Table Of ContentEXPERIMENTAL ENDOCRINOLOGY
A SOURCEBOOK OF BASIC TECHNIQUES
by
M. X. ZARROW J. M. YocfflM
Department of Biological Sciences Department of Comparative
Purdue University Biochemistry and Physiology
Lafayette, Indiana University of Kansas
Lawrence, Kansas
J. L. MCCARTHY
Department of Biology
Southern Methodist University
Dallas, Texas
WITH A CHAPTER ON INVERTEBRATE HORMONES BY
R. C. SANBORN
Department of Biological Sciences
Purdue University
Lafayette, Indiana
1964
ACADEMIC PRESS
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First Printing, 1964
Second Printing, 1965
PRINTED IN THE UNITED STATES OF AMERICA.
Dedicated to our respective wives, whose aid and encouragement
was of inestimable value in the completion of this book.
Preface
Endocrinology is a subject that has come to the fore only since the
third decade of the twentieth century. As a result, this discipline has
borrowed concepts and methods from areas in the biological sciences
that were well established at the time of its infancy. At one time or
another endocrinology has been taught as part of formal courses in
anatomy, physiology, biochemistry, pharmacology, internal medicine,
and other subjects. Indeed, it is part of all of these areas, but our
knowledge has so increased that endocrinology can no longer be taught
adequately as part of another course. To permit the student to synthesize
concepts from the vast array of experimental data requires a study of
the subject as such, and not as a part of one of the older disciplines. One
might say that endocrinology has come of age.
Houssay, Nobel Laureate in Medicine, has stated that physiology can
be taught properly only by practical and individual teaching. Indeed,
this is also true of endocrinology. A discipline that has borrowed tech
niques from so many areas of science offers a wide variety of experiences
and experiments for the student. Only by working in the laboratory with
the hormone, the animal, the cell, and the test tube can the student truly
learn to appreciate the role of the hormones in the regulation of physio
logical events. In this respect, the present text is dedicated to the appre
ciation and understanding of the whole organism and its interrelationship
with the environment.
This book, based on a laboratory course that was first taught in
Professor F. L. Hisaw's laboratory in 1946, is designed as a textbook
and manual for use in an advanced course or for use by the individual
student in a project course. Therefore, the student should possess some
basic knowledge in the areas of physiology, biochemistry, histology, and
endocrinology. Furthermore, the inclusion of an extensive list of refer
ences is designed to give the student an opportunity to examine the
literature pertinent to the laboratory work he is doing, and to utilize
this literature in the preparation of his written reports.
The practical goal of the book has been to include, in addition to
vii
viii PREFACE
those exercises which have been tried at classroom level, some tech
niques not commonly used in the classroom, but nonetheless applicable.
An attempt also has been made to include more exercises than can be
used in a one-semester course. This should permit the student or instruc
tor to select those exercises best suited to the student's goal and within
the means of the laboratory.
Finally, it should be noted that not all of these experiments will be
successful in different laboratories. Such factors as strain of animals,
environmental conditions, nutritional state, etc., may influence results.
It may be necessary to change treatment schedules, dosages, etc., but
regardless of results, the laboratory experience should be of inestimable
value. Under the direction of the authors, about 85 per cent of these
experiments have worked successfully at the student level.
M. X. ZARROW
April, 1964
CHAPTER 1
Introduction
During the early history of endocrinology, it became apparent that
a similar approach was used by many investigators in studies on the
identification of glands of internal secretion and their secretory products.
This classical approach consisted of three steps: ablation, replacement
therapy, and chemical isolation and purification.
