e r o f l a b e l e d g l u c o s e f r o m m o t h e r t o f e t u s r e s u l t e d f r o m s t e r o i d e f f e c t s o n m a t e r n a l c a r b o h y d r a t e m e t a b o l i s m . 52 TABLE IX. G l u c o s e c o n c e n t r a t i o n and r e l a t i v e r a d i o a c t i v i t y o f m a t e r n a l b l o o d f r o m s t e r o i d - t r e a t e d m i c e Mouse Dexameth- Glucose R e l a t i v e r a d i o a c t i v i t y s t r a i n asone c o n c e n t r a t i o n (dpm/microgram g l u -dose (ug) (mg/100 ml blood) cose) A / J 0 4 4 , 3 5 , 6 1 7 4 0 0 , 6 6 7 0 , 9 9 0 0 50 9 2 , 1 3 6 , 1 0 7 3 1 1 0 , 3 7 7 0 , 4 9 4 0 1 0 0 1 3 0 , 7 4 , 8 7 5 7 8 0 , 3 4 4 0 , 3 8 6 0 2 0 0 1 1 0 , 2 2 1 1 1 6 0 , 5 4 2 0 300 9 7 , 9 3 2 1 8 0 , 3 4 1 0 SWV 2 0 0 1 3 0 , 1 4 1 , 1 9 8 5 2 7 0 , 5 7 4 0 , 7 1 4 0 C 5 7 B L / 6 J 2 0 0 1 5 5 , 1 7 3 7 1 0 0 , 5 1 1 0 Pregnant mice ( g e s t a t i o n a l day 18) r e c e i v e d an i . p . dose of dexamethasone suspensed i n 0 . 1 ml sesame o i l . They were t r e a t e d one hour l a t e r with a s.c. i n j e c t i o n of l a b e l e d g l u c o s e and a f t e r another 15 min 0 . 4 ml blood samples were o b t a i n e d from the t h r o a t and mixed th o r o u g h l y with 0 , 1 ml sodium c i t r a t e s o l u t i o n . Blood g l u c o s e was assayed on prepared Somogyi f i l t r a t e s u s i n g the semi-micro procedure d e s c r i b e d i n Methods, w h i l e an a l i q u o t of the f i l t r a t e was a l s o assayed f o r r a d i o a c t -i v i t y . 53 F . E f f e c t o f d e x a m e t h a s o n e o n a m i n o a c i d i n c o r p o r a t i o n i n t o t h e a c i d - s o l u b l e f r a c t i o n o f f e t a l t i s s u e s  S i n c e g l u c o s e t r a n s p o r t t o t h e f e t u s a p p e a r s t o be i n h i b i t e d b y d e x a m e t h a s o n e i n j e c t i o n i n t o t h e p r e g n a n t m o u s e , i t i s p o s -s i b l e t h a t t h e i n h i b i t i o n r e s u l t e d f r o m a g e n e r a l e f f e c t , o n p l a -c e n t a l t r a n s p o r t . T o t e s t t h i s , t h e e f f e c t o f d e x a m e t h a s o n e u p o n t h e i n c o r p o r a t i o n o f a n a m i n o a c i d w a s i n v e s t i g a t e d u s i n g u r e i d o - * G l a b e l e d c i t r u l l i n e , a n a m i n o a c i d e x p e c t e d t o h a v e a s l o w t u r n o v e r i n t h e f e t u s . A t t h e t w o d o s e s o f d e x a m e t h a s o n e u s e d ( T a b l e X ) , t h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e i n t h e r e -c o v e r y o f t h e l a b e l f r o m s t e r o i d - t r e a t e d o r c o n t r o l f e t u s e s ( P = 0.6 t o 0.7 u s i n g t t e s t ) . S i n c e t h e r e w a s d e f i n i t e i n h i -14 b i t i o n o f C - g l u c o s e t r a n s f e r a t t h e s e s t e r o i d d o s e s , t h e e f f e c t a p p e a r s t o b e r e l a t i v e l y s p e c i f i c f o r g l u c o s e . S t e r o i d - r e c e p t o r C o m p l e x e s i n M o u s e P l a c e n t a l T i s s u e A l l s t e r o i d - s e n s i t i v e t i s s u e s e x a m i n e d t o d a t e h a v e b e e n d e m o n s t r a t e d t o c o n t a i n i n t r a c e l l u l a r s t e r o i d b i n d i n g p r o t e i n r e c e p t o r m o l e c u l e s o f h i g h a f f i n i t y . T h e c o r r e l a t i o n o f r e c e p t o r -b i n d i n g w i t h t h e i n v i v o a c t i v i t y o f t h e g l u c o c o r t i c o i d s h a s b e e n e s t a b l i s h e d f o r c u l t u r e d h e p a t o m a c e l l s (71, 72), l y m p h o i d t i s s u e (73), a n d f i b r o b l a s t s (74). E x t e n s i v e w o r k w i t h t h y m u s t i s s u e (65, 66, 67), h a s a l s o i n d i c a t e d t h a t t h e g l u c o c o r t i c o i d — s p e c i f i c r e c e p t o r s f o u n d i n t h e s e t i s s u e s p l a y a p h y s i o l o g i c a l r o l e i n m e d i a t i n g a l l t h e m e t a b o l i c e f f e c t s o f t h e s t e r o i d , i n -c l u d i n g t h e i n h i b i t i o n o f g l u c o s e u p t a k e , w h i c h h a s b e e n p r o -p o s e d a s o n e o f t h e p r i m a r y c o r t i c o i d e f f e c t s (38). I f t h e d e c r e a s e d i n c o r p o r a t i o n o f g l u c o s e i n t o f e t u s e s o f g l u c o c o r t i c o i d - t r e a t e d a n i m a l s w e r e a s t e r o i d - m e d i a t e d e f f e c t , 5^ TABLE X. E f fec t of dexamethasone upon the incorporat ion of labeled c i t r u l l i n e into SWV mouse fetuses Dexamethasone Ac id- so lub le Corrected counts dose counts (dpm) (dprn/mg wet wt fetus) Control 76,500 + 9 i 6 0 0 2 5 . 5 + 2 .1 500 ug 8 9 , 0 0 0 + 1 3 , 2 0 0 2 7 . 5 + 4 . 4 1 mg 1 0 4 , 2 0 0 + 1 0 , 2 0 0 3 3 - 4 + 4 . 0 Pregnant SWV mice were in jec ted with dexamethasone and labeled c i t r u l l i n e as described i n Methods. Ac id- so lub le r a d i o -a c t i v i t y was determined for each fetus and f igures represent mean + SEM for 4 to 6 fetuses from s ing le animals. 55 t h e r e s h o u l d b e r e c e p t o r s i n v o l v e d i n t h e i r m o d e o f a c t i o n . S i n c e t h e t r a n s m i s s i o n o f m a t e r i a l f r o m m o t h e r t o f e t u s o c c u r s v i a p l a c e n t a l t r a n s f e r , i t s e e m e d f e a s i b l e t h a t t h e p l a c e n t a m i g h t c o n t a i n i n t r a c e l l u l a r m o l e c u l e s w h i c h i n t e r a c t s p e c i f i c a l l y w i t h t h e g l u c o c o r t i c o i d s . A n a t t e m p t w a s t h e r e f o r e m a d e , u s i n g l a b e l e d s t e r o i d s o f h i g h s p e c i f i c a c t i v i t y , t o i s o l a t e a s t e r o i d -m a c r o m o l e c u l e c o m p l e x f r o m w i t h i n p l a c e n t a l c e l l s . A . P r e l i m i n a r y i s o l a t i o n o f n u c l e a r c o m p l e x e s H o m o g e n a t e s o f p l a c e n t a l t i s s u e w e r e p r e p a r e d , i n c u b a t e d w i t h l a b e l e d c o r t i c o s t e r o n e a n d t h e n u c l e a r f r a c t i o n i s o l a t e d a s d e s c r i b e d i n M e t h o d s . T h e n u c l e a r f r a c t i o n w a s t h e n e l u t e d t h r o u g h S e p h a d e x G - 2 5 t o s e p a r a t e t h e f r e e c o r t i c o s t e r o n e f r o m t h a t b o u n d t o m a c r o m o l e c u l e s . A t y p i c a l e l u t i o n p r o f i l e i s s e e n i n F i g . 5» w i t h t w o p e a k s o f r a d i o a c t i v i t y a n d a s i n g l e p r o t e i n p e a k . T h e s h a r p r a d i o a c t i v e p e a k w h i c h c o i n c i d e d w i t h t h e p r o -t e i n p e a k , o c c u r r e d i n t h e v o i d v o l u m e , i n d i c a t i n g a n a s s o c i a t i o n o f the r a d i o a c t i v e c o r t i c o s t e r o n e w i t h a m a c r o m o l e c u l e . T h e o t h e r p e a k w h i c h t r a i l s , i s i n c l u d e d i n t h e g e l v o l u m e a n d r e p r e -s e n t s u n b o u n d s t e r o i d . B . S p e c i f i c i t y o f n u c l e a r r e c e p t o r b i n d i n g S e v e r a l e x p e r i m e n t s w e r e p e r f o r m e d t o d e t e r m i n e i f t h e b i n d i n g i n the v o i d v o l u m e r e p r e s e n t e d s p e c i f i c b i n d i n g , p o s s i b l y o f p h y -s i o l o g i c a l i m p o r t a n c e . H o r m o n e s p e c i f i c i t y i s o n e o f t h e p r i m e c r i t e r i a f o r d i s t i n g u i s h i n g b e t w e e n s p e c i f i c a n d n o n s p e c i f i c b i n d -i n g ( 9 3 ) - I n c u b a t i o n s w e r e p e r f o r m e d u s i n g l a b e l e d C o r t i s o l , p r o -g e s t e r o n e , a n d c o r t i s o n e t o a s c e r t a i n i f t h e s e s t e r o i d s a l s o s h o w e d a f f i n i t y f o r t h e r e c e p t o r . O f t h e t h r e e s t e r o i d s , o n l y C o r t i s o l r e s e m b l e d c o r t i c o s t e r o n e i n b e i n g a n a c t i v e g l u c o c o r t i -F i g . 