Monographs on Endocrinology Volume 10 Edited by F. Gross, Heidelberg· M. M. Grumbach, San Francisco· A. Labhart, Zlirich M. B. Lipsett, Bethesda . T. Mann, Cambridge 1. T. Samuels, Salt Lake City . J. Zander, Mlinchen WI.P Mainwaring The Mechanism of Action of Androgens With 12 Figures Springer-Verlag New York· Heidelberg· Berlin w. IAN P. MAINWARING Androgen Physiology Department, Imperial Cancer Research Fund London WC2A 3 PX/Great Britain ISBN-13: 978-3-642-88431-3 e-ISBN-13: 978-3-642-88429-0 DOl: 10.1007/ 978-3-642-88429-0 Library of Congress Cataloging in Publication Data. Mainwaring, W.LP. The mechanism of action of androgens. (Monographs on endocrinology; v. 10). Bibliography: p. Includes index. 1. Androgens. 1. Title. QP572.A5M34 599'.01'927 76-29057 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. © by 1977 Springer-Verlag New York Inc. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. This monograph is dedicated to Dr. G. F. Marrian, C. B. E., F. R. S. His work and words largely forged my interest in steroids. Preface My introduction to androgens was neither auspicious nor impressive. I was sitting my viva voce examination for a degree in physiology and had haltingly intimated to my examiner (name decorously withheld) that I intended to pursue a career in re search. "On what topic?" was the reply. I had been deeply impressed by the work of C. Huggins and C. V. Hodges (Cancer Res. 1, 293, 1941) on the dramatic arrest of canine prostatic hyperplasia by the administration of stilboestrol. With some en thusiasm, I responded, "On steroid hormones, because I am struck by the profound effects that may be achieved by relatively small numbers of molecules." The examiner sank into deep contemplation before replying, "Young man, have you considered go ing into teaching?" Suitably chastened, I finally began my research career investigating the effects of steroids on the nucleic acid metabolism of experimental tumours and on the process of senescence in animal cells. Reaching an impasse in this work, I mentioned one day to Dr. G. F. Marrian that, somewhat surprisingly, we had no understanding of the fundamental mechanism of action of steroid hormones, especially the androgens. He encouraged me to tackle this problem, particularly since exciting new insights were then being made into the interaction of radioactively labelled oestradiol-l?,8 with such tissues as rat uterus. My first faltering experiments on the mechanism of action of androgens began in the late summer of 1968 and have continued ever since, with a particular fillip in 1971 when I was fortunate enough to be made head of a Department of Androgen Physiology. In retrospect, the experience has been extremely rewarding, for after all, I am doing what I initially resolved to do. While at times, the road has been tortuous and beset with deep potholes, at others it has led to clear and exciting heights. In preparing this monograph, I am deeply obligated to the many people con cerned, directly and indirectly. First, to past and present members of my Department, without whose interest, enthusiasm and diligence, nothing could have been accom plished. Second, to the Director of Research of this Institute, Dr. Michael Stoker, C.B.E., F.R.S., for his continued interest in the work and the provision of outstanding research facilities. Third, to my overworked typist and secretary, Cilla Conway, and to Dr. Heinz Gotze and his staff at Springer-Verlag for helping to make my transi tion from random thoughts into lucid words a practical reality. Finally, to my family and friends, for forgiving a few irascible outbursts during the process of writing and giving the encouragement and strength to complete the task. London, September 1976 W. I. P. MAINWARING Contents Introduction 1 1. A Contemporary Model for the Mechanism of Action of Androgens. 5 1. Definitions .................. 6 2. The Model for the Mechanism of Action of Androgens 8 3. Experimental Support for the Model . . . . . . . 10 a) Antiandrogens .............. 10 b) Experimental Testicular Feminisation Syndromes . 10 c) Specificity of Induction and Dose-Response Phenomena 11 n d) Temporal Integration of Steroid-Mediated Responses e) Tumours with Different Sensitivities to Androgens. 14 4. Limitations of the Model of Androgen Action. . . 16 a) Fundamental Mechanisms of Hormone Action. 16 b) Redundant (Aberrant) Binding . . . . . . 16 c) General Applicability of the Model . . . . . 17 d) Acute Tissue-Specificity of Androgenic Responses 17 e) Androgen Responses Independent of the Receptor System 18 f) Hormonal Synergism . . . . . . . . 18 g) Responses Independent of Transcription 19 5. Conclusions ............. 21 II. The Metabolism of Androgens in Relation to Their Mechanism of Action. 23 1. Relative Biological Activities of Testosterone and Its Metabolites . 25 2. General Comments on the Metabolism of Androgens. . . 