5- I s o l a t i o n of a -TI-corticosterone-receptor complex on Sephadex G-25 A placental homogenate was incubated with labeled corticosterone (1 uM) and the nuclear steroid-receptor complex was then i s o l a t e d as described i n Methods. 57 coid per se. Progesterone has no g l u c o c o r t i c o i d a c t i v i t y and cortisone i s only active i n vivo by v i r t u e of i t s conversion to C o r t i s o l by the 11 /3 -hydroxysteroid: NADP oxidoreductase en-zyme. The i n vivo g l u c o c o r t i c o i d a c t i v i t y of these steroids correlated with the binding assay (Fig, 6 ) since only C o r t i s o l gave a radioactive p r o f i l e on Sephadex s i m i l a r to c o r t i c o s t e -rone. When cortisone or progesterone was used, there was no s i g n i f i c a n t radioactive peak occurring i n the void volume, only a peak representing the unbound s t e r o i d . Mammalian plasma contains corticosteroid-binding g l o b u l i n ( t r a n s c o r t i n or CBG), a pr o t e i n which w i l l bind c o r t i c o i d s and progesterone with high a f f i n i t y ( 9 k ) . The p o s s i b i l i t y that a high concentration of t h i s protein could contaminate the nuclear preparation and account f o r the s p e c i f i c binding was investigated. A preparation of placental homogenate was washed three times with 2 ml cold buffer before incubating with the labeled stero i d . If the binding were due to contamination of the nuclear prepar-ation with serum proteins, then the washed homogenate should show l e s s binding of r a d i o a c t i v i t y . This was not observed since the washed homogenate, even though i t contained much l e s s protein than the control, had a higher r e l a t i v e binding. (Table XI). In another experiment, 1 ml of maternal blood (which has a high concentration of transcortin) was added to the buffer with which the placental tissue was homogenized. The added transcor-t i n , i f i t were responsible f o r the binding, would be expected to increase the s p e c i f i c binding of the sample. However, the sample with maternal blood added had a lower s p e c i f i c binding 5 8 VOLUME (ml)" F i g . 6. Steroid s p e c i f i c i t y i n the i s o l a t i o n of a nuclear complex Placental homogenates were incubated with the indi-cated labeled s t e r o i d (approximately 1 uM) and the nuclear steroid-receptor complex was then i s o l a t e d as described i n Methods. O — O H-Corticosterone 14 C - C o r t i s o l 14 C-Progesterone ^H-Cortisone 59 TABLE XI. E f f e c t of washing and a d d i t i o n of b l o o d on the i s o l a t i o n of a l a b e l e d s t e r o i d complex f r o m n u c l e i Sample S p e c i f i c P r o t e i n R e l a t i v e r a d i o -b i n d i n g (dpm) (mg) a c t i v i t y (dpm/mg p r o t e i n ) C o n t r o l 14 , 5 1 0 2 . 4 8 5 8 6 0 A (washed) 1 0 , 6 7 0 1 . 5 7 6800 C o n t r o l 8 , 2 9 5 1 . 6 8 4940 B ( b l o o d added) 5 , 1 0 0 1 . 6 3 3 1 3 0 The p l a c e n t a l homogenates were i n c u b a t e d w i t h J H - c o r t i -c o s t e r o n e ( 1 3 - 8 nM) and the n u c l e a r s t e r o i d - r e c e p t o r complex i s o l a t e d as d e s c r i b e d i n Methods. Sample A (washed) was washed t h r e e t i m e s w i t h 2 ml c o l d KEP b u f f e r b e f o r e a d d i n g l a b e l e d s t e r -o i d . Sample B had 1 ml o f whole b l o o d added t o t h e homogenate b e f o r e a d d i t i o n of the l a b e l . R a d i o a c t i v i t y a s s o c i a t e d w i t h the p r o t e i n peak was used as a measure of s p e c i f i c b i n d i n g . 60 r e l a t i v e to c o n t r o l e v e n t h o u g h t h e a m o u n t of p r o t e i n i s o l a t e d w a s t h e same as f o r t h e c o n t r o l . From t h i s d a t a i t a p p e a r e d that t h e s e r u m p r o t e i n s do n o t a c c o u n t f o r the s p e c i f i c b i n d i n g of the n u c l e a r p r e p a r a t i o n . Munck a n d Brinck - J o h n s e n (65) f o u n d t h a t a l l t h e m e t a b o l i c e f f e c t s of C o r t i s o l on t h y m u s c e l l s in v i t r o c o u l d be p r e v e n t e d b y a d d i t i o n of t h e m e t a b o l i c a l l y i n a c t i v e s t e r o i d , c o r t e x o l o n e , w h i c h has b e e n s h o w n to c o m p e t e w i t h a c t i v e g l u c o c o r t i c o i d s f o r the s p e c i f i c b i n d i n g s i t e s ( 65 ) . This c o m p o u n d w a s t e s t e d w i t h p l a c e n t a l b i n d i n g a n d , as Fig. 7 s h o w s , a d d i t i o n of a t e n f o l d e x c e s s of u n l a b e l e d c o r t e x o l o n e to t h e p l a c e n t a l i n c u b a t i o n m i x -ture r e s u l t e d in the v i r t u a l e l i m i n a t i o n of the specific b i n d i n g peak of Cortisol. The same e f f e c t w a s a l s o o b s e r v e d w i t h l a b e l e d c o r t i c o s t e r o n e (Table XII). The a b o l i t i o n of t h e b i n d -i n g peak by c o r t e x o l o n e i s e v i d e n c e t h a t t h i s peak r e p r e s e n t s s p e c i f i c b i n d i n g s i n c e c o r t e x o l o n e c o m p e t e s f o r t h e s p e c i f i c b i n d i n g w i t h g l u c o c o r t i c o i d s in o t h e r t i s s u e s ( 64 ) . The s t e r e o s p e c i f i c i t y of the c o r t i c o s t e r o n e b i n d i n g w a s i n -v e s t i g a t e d by t e s t i n g t h e e f f e c t s of a d d i t i o n of u n l a b e l e d C o r -t i s o l a n d i t s b i o l o g i c a l l y i n a c t i v e e p i m e r , 1 1 - e p i c o r t i s o l . Since e p i c o r t i s o l h a s no m e t a b o l i c a c t i v i t y as a g l u c o c o r t i c o i d a n d i s n o t an a n t a g o n i s t , i t w o u l d n o t be e x p e c t e d to a f f e c t t h e specific b i n d i n g , w h e r e a s C o r t i s o l s h o u l d c o m p e t e w i t h c o r -t i c o s t e r o n e f o r t h e s p e c i f i c b i n d i n g s i t e s . As s h o w n in Fig. 8, a d d i t i o n of t h e a c t i v e e p i m e r a b o l i s h e d t h e s p e c i f i c b i n d i n g c o m p l e t e l y w h e r e a s 1 1 - e p i c o r t i s o l r e d u c e d t h e t o t a l s p e c i f i c b i n d i n g p e a k by o n l y 7%. Other u n l a b e l e d s t e r o i d s w e r e a l s o u s e d in t h e same m a n n e r 61 — © — o — Cort isol • — - B - n — Cortisol + Cortexolone VOLUME (ml) F i g . 7. E f f e c t of cortexolone on the i s o l a t i o n of a C-c o r t i s o l nuclear complex Homogenates of placental tissue were incubated with labeled C o r t i s o l (O .96 uM) alone or together with 10 uM unlabeled cortexolone. The nuclear steroid-receptor complex was- then i s o l a t e d as described i n Methods, 62 TABLE X I I . Ef f e c t , of c o r t e x o l o n e on the i s o l a t i o n of a l a b e l e d s t e r o i d complex from p l a c e n t a l n u c l e i Expt. L a b e l e d Cortexolone S p e c i f i c % of No. s t e r o i d added b i n d i n g c o n t r o l (dpm) C - c o r t i s o l 0 2 , 5 2 0 100 (O . 9 6 uM) 10 uNl 920 3? k C - c o r t i s o l 0 1 , 9 9 5 1 0 0 (O . 9 6 uM) 10 u M 350 18 'H-corticosterone 0 1 7 , 1 0 0 1 0 0 ( 1 . 0 uM) 10 uM 5 . 6 9 0 33 Homogenates of p l a c e n t a l t i s s u e were incubated w i t h l a b e l e d s t e r o i d , alone or with u n l a b e l e d c o r t e x o l o n e added. The n u c l e a r s t e r o i d - r e c e p t o r complex was then i s o l a t e d as d e s c r i b e d i n Methods. R a d i o a c t i v i t y i n the v o i d volume was taken to be a s s o c i a t e d w i t h the macromolecular peak and used as a measure of s p e c i f i c b i n d i n g . 6 3 TOTAL ( t u b e s 4-10)  COUNTS(dpm) PROTEIN(mg) A ^ H - c o r t i c o s t e r o n e 1,250 2.06 " " " + Cortisol H - c o r t i c o s t e r o n e 11,500 2.00 + e p i - c o r t i s o l ^ H - c o r t i c o s t e r o n e 12,400 2.10 a l o n e Volume (ml) 8 . S p e c i f i c i t y of JH-corticosterone binding i n placental homogenates Homogenates of placental tissue were incubated with labeled corticosterone (13.8 nM) alone or together with either 1 1 - e p i c o r t i s o l or C o r t i s o l at 2 uM concentration. The nuclear steroid-receptor complex was then i s o l a t e d as described i n Methods. 64 to test their effectiveness in displacing JH-corticosterone from the specific binding sites (Table XIII). Corticosterone, cortexolone, Cortisol, and progesterone were a l l about equally effective, whereas cortisone was p a r t i a l l y effective and 11-epicortisol had no significant effect. C. Characterization of the bound steroid The radioactive steroid specifically bound was analyzed to ascertain i f i t had been metabolized to any extent. Eoth ^H-14 corticosterone and C-cortisol were chromatographed as the ex-tracted free steroid, or by f i r s t preparing the C-21 acetate derivative. As summarized in Table XIV, greater than 80$ of the radioactivity in a l l cases was present in the UV absorbing zones corresponding to the added authentic carrier compounds. Further characterization of ^H-corticosterone-21-acetate was ac-complished by recrystallizing to constant specific a c t i v i t y (Table XV). These data indicate that the radioactivity bound by the nuclear receptor(s) represents the unmetabolized hormone, corticosterone (or Cortisol) which was added. D. Isolation of cytoplasmic receptor The isolation of a specific steroid-macromolecule complex from the nuclear fraction of placental ce l l s then prompted the examination of the cytosol for the presence of cytoplasmic bind-ing proteins. The current theory postulates that the nuclear receptor originates from transmission of a cytoplasmic molecule to the nucleus. Placental tissue was fractionated after incu-bation with the label. As seen i n Fig. 9» a large proportion of the counts were in the cytoplasm (fraction C) as well as 65 T A B L E X I I I . C o m p e t i t i v e i n t e r a c t i o n o f s t e r o i d s w i t h H -c o r t i c o s t e r o n e b i n d i n g S t e r o i d a d d e d R e l a t i v e r a d i o a c t i v i t y % o f ( d p m / m g p r o t e i n ) c o n t r o l C o n t r o l 5120 250 1 0 0 E p i c o r t i s o l 4940 + 550 97 C o r t i s o n e 2190 + 530 43 P r o g e s t e r o n e 820 + 210 16 C o r t i s o l 650 + 1 2 0 13 C o r t i c o s t e r o n e 510 + 110 10 C o r t e x o l o n e 505 + 115 10 o H o m o g e n a t e s o f p l a c e n t a l t i s s u e w e r e i n c u b a t e d w i t h • ^ H - c o r t i c o s t e r o n e (13'8 n M ) a l o n e o r t o g e t h e r w i t h v a r i o u s u n -l a b e l e d s t e r o i d s (2 u M ) , a n d t h e n u c l e a r s t e r o i d - r e c e p t o r c o m -p l e x i s o l a t e d a s d e s c r i b e d i n M e t h o d s . R a d i o a c t i v i t y a s s o c i a t -e d w i t h t h e p r o t e i n p e a k w a s u s e d a s a m e a s u r e o f s p e c i f i c b i n d i n g a n d f i g u r e s r e p r e s e n t m e a n + S E M f o r 3 t o 4 t r i a l s . 66 TABLE XIV, Chr o m a t o g r a p h i c c h a r a c t e r i z a t i o n of l a b e l e d s t e r o i d from i s o l a t e d n u c l e a r complexes Compound % t o t a l c o u n t s r e c o v e r e d .in UV a b s o r b i n g zone 3 H - c o r t i c o s t e r o n e 80.6 3x H - c o r t i c o s t e r o n e -2 1 - a c e t a t e 81.0 14 C - c o r t i s o l -2 1 - a c e t a t e 83.1 Samples of the n u c l e a r s t e r o i d - r e c e p t o r complex from s e v e r a l e x p e r i m e n t s were p o o l e d and e x t r a c t e d w i t h 6 volumes of d i c h l o r o m e t h a n e . The e x t r a c t s were e v a p o r a t e d under n i t r o -gen, and where i n d i c a t e d , a c e t y l a t i o n was a c c o m p l i s h e d u s i n g a c e t i c a n h y d r i d e (95). A u t h e n t i c c a r r i e r s t e r o i d s were added, and the samples were s p o t t e d onto Eastman TLC s h e e t s . A f t e r development f o r 4 h r s i n a system composed o f t o l u e n e : c h l o r o -form:methanol:water ( 1 2 : 6 : 2 : 0 . 1 ) , the s t e r o i d zones were l o -c a t e d by UV absorbance. A u t o r a d i o g r a m s were p r e p a r e d , and the UV a b s o r b i n g zones were t h e n c u t o u t , e l u t e d , and counted. The autoradiograms in a l l cases (except for a faint spot in the Cortisol-21-acetate chromatogram corresponding to Cortisol) showed no trace of radioactivity except for the spot corres-ponding to the UV absorbing zone. 67 TABLE XV. I d e n t i f i c a t i o n by c r y s t a l l i z a t i o n of l a b e l e d s t e r o i d from i s o l a t e d n u c l e a r complexes S p e c i f i c a c t i v i t y (dpm/ug) C r y s t a l l i z a t i o n Mother l i q u o r C r y s t a l s F i r s t 14. .4 11, .3 Second 1 9 . ,6 12, ,1 T h i r d 12. ,1 11, .5 F o u r t h 11. ,0 11. .7 J H - c o r t i c o s t e r o n e , i s o l a t e d f r o m p o o l e d samples of n u c l e a r s t e r o i d - r e c e p t o r complex by e x t r a c t i o n w i t h 6 volumes of d i c h l o r o m e t h a n e , was e v a p o r a t e d under n i t r o g e n and t h e n a c e t y l a t e d (95)- The l a b e l e d compound was mixed w i t h a s m a l l amount of c a r r i e r c o r t i c o s t e r o n e - 2 1 - a c e t a t e and t h e n chroma-t o g r a p h e d as b e f o r e ( T a b l e X I V ) . The c o r t i c o s t e r o n e zone was e l u t e d and s e v e r a l mg of f r e s h l y r e c r y s t a l l i z e d c o r t i c o s t e r -o n e - 2 1 - a c e t a t e were added to the sample i n methanol. The com-pound was c r y s t a l l i z e d f o u r t i m e s by a d d i t i o n of w a t e r t o the m e t hanol. The c r y s t a l s and mother l i q u o r were a s s a y e d by UV absorbance a t 240 run and r a d i o a c t i v i t y d e t e r m i n a t i o n s . 