29 3. Developmental Aspects of the Metabolism of Testosterone 32 a) Foetal Development . 32 b) Neonatal Development . . . . . . . . . . . . . 33 c) Adult Development . . . . . . . . . . . . . . 36 d) Fluctuations in the Circulating Concentrations of Androgens. 37 4. Androgenic Responses Not Mediated by 5a-dihydrotestosterone 38 5. Interconversion of Steroid Hormones 40 6. Conclusions . . . . . . . . . . . . . . . . . . . . . 42 x Contents III. Initial Events in the Mechanism of Action of Androgens. 43 1. The Uptake, Retention, and Release of Androgens: Studies on Rat Ventral Prostate Gland . . . . . . . . . . . . . 44 a) Transport of Testosterone and Related Steroids in Plasma 44 b) Entry of Testosterone into Cells. . . 45 c) Cytoplasmic Receptors for Androgens . . . . 46 d) Antiandrogens ............. 53 e) The Nuclear Binding of 5a-dihydrotestosterone 54 f) Reconstituted, Cell-Free Systems for the Nuclear Transfer of 50!- dihydrotestosterone: the Acceptor Hypothesis . . . . . . . .. 57 g) The Structure of Chromatin and the Nature of Nuclear Acceptor Sites . . . . . . . . . . . . . .. ........ 62 h) The Exit of 5a-dihydrotestosterone from the Prostate Gland: Re- ceptor Cycling. . . . . . . . . . . . . . . . . 72 i) Binding of Steroids Other than 5a-dihydrotestosterone 73 i) Purification of Receptor Proteins . . . . . . . . . 74 2. The Binding of Androgens in Other Target Cells . . . . 78 a) Distribution and Specificity of Receptors for Androgens and Related Steroids . . . . . . . . . . . . . . . . . . . . 78 b) Physical Properties of Cytoplasmic and Nuclear Receptors 87 c) Binding of Hormones Other than Androgens . 88 d) Specificity of Androgenic Responses . . . . 89 3. Initial Biochemical Events Stimulated by Androgens 92 a) Protein Synthesis . . . . . . . . . . . . 92 b) Activation of Protein Phosphokinases . . . . 92 c) Stimulation of the Initiation Step of Protein Synthesis 93 d) The Androgenic Regulation of RNA Synthesis: Receptor Effects in t1itro 94 4. Conclusions 97 IV. Early Events Stimulated by Androgens . . . 99 1. Early Biochemical Changes Stimulated by Androgens 100 2. A More Detailed Enquiry into Enzyme Induction by Androgens: Aldolase in Rat Prostate . . . . . . . . . . . . . . 106 3. Experimental Models for Studying Tissue-Specific Responses 108 a) Rat Seminal Vesicle . . . . . . . . . . . . 108 b) Mouse Kidney . . . . . . . . . . . . . . 111 4. Morphologic Aspects of Androgenic Stimulation 116 5. Conclusions . . . . . . . . . . . . . . . 119 V. Late Events in the Mechanism of Action of Androgens. . . 121 1. Cell Renewal and Morphologic Studies on the Mitogenic Activity of Testosterone and Its Metabolites . . . . . . . . . . . . . . .. 122 Contents XI 2. Biochemical Approaches to the Regulation of Mitosis in Rat Ventral Prostate Gland . . . . . . . . . . . . 125 a) Enzymes Engaged in DNA Replication 125 b) Proteins Regulating the Onset of Mitosis 128 c) DNA-Unwinding Proteins 129 d) Synthesis of Histones 132 3. Conclusions ..... 133 Overall Conclusions 135 References 142 Subject Index 173 Introduction From the dawning of civilised time, when man forsook the life of the nomadic hunter and adopted the mantle of the communal farmer, his interest in androgens or, more correctly, testicular secretions, has continued unabated. As early man strove to establish his supremacy over the animal world, the extirpation of certain organs from domesticated birds and beasts was widely practised. The capon was recognized as a palatable culinary delight, the ox provided an exceedingly strong but biddable beast of burden and the gelding possessed advantageous features as a dependable yet fast mode of transport. Despite the gradual acquisition by man of an ever-thickening veneer of civilisation and sophistication, these castrated animals remain a feature of life even today. As man developed a more inquisitive and perceptive attitude toward himself, his environment and his procreation, interest in the gonads escalated in the form of complex sexual taboos and fertility rites. In attempts to heighten libido and reproductive prowess, extracts of moulds, plants, and molluscs achieved a measure of mystique as aphrodisiacs and the gonads of slain adversaries were often ingested in the hope of perpetuating sexual activity. Contemporary man tends to place himself above such misguided beliefs, yet for these very reasons the white rhinocerus is nearly extinct and the enigmatic Yeti, the Abominable Snowman of the Himalayas, was claimed to have removed the gonads from its victims. All aspects of this bizarre affair remain to be substantiated, however. Since the time of the ancients, ablative surgery on male reproductive organs was practised for certain putatively expedient purposes. The barbaric custom of mollifying slaves and captives by castration began in the southern areas of the Aegean and persisted in a wider sphere up to the time of the Inquisition. In Russia, the celibacy of extreme religious