68 MINCED PLACENTAL TISSUE (2 gm) f 1. 2. 3. 4. Wash three times with 10 ml cold "buffer. Resuspend in 10 ml buffer and incubate 10 min at 37°C with -^H-corticosterone, (1 uCi). Discard buffer and wash three times with 10 ml cold buffer, then resuspend in 10 ml buffer and homogenize. Centrifuge at 1200xg for 10 min. PELLET 1 f SUPERNATANT 1. Wash twice with 5 nil cold buffer and resus-pend in 10 ml buffer. (A) 2. Centrifuge at 1200xg for 30 min. PELLET SUPERNATANT (B) f Centrifuge 105,000xg for 60 min. PELLET Wash twice with 5 ml cold buffer. (D) SUPERNATANT (C) Wash twice with 5 ml cold buffer. (E) WASHED PELLET (P) WASHED PELLET (G) Fraction Total counts (dpm) % of total recovered A B C D E P G 57,000 5,800 222,000 3,700 8 ,600 55,400 124,000 12.0 1.2 46.6 0.8 1.8 11.6 26.0 Fig. 9. Fractionation of placental tissue A l l procedures carried out between 0 and 6°C. Buffer used in a l l operations is KEP buffer, pH 7.k» containing 2.8 mM glucose. 69 the microsomal and mitochondrial f r a c t i o n ( G ) . When an aliquot of the cytosol f r a c t i o n was passed through Sephadex G-25 there was found again to be a radioactive peak associated with the protein peak i n the void volume which resembled the s p e c i f i c nu-clear binding. However, epicorticosterone was found to decrease the amount of r a d i o a c t i v i t y recovered i n t h i s peak, i n d i c a t i n g that there was nonspecific binding present. This was tested using the cytosol exchange assay as indicated i n Methods (Table XVI)and i t was found that 200 uM epicorticosterone decreased the amount of r a d i o a c t i v i t y recovered i n the area under the protein peak by 70%. However, the presence of the same concentration of unlabeled corticosterone decreased i t by 96%. I t would be reasonable to suppose that both stereoisomers of corticosterone would compete almost equally f o r nonspecific binding s i t e s of the ^H-corticosterone. But since only 11 /3 -corticosterone has g l u c o c o r t i c o i d a c t i v i t y , only t h i s isomer should compete with the labeled s t e r o i d f o r s p e c i f i c binding. Thus, the difference i n displacement caused by these two isomers i s an i n d i c a t i o n of the s p e c i f i c binding of -\K-corticosterone i n the cytosol ex-change assay (74, 90). E. Binding properties of the cytoplasmic receptor Using the cytosol exchange assay the time course of the binding i n t e r a c t i o n was investigated at both 37° and 0°C. At 0° the s p e c i f i c binding reached a maximum by 30 min and then l e v e l l e d o f f . As shown i n F i g . 10, the rate of association was greater at 37°C than at 0°C since the maximum binding was reach-ed at the higher temperature within 5 min of incubation. How-70 TABLE XVI. S p e c i f i c and n o n s p e c i f i c b i n d i n g u s i n g the c y t o s o l exchange assay S t e r o i d added B i n d i n g (dpm) P r o t e i n (mg) R e l a t i v e r a d i o a c t i v i t y (dpm/mg p r o t e i n ) C o n t r o l E p i c o r t i c o s t e r o n e C o r t i c o s t e r o n e 2 5 , 5 0 0 7 , 7 0 0 900 0 . 70 0 . 7 2 0 . 6 6 3 6 , 4 0 0 1 0 , 7 0 0 1 , 3 6 0 The c y t o s o l f r a c t i o n was prepared as d e s c r i b e d i n Methods and the c y t o s o l exchange assay c a r r i e d out a t 0°C f o r 35 min. The t o t a l r a d i o a c t i v i t y a s s o c i a t e d w i t h the p r o t e i n peak was used as a measure of b i n d i n g . 71 \ Length of i n c u b a t i o n (min) \ F i g . 10. Time course of binding using the cytosol exchange assay The cytosol f r a c t i o n was i s o l a t e d and the exchange assay car r i e d out at 37 C and 0 C f o r various times. The amount of s p e c i f i c binding (see Methods) was used to calculate the r e l a t i v e r a d i o a c t i v i t y (dpm/mg protein). 72 ever, at the higher temperature, the amount of bound steroid decreased after more than a 5 min incubation, implying a dis-sociation of the complex. For this reason further experiments using the cytosol exchange assay were performed at 0°C for 35 min to obtain maximum s t a b i l i t y of the complex. Since the protein concentration of the isolated cytosol fraction can vary according to variation in placental size, i t was necessary to demonstrate that the amount of spec i f i c a l l y bound steroid i s related linearly to the concentration of cyto-plasmic extract. This was investigated by making dilutions of the isolated high speed cytosol and then performing the cytosol exchange assay on the aliquots. The linear relationship which results (Fig. 11), shows that there i s no ar t i f a c t i n the bind-ing due, for example, to aggregation of protein molecules at high concentrations (72). The effect of various treatments on this i n i t i a l cytoplasmic interaction between steroid and binding molecule was examined using the cytosol exchange assay. Along with the labeled ste-roid, hydrolytic enzymes or sulfhydryl reagents were added and the samples incubated at 37°C for 10 min (except for the heat-treated sample). As shown in Table XVII, pronase at a concent-ration of 1.5 mg/ml decreased the specific binding by 90$ where-as deoxyribonuclease and ribonuclease had no effect. Heat treat-ment also had a drastic effect on the st a b i l i t y of the complex but the sulfhydryl reagents had only a minor effect. F. Kinetic examination of the binding A study of the kinetics of corticosterone binding to the 73 \ Fig. 11. Homogeniety of cytosol receptor distribution The cytosol fraction was isolated as before and various dilutions were prepared using KEP buffer (pH 7.4, with 2.8 mM glucose). The cytosol exchange assay was then carried out at 0°C for 35 rain. The radioactivity recovered i n the specific binding fraction was used to calculate the amount of bound corticosterone. 7 4 TABLE X V I I . S e n s i t i v i t y o f the c y t o s o l s t e r o i d - r e c e p t o r complex to h e a t , h y d r o l y t i c enzymes, and s u l f h y d r y l r e a g e n t s Treatment R e l a t i v e a c t i v i t y $ of c o n t r o l (dpm/mg p r o t e i n ) C o n t r o l 8950 + 2 3 0 a 100 Heat (65°C, 10 min) 1 0 1 0 b 11 Pronase 8 9 0 b 10 DNAse 9 5 0 0 106 RNAse 9 2 0 0 b ' 1 0 3 N - e t h y l m a l e i m i d e 8260° 92 p - c h l o r o m e r c u r i b e n z o a t e 8460° 95 D i t h i o t h r e i t o l 8210° 92 The c y t o s o l f r a c t i o n was p r e p a r e d and 50 u l h y d r o l y t i c enzyme o r s u l f h y d r y l r e a g e n t was added t o 0 . 4 5 ml a l i q u o t s . A f t e r a d d i t i o n of the l a b e l e d c o r t i c o s t e r o n e and the u n l a b e l e d competing s t e r o i d s , the exchange a s s a y was c a r r i e d out a t 0'C f o r 35 min ( e x c e p t f o r the h e a t - t r e a t e d sample) and th e n e l u t e d t h r o u g h Sephadex. The d i f f e r e n c e i n r a d i o a c t i v i t y a s s o c i a t e d w i t h the p r o t e i n peak f o r the two s e r i e s (see Methods) r e p r e -s e n t s s p e c i f i c b i n d i n g . The pronase c o n c e n t r a t i o n was 1 . 5 mg/ml; DNAse and RNAse, 1 . 0 mg/ml; and the s u l f h y d r y l r e a g e n t s were a l l used a t 1 mM c o n c e n t r a t i o n . mean + SEM of 6 samples b average of 2 d e t e r m i n a t i o n s mean of 3 samples 75 cytosol receptors was performed in order to estimate the total receptor content of the tissue as well as the a f f i n i t y of the hormone for the receptor. This was done by preparing aliquots of the cytosol fraction and incubating them with varying concen-3 trations of ^H-corticosterone. If the amount of specific bind-ing is determined as before using the cytosol exchange assay, then this represents the amount of bound corticosterone. The method of Scatchard (96) was used for plotting this data since a more accurate analysis i s permitted by this method (97). The results are plotted as the ratio of the bound to unbound -^H-corticosterone against the amount of bound -^H-corticosterone. From such a plot the number of high a f f i n i t y binding sites as well as the a f f i n i t y of corticosterone for these sites can be obtained. In the equations B/u = 1/Kd (n - B) (i) 3 3 B represents bound vH-corticosterone, u is unbound ^H-corticos-terone, Kd is the dissociation constant for the steroid-receptor complex, and n represents the total number of high a f f i n i t y bind-ing sites specific for ^ H-corticosterone. The reciprocal of the slope w i l l therefore represent the dissociation constant (-Kd) and extrapolation of the slope to the abscissa w i l l give the value for n directly. The Scatchard analysis of the binding assay using -^H-corti-costerone revealed a plot with two components (Fig. 1 2 ) . Slope II, which has low a f f i n i t y and an almost i n f i n i t e number of bind-ing sites, represents nonspecific binding which results when high 76 F i g . 12. Scatchard p l o t of -^H-corticosterone binding i n SWV mouse placental cytosol The cytosol f r a c t i o n was i s o l a t e d and increasing concentrations of labeled corticosterone were added to ali q u o t s . The exchange assay was then c a r r i e d out at 0 C f o r 35 min. The r a d i o a c t i v i t y recovered i n the s p e c i f i c binding f r a c t i o n was used to calculate the amount of bound corticosterone (B). 77 concentrations of -^H-corticosterone are used. The steeper slope (I) most l i k e l y represents high a f f i n i t y specific binding with a limited number of receptor sites. Using the extrapolated slope (I), the calculated Kd = 17.5 nM and n = 0.26 pmoles/mg protein. These values compare favorably with those obtained by other workers using similar systems. Rousseau, Baxter, and Tomkins (72) using Cortisol and cultured hepatoma cells obtained values of Kd = 11.0 nM and n = 0.63 pmoles/mg protein. Ballard and Ballard (98) using dexamethasone with rabbit placental t i s -sue reported Kd = 3-7 nM and n = 0.26 pmoles/mg protein. The latter report, which appeared while this work was in progress, is the only reference in the literature pertaining to the iso-lation of receptors from placental tissue. G. Sucrose density gradient analysis A sucrose density centrifugation of the cytosol steroid-re-ceptor complex was done to identify the number of binding compo-nents present in the specific binding fraction from Sephadex. A crude estimate of the molecular weight for the receptor can be made using the method of Martin and Ames (99). The gradients were prepared using a method modified by Nagy (100) in which the lin e a r i t y of density of the sucrose gradient was checked by de-termining the refractive index of each fraction. If the gradient is linear, then there w i l l be linear migration by the molecules and for molecules of roughly the same partial specific volume, the following equation w i l l be trues ( i i ) 78 In the above equation, d is the distance the molecule has migrat-ed from the top of the gradient and MW represents the molecular weight (99). Using this equation and the radioactivity profile for the sedimentation as shown in Fig. 13t the molecular weight of the cytoplasmic receptor was estimated to be approximately 55.800. The sucrose density centrifugation also demonstrated the presence of a single binding peak which was abolished by the ad-dition of excess unlabeled dexamethasone or corticosterone. The fact that dexamethasone w i l l compete with ^H-corticosterone for binding on the receptor i s significant. Corticosteroid-binding globulin has been shown to have no a f f i n i t y for this synthetic corticosteroid (72). The complete abolition of the binding peak by dexamethasone, therefore, provides further evidence that these binding components are clearly different from plasma transcortin. H. Relating the presence of placental receptors to the i n -hibition of glucose transfer  An attempt was made to interrelate the two areas of experi-mental work in this study. Transport across a tissue can be con-sidered as an extension of uptake by cells of the particular t i s -sue (101). Therefore, inhibition of uptake by placental cells could lead to inhibition of transport of the molecule under study. If glucose uptake by placental tissue is inhibited due to gluco-corticoid interaction with receptors resulting in a response from the nucleus, then inhibition of this response w i l l abolish the steroid effect. However, i f only the maternal hyperglycemia is inhibited and not the effects on fe t a l uptake of glucose, then the recovered counts in the treated fetuses would be expected to 7 9 Fig,. IJ). S u c r o s e d e n s i t y c e n t r i f u g a t i o n of the c y t o s o l s t e r o i d -r e c e p t o r complex i s o l a t e d from Sephadex chromatography Sucrose g r a d i e n t s of 5 t o 20$ i n KEP b u f f e r (pK 7.4, w i t h 2.8 mM g l u c o s e ) were p r e p a r e d w i t h a. Beckman g r a -d i e n t f o r m e r ( 1 0 0 ) . The c y t o s o l f r a c t i o n was p r e p a r e d and i n c u b a t e d w i t h l a b e l e d c o r t i c o s t e r o n e (13.8 nM) a l o n e or w i t h u n l a b e l e d s t e r o i d s (see b e l o w ) , f o r 35 min a t 0 C. The samples were th e n r u n through Sephadex G - 2 5 a t 6°C to o b t a i n the f r a c t i o n s e l u t i n g i n the v o i d volume. Samples of t h e s e f r a c t i o n s (0.2 ml) were l a y e r e d onto p r e -c o o l e d s u c r o s e g r a d i e n t s . The g r a d i e n t s were c e n t r i f u g e d a t 4 C f o r 29 h r s a t 2 8 4 , 0 0 0 x g i n a Beckman L2-65B u l t r a -c e n t r i f u g e u s i n g a SW 40 T i s w i n g i n g b ucket r o t o r . Marker p r o t e i n s , 1 . 5 mg i n 0.2 ml of KEP b u f f e r (pH 7 . 