sects, notably the Skopetsy, was given an incontestable finality by the removal of the testes of novitiate disciples. Castrati were also common in cathedral choirs until the end of the eighteenth century. The origins of a more scientific enquiry into the male reproductive glands are hidden in the mists of time but in their authoritative reference treatise, DORFMAN and SHIPLEY (1956) suggest that Aristotle (quoted in Historia Animalium, vol. 4, Book 9) was the first to record the profound changes evoked in the cockerel after castration. As with other aspects of cultural and scientific endeavour, further progress and research was lost in the abyss of the Dark Ages, only to begin again in the welcome light of the Renaissance. Then, scientific and aesthetic appreciation of the human body emerged anew. New medical schools were instituted, especially in Italy, where the new concepts of human anatomy laid down by Vesalius in Padua and Pavia brushed away the cobwebs of the past and in the arts, the dignity of the human form reached a pitch of breathless perfection in Michelangelo'S statue of David. 2 Introduction These were the trends developing in European cultures, but the remarkably per ceptive researches of NEEDHAM and Lu (1968) indicate that the Chinese may well have isolated and purified steroids by this time. This is a further example of the inventiveness and skill of the alchemists of ancient Cathay. Slowly but surely, scientific progress kept pace with this new and invigorating pursuit of knowledge and the following contributions, all acknowledged classics of endocrinological research, laid the foundations of our understanding of the mecha nism of action of androgens. Pott, in a paper redolent with the social injustices of his time, remarked on the extremely high incidence of cancer of the scrotum in those desperate unfortunates of the industrial revolution, the chimney boys and noted the impairment of sexual activity associated with this disorder. In one master stroke, the foundations of chemical carcinogenesis and testicular function had been established. Among many contributions from a remarkably colourful and illustrious career, Hunter noted that on removal of the gonads from male animals, many organs distant from the testis atrophied. Hunter surmised that the growth of certain glands, the accessory or secondary sexual organs, was uniquely dependent on the presence of the testis. In a series of bold and imaginative experiments involving the transplantation of the gonads in cockerels, Berthold made a revolutionary contribution. Provided that the blood supply to the gonads was restored, despite their transplantation to other bodily sites, then the reduction in the growth of the comb, so characteristic of the castrated capon, was not evident. However, ligation of the blood supply from the gonads, irrespective of their anatomical location, was manifest in a prompt reduction in comb growth. Taken in concert, Berthold's results indicated that the growth of the secondary sexual glands was maintained by some testicular component that was dis tributed by the general circulation. Just before the turn of the last century, two enter prising surgeons, Ramm in Norway and White in the United States of America, independently advocated castration for the palliation of human benign prostatic hyperplasia, a disorder of then unproven testicular aetiology. Finally, bringing male reproductive physiology into the present era, Brown-Sequard capitalised on the earlier teachings of the Indian Ayur-Veda and claimed that the injection of testicular extracts, in his case self-administered, was beneficial for the rejuvenation of the ageing human male. All of these historic contributions were brought into close har mony when STARLING (1905) formulated his concept of hormones (in Greek, OPP.UOJ, to arouse or excite), the chemical messengers secreted by discrete glands di rectly into the blood stream in order to regulate the function and growth of organs elsewhere. The unequivocal demonstration of hormonal activity in testicular extracts was first achieved by PEzARD (1911) and thus he may be considered the father of the contemporary physiology of male sex hormones. Between 1920 and 1940, impressive advances were made into the physiology and chemistry of hormones, especially those with the basic steroid structure related to pentaperhydrophenanthrene. Many factors were responsible for this success. The preservation and sectioning of tissues was now an advanced skill, being aided by a wealth of precise histochemical stains whereby intracellular components could be specifically located at low concentrations. By such means, the structure of many hor mone-producing glands such as the adrenals and ovaries was unequivocally established. Experimental physiology also became a sophisticated discipline and the development of techniques for the selective ablation of endocrine or hormone-producing glands,