4 , w i t h 2.8 mM g l u c o s e ) , were l a y e r e d onto s u c r o s e g r a d i e n t s and c e n t r i f u g e d a l o n g w i t h the samples. The markers used were human y g l o b u l i n ( 7G) and bovine serum a l b u m i n (BSA) w i t h m o l e c u l a r w e i g h t s of 160,000 and 6 7,000. A f t e r c e n t r i f u g a t i o n , f r a c t i o n s were c o l l e c t e d by p i e r c i n g the bottom of the c e l l u l o s e n i t r a t e tube c o n t a i n -i n g the g r a d i e n t w i t h a Beckman manual f r a c t i o n c o l l e c t o r . A f l o w r a t e o f 1 to 2 drops p e r second was m a i n t a i n e d and a t o t a l of 20 f r a c t i o n s , each c o n t a i n i n g 22 d r o p s , was c o l l e c t e d i n t o s c i n t i l l a t i o n v i a l s or t e s t t u b e s . D e t e r -m i n a t i o n s f o r r a d i o a c t i v i t y and Lowry p r o t e i n were assay e d as i n d i c a t e d i n Methods. E p i c o r t i c o s t e r o n e , Dexamethasone, C o r t i c o s t e r o n e , 200 uM o — o 200 uM * * 200 uM • • 80 81 be much lower (see Fig. 2). Injection of actinomycin D just be-fore administration of dexamethasone resulted in recovery of counts in the treated fetuses equivalent to untreated controls (Table XVIII). This indicates that a l l effects of the steroid were prevented by the actinomycin D and suggests that the steroid induced inhibition of glucose uptake requires a nuclear response which is sensitive to metabolic inhibitors. The competing steroid, cortexolone, was also administered in an attempt to block the effect of dexamethasone in vivo. How-ever, treatment with cortexolone had no effect on the dexametha-sone-induced inhibition (Table XIX). It i s not known, however, to what extent cortexolone can compete with dexamethasone and displace the latter from specific binding sites. Thus, although these experiments are not conclusive, there is some indication that the processes involved show similarities to those in other tissues where steroid-receptor interactions have been studied. 82 TABLE XVI J.I. I n f l u e n c e of a c t i n o m y c i n D on dexamethasone-in d u c e d e f f e c t s i n pregnant mice Mouse s t r a i n C o n t r o l Dexamethasone a l o n e Dexamethasone & a c t i n o m y c i n D C57BL/6J 36.O + 3 . 2 ( 9 , 3 ) 5 3 - 9 + 5 . 7 ( 9 , 3 ) A/J 3 2 . 3 + 2 . 2 ( 8 , 3 ) 3 1 - 9 + 0 . 4 ( 6 , 2 ) 2 9 . 5 + 2 . 0 ( 9 , 3 ) 3 1 . 9 + 1 . 6 ( 9 , 3 ) P r e g n a n t mice ( g e s t a t i o n a l day 18) r e c e i v e d an i . p . i n j e c t i o n of 20 ug a c t i n o m y c i n D i n 0.1 ml p h y s i o l o g i c a l s a l i n e o r s a l i n e a l o n e . They were i n j e c t e d one min l a t e r w i t h an i . p . dose of 200 ug dexamethasone suspended i n 0.1 ml sesame o i l . One hour l a t e r a s.c. i n j e c t i o n o f l a b e l e d g l u c o s e was made and a f t e r a n o t h e r 1,5 min the f e t u s e s were removed, weighed, and p l a c e d on i c e . A c i d - s o l u b l e r a d i o a c t i v i t y was d e t e r m i n e d f o r each f e t u s and f i g u r e s r e p r e s e n t mean of c o r r e c t e d c o u n t s (dpm/ mg wet wt f e t u s ) + SEM. The f i g u r e s i n b r a c k e t s r e p r e s e n t the number of fetuses~a.nd a n i m a l s , r e s p e c t i v e l y . 8 3 TABLE XIX. E f f e c t of c o r t e x o l o n e on dexamethasone i n h i b i t i o n of the i n c o r p o r a t i o n of l a b e l e d g l u c o s e i n t o SWV mouse f e t u s e s E x p t . Dexamethasone Dexamethasone C o r t e x o l o n e No. a l o n e & c o r t e x o l o n e a l o n e 1 11.3+0.1+ 1 0 . 8 + 0 . 4 2 1 1 . 2 + 0 . 5 1 1 . 0 + 0 . 5 1 2 . 2 + 0 . 4 P r e g n a n t SWV mice r e c e i v e d an i . p . dose o f 6 mg c o r -t e x o l o n e i n sesame o i l or 0 . 6'ml sesame o i l s p r e a d over 6 i n -j e c t i o n s 15 min a p a r t . They were t h e n t r e a t e d w i t h a s i n g l e i . p . dose of 200 ug dexamethasone 20 min a f t e r the f i r s t c o r -t e x o l o n e i n j e c t i o n . One hour a f t e r dexamethasone t r e a t m e n t a s.c. i n j e c t i o n of l a b e l e d g l u c o s e was made and a f t e r a n o t h e r 15 min the f e t u s e s were removed and weighed. A c i d - s o l u b l e r a d i o a c t i v i t y was d e t e r m i n e d f o r each f e t u s and f i g u r e s r e p r e -s e n t mean of c o r r e c t e d c o u n t s (dpm/mg wet wt f e t u s ) + SEM f o r 4 t o 6 f e t u s e s f rom s i n g l e a n i m a l s . 84 DISCUSSION The metabolic effects of glucocorticoid hormones on l i v e r , adipose, and muscle tissue have been studied extensively. How-ever, very l i t t l e is known concerning the influence of these hormones on fe t a l development or their possible role during ges-tation. Corticosteroid hormone levels in the mouse decrease during the f i r s t half of pregnancy but increase about thirteen-f o l d by the late stages of gestation (102). The passage of cor-ticosteroids across the mouse placenta has been demonstrated (80, 81, 103)» although the fetus may be protected from high concen-trations of corticosteroids. Pregnancy in the mouse, as in most other mammals, markedly increases the transcortin level (102). The binding of the c o r t i -coids by transcortin would effectively lower the concentration of free steroid, and only the free unbound corticosteroids have biological activity (102). Also, the 11 /3-hydroxysteroids NADP oxidoreductase enzyme, present in both placenta and i n f e t a l tissues (56), w i l l convert glucocorticoids to their inactive 11-dehydro metabolites (103). This could explain the failure of the natural corticosteroids to e l i c i t in vivo responses except with supraphysiological doses (56, 104). By using dexamethasone, a synthetic corticoid which does not undergo extensive metabolism and is not bound by trans-cortin (94), acute effects upon the feto-placental unit might be more easily demonstrated. Maternal corticosteroids normally do not affect f e t a l growth directly, whereas glucose i s the most c r i t i c a l factor (105). 85 There i s c l i n i c a l evidence that maternal glucose loading has favorable effects on management of fe t a l distress due to placen-ta l "insufficiency or dysfunction" (106, 107). Animal experi-ments have also shown that hypertonic glucose can increase both fet a l and neonatal survival and may also have favorable effects on the fetus under conditions of hypoxia (106). In an attempt to explain the toxic and teratogenic effects induced by dexamethasone (Table II), the transfer of important precursors from mother to fetus was examined. From the known effects of glucocorticoids on peripheral u t i l i z a t i o n of glucose, i t was expected that dexamethasone administration would result in an elevation of the maternal blood glucose level. Since under most circumstances the placental transport of glucose is a pas-sive process dependent only on the concentration gradient, an i n -crease in the maternal blood glucose level should result in an increased flow of glucose to the fetus. What was unexpected, however, was that inhibition of glucose transfer also occurred when the animals were treated with dexamethasone (see Fig. 2 and Table III). This inhibition, which required a higher level of dexamethasone than the induction of maternal hyperglycemia, counteracted the increased levels of maternal blood glucose at the higher doses. Separation of these two effects was evident in the experi-ments where the dexamethasone was injected directly into the fetus in utero (Fig. 4 and Table VIII). At the low doses, only inhibition was evident whereas above a c r i t i c a l level (between 25 and 50 ug dexamethasone) the steroid presumably reached mater-86 nal tissues at a sufficient level to induce hyperglycemia, just as i f the steroid had been injected into the mother. The deleterious effects of excess corticoids which have been observed (79), can be attributed to the reduction of glucose available to the fetus. The inhibition by corticosteroids of glucose uptake by peripheral tissue has been well documented. However, steroid-mediated effects on feto-placental uptake or transport of glucose have not previously been demonstrated. It is possible, i f corticosteroids inhibit feto-placental uptake of glucose, to reconcile the paradoxical observations of Hoar (108) that c l e f t palate in guinea pigs can be induced by adrenalectomy of mothers (109) as well as by administration of excess corticos-teroid. The pattern of abnormalities manifested by embryos sub-jected to these opposite levels of adrenal a c t i v i t y was found to be strikingly similar (108). In the l i g h t of the present work, however, this could be explained as follows: adrenalectomy re-sults in low maternal glucose levels,thereby lowering the amount available to the fetus. An excessive level of corticosteroid, although i t would induce maternal hyperglycemia, would also re-sult in inhibition of uptake of glucose by the fetus. Thus the same net effect is produced on the fetus when the animals are subjected to either hypo- or hyperadrenocorticalism: in both cases i t i s the glucose which is c r i t i c a l . There is a correlation between fet a l and placental size in almost every species examined (110). However, fe t a l growth and placental growth do not always go hand in hand. In these cases i t may be that an increase in placental weight would not be 87 accompanied by any corresponding increase in i t s capacity to transfer nutrients to the fetus. Other factors, such as the pressure at which maternal blood arrives at the placenta, may affect both placental and fet a l growth, although independently (110). Wigglesworth (111) has stated that in many cases of fe t a l growth retardation the primary placental abnormality has been impairment of the blood supply, followed by reduced growth of the placenta. Reduction of the blood flow i n the placenta would be detrimental to the supply of many of the v i t a l nutrients re-quired by the fetus. Corticosteroids have been shown to reduce placental blood flow (112) which could influence placental trans-fer function, although no work has been done to ascertain the c r i t i c a l nutrients affected. The biological role of this steroid-induced inhibition could involve control over f e t a l growth and development. The gluco-corticoids cause increased maternal blood glucose levels which w i l l be reflected in f e t a l hyperglycemia (105). Conditions of stress cause increased secretion of adrenal steroids and increas-ed glucose being available to the fetus. Since glucose is the c r i t i c a l factor for f e t a l growth} i f i t s entry were unrestric-ted, then the result could be uncontrolled growth of the fetus. This i s believed to be the case in "heavy" infants of diabetics where the elevated glucose level results in accelerated f e t a l growth (105). If uncontrolled, this may have deleterious effects on f e t a l development and survival. The f e t a l death which resul-ted from dexamethasone treatment (Table II) probably represents severe inhibition since the synthetic steroid would escape the 86 biological controls ( i . e . , binding and metabolism) which normally function. A possible mechanism whereby corticosteroids could inhibit the transfer of glucose from mother to fetus i s indicated by the presence of receptors in placental tissue. The hormone sp e c i f i -c i t y of the placental nuclear binding f u l f i l s one of the c r i t e r i a for receptor binding and also provides information concerning the nature of the cytoplasmic interaction. Cortisol and c o r t i -costerone interact with the active conformation of the receptor, forming complexes which are translocated into the nucleus. Pro-gesterone, cortisone, and cortexolone in this system behave as competitors with labeled glucocorticoids for binding. However, their i n i t i a l interactions must dif f e r since cortisone and pro-gesterone do not form complexes that can be isolated from the nu-cleus (Pig. 6)t whereas cortexolone w i l l apparently form a nu-clear complex (38). This complex, however, has no glucocorticoid a c t i v i t y since i t is not apparently recognized by the nuclear acceptor site(s) (113). The cortexolone therefore possesses suf-f i c i e n t structural s p e c i f i c i t y to bind to specific sites but can-not interact with the receptor to produce the "active complex" necessary to i n i t i a t e metabolic effects. Epicortisol does not compete with specific glucocorticoid binding and therefore has no a f f i n i t y for any of the conformations of this receptor system. Corticosteroid-binding globulins have been isolated from v i r t u a l l y a l l vertebrate species examined, including the mouse (9*+). These proteins are present in low concentration (less than 1 uM) but exhibit a high binding a f f i n i t y for the corticoids 89 (Ka = 10 to 10 v M~ ) as well as progesterone. Since progester-one does not form a nuclear complex in placenta this was one in-dication that the binding was not due to transcortin. The expe-riments with washed homogenate and with addition of maternal blood represent further checks on this point. Transcortin w i l l compete with the receptors for binding to the labeled steroid but very l i t t l e of the isolated nuclear binding could represent a steroid-transcortin complex. This data also indicates that N the isolated nuclear binding represented binding to a receptor which may have physiological significance. The presence of receptor molecules in the nucleus arises from translocation of these molecules from the cytoplasm, where the i n i t i a l interaction occurs. Therefore, examination of the cytosol for the presence of these receptors is a fundamental c r i -t e r i a for establishing a physiological role for the nuclear bind-ing. The binding properties of the cytoplasmic receptor indicates that the complex was unstable during long incubations at 37°C. This i n s t a b i l i t y has also been reported for crude extracts from thymocytes, even at k o C ( l l k ) . Many factors such as low protein concentration, the absence of stabilizing compounds which might be present in intact c e l l s , or the presence of proteolytic en-zymes could influence the stabilization (115). The nature of the corticosterone-receptor complex cannot be analyzed in great detail with a crude extract. However, the sensitivity of the binding to heat denaturation and proteolytic digestion suggests that the integrity of the protein is important for the binding. The fact that neither deoxyribonuclease nor 90 riboriuclease w i l l hydrolyze the corticosterone-receptor complex suggests that nucleic acid has no role in the. cytoplasmic binding. The sulfhydryl reagents also seem to have only a minor effect on the cytosol binding, which is different from the effects reported for the thymocyte receptor ( 1 1 5 ) . The kinetic analysis of the binding reveals that the speci-f i c receptors can be saturated but there are also present non-specific sites which are not saturated even at the highest con-centrations of corticosterone used. This pattern of steroid binding has also been reported for thymus tissue, in which cyto-plasm and nuclei both show two classes of receptors ( 1 1 6 ) . The non-saturable fraction most l i k e l y represents the nonspecific binding that Munck has reported (38, 6 5 ) , and is found with a l l steroids i f used at high concentrations ( 6 2 ) . This was substantiated by the sucrose density analysis of the specific binding fraction from Sephadex (Fig. 1 3 ) . The lower steroid concentration used, along with the lengthy centrifugation time, would vi r t u a l l y eliminate any nonspecific binding. The single radioactive peak, which sedimented at approximately 4 s , has a similar elution profile on sucrose gradients to the gluco-corticoid-binding macromolecule that Kaiser, Milholland, and Rosen have isolated from rat thymocytes ( 1 1 7 ) , Plasma transcortin appears to be ruled out as one of the glu-cocorticoid receptors in mouse placenta. In contrast to the tight binding of Cortisol and corticosterone, transcortin does not bind dexamethasone (72, 9*0. Therefore, dexamethasone would not be expected to compete with labeled corticosterone for binding i f 91 the "binding component were transcortin. Since excess unlabeled dexamethasone prevents the binding of -^H-corticosterone by the cytosol receptor (Fig. 1 3 )» this indicates that transcortin (as in the case with the receptor from nuclei) does not account for any of the cytosol receptor binding. Many of the properties of this receptor from the placenta are similar to those properties reported for glucocorticoid-bind-ing macromolecules from other tissues. Thus, there i s good cor-relation between the in vivo hormone activity and the binding properties of steroids i n this system. The sensitivity of the cytosol complex to heat and pronase dissociation and the lack of effect of deoxyribonuclease and ribonuclease also demonstrates that this cytosol receptor, as with a l l receptors examined to date, i s at least in part protein. A notable difference i n the properties, however, is the apparent lack of effect by the sulf-hydryl reagents on the binding. Whether or not this has any sig-nificance in the physiological function of this receptor remains to be established. If the glucocorticoid-induced inhibition of glucose uptake by mouse fetuses is related to the presence of cytoplasmic and nuclear receptors in placental tissue, then the two processes should respond to similar treatments. The apparent abolition of both the maternal hyperglycemia and the inhibition of f e t a l up-take by actinomycin D indicates that these processes require de  novo nucleic acid synthesis. The interaction of glucocorticoids with target tissues has been shown to be sensitive to metabolic inhibitors. To establish this link conclusively would require 92 further investigation using specific metabolic inhibitors to. demonstrate that the inhibition of glucose uptake by steroids proceeds along some chain of metabolic events similar to other glucocorticoid-induced effects (63, 118, 119). Since the fluorinated corticosteroids bind with much grea-ter a f f i n i t y to glucocorticoid receptors than the natural ster-oids (117); examining the competing effects of, for example, a 9#-fluoro derivative of cortexolone may indicate more con-clusively the physiological significance of this receptor. This might be accomplished by using such a compound to block the dexamethasone-induced inhibition in vivo. The ultimate proof to link the two processes, however, would involve isola -tion of a specific factor induced in placental tissue via the receptor system in a manner analogous to the induction of specific enzymes in adult l i v e r . If this factor then played a role in the transport or uptake of sugars by placenta, the role of the receptors in this action of the glucocorticoids could be established. 93 BIBLIOGRAPHY 1. B r i t t o n , S.W., and S i l v e t t e , H., Amer. J. Med. 100:693 (1932). 2. C o r i , C.F., and C o r i , G.T., J. B i o l . Chem. 24*743 (1927). 3. 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(U.S.A.) 68:1269 (1971). 100 APPENDIX I Preparation of Krebs-Eggleston Phosphate Buffer, pH 7.4 Stock solutions Concentration (M) Relative volume used NaCl 0.154 100 KC1 0.154 4 C a C l 2 0.110 3 KHgPO^ 0.154 1 MgSO^ • 7H 20 0.154 1 NagHPO^ • 2H 20* 0.100 12 * adjusted to pH 7.4 with IN HCl 101 APPENDIX I I . S t r u c t u r e s of the S t e r o i d s Used llp-cortisol lla-cortisol cortisone llp-corticosterone lla-corticosterone cortexolone 102 APPENDIX III E f f e c t of Stress on Pregnancy i n A/J and C57BL/6J Mice Although there i s no documented evidence f o r a s t r a i n difference i n the s u s c e p t i b i l i t y to stres s between the A/J and C57BL/6J mice, there are indications that a difference e x i s t s . The "Handbook on G e n e t i c a l l y Standardized Jax Mice", issued by The Jackson Laboratory, notes that there i s a 10% difference i n the number of f e r t i l e matings between the two stra i n s (C57BL/6J higher). When pregnant mice are ordered there i s also a difference i n the percentage of pregnant a n i -mals i n each s t r a i n , The C57BL/6J had greater than 90% preg-nancy whereas the A/J occasionally a r r i v e d with l e s s than 30% of the animals pregnant. Even though the "two s t r a i n s did not a r r i v e together, there were indications that a difference i n t h e i r response to stress during shipping d i d e x i s t . To t e s t t h i s , pregnant animals of both s t r a i n s were order-ed at the same time. These animals were then treated from gestational day 13 to 17» i n c l u s i v e , by p l a c i n g them i n a sha-ker f o r 3 hrs each day and shaking them a t a frequency of 180 cycles per min. The animals were then examined on day 17 f o r evidence of pregnancy. Out of Ik animals each, the A/J had two d e f i n i t e pregnancies with viable fetuses and two animals with d e f i n i t e indications of resorption. The G57BL/6J had f i v e d e f i n i t e animals pregnant and two others with evidence of resorption. Therefore, out of lk animals each, the A/j had a t o t a l of four possible pregnancies; and the C 5 7 B L / 6 J had seven possible. 

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