Ablation, the surgical removal of the tissue or gland in question, was
the initial step and, if successful, usually resulted in a specific syndrome
or deficiency state directly ascribable to the loss of the gland. Once the
deficiency state was induced experimentally, replacement therapy was
instituted in an effort to prevent the syndrome. This was effected by the
reimplantation of the gland in question back into an animal. Thus, the
testes, for example, removed from their normal location, were immedi
ately reintroduced into another part of the body, thus preventing -the
appearance of castration symptoms. Such an experiment was first de
scribed in the classical work of Berthold. It is now possible, by utilizing
highly inbred strains of animals, to permit the deficiency symptoms to
develop following ablation and then to alleviate the state by the implan
tation of a similar gland from a genetically similar animal. A final stage
in this step was to prepare a crude extract of the gland which could be
injected into the animal after ablation. The third step in the identification
of an endocrine gland and its secretory products was the isolation of the
stimulating substance from the gland, and its purification.
Sources of the Hormones
The classical development of the experimental method described
above provided all the necessary criteria by which a substance could be
identified as a hormone. According to the original definition, a hormone
is a specific chemical entity that is secreted into the blood by a well
defined group of cells and is transported via the vascular system to a site
distant from its point of origin, where its action takes place. Similarly,
the endocrine gland was described as a well defined group of cells that
1
2 1. INTRODUCTION
secretes a unique organic chemical product directly into the circulation
rather than through a series of ducts, as do digestive glands such as the
salivary gland or the exocrine pancreas.
Thus, the endocrine system and its hormones was thought to be a
system for control and integration of physiological processes analogous
to, but separate and distinct from, the other great integrative mechanism
in the organism—the nervous system. Unlike the nervous system, which
is almost entirely of ectodermal origin, the endocrine system is com
posed of organs that vary widely in embryological origin, being derived
from all three primitive layers, ectoderm, mesoderm, and endoderm
(see Table 1-1). Again unlike the nervous system, whose action is rapid
and ephemeral, the endocrine system exerts its effects over prolonged
periods of time, sometimes for weeks or months.
TABLE 1-1
THE EMBRYONIC DERIVATION OF THE DUCTLESS GLANDS AND THE
TYPES OF HORMONES PRODUCED
Embryonic
source of Derivation Type of
gland of gland Gland secretion
Endoderm Pharyngeal pouches Thyroid Iodinated amino acids
Parathyroid Proteins or polypeptides
Intestine Islets of Langerhans Protein
Mesoderm Mesenchyme from Adrenal cortex Steroids
dorsal mesentery
Genital ridge Ovary, testis Steroids, protein, or poly
peptide
Ectoderm Neural crest material, Adrenal medulla Cathechol amines
chromaffin tissue of
primitive sympa
thetic nervous
system
Stomadeum Adenohypophysis Proteins
Diencephalon Neurohypophysis Polypeptides
However, it soon became apparent that both the nervous system and
the endocrine system were not entirely distinct and that each utilized
"methods" of the other. For example, the adrenal medulla might be con
sidered as an extension of the nervous system. This group of cells (within
the adrenal gland in mammals) originates from nervous tissue, receives
an abundant preganglionic autonomic nerve supply, and when stimu
lated secretes into the circulation epinephrine and norepinephrine. Nor
epinephrine is identical in structure and function to the substance
3
SOURCES OF THE HORMONES
released by most postganglionic sympathetic nerve fibers. If, however,
norepinephrine is accepted as a hormone, its alternate tissue source,
the postganglionic sympathetic nerve ending should be considered an
endocrine structure; and if so, one is tempted in addition to admit acetyl
choline into the realm of hormones.
Indeed, recent findings indicate that the hormones vasopressin and
oxytocin are stored in the posterior pituitary gland but originate in
certain neurons of the hypothalamus. These hormones then pass from
the hypothalamus to the neurohypophysis by way of the axons of the
hypothalamic-hypophyseal nerve tract. Such an origin of certain hor
mones in neural tissue has long been recognized in insects and crusta
ceans. In mammals, it is now postulated that a number of hypophyseal
releasing factors are formed in the hypothalamus, pass directly to the
anterior pituitary gland via a portal circulatory system, and influence the
release of such hormones as the gonadotropins, adrenocorticotropin, and
thyrotropin. It is apparent that the cells of the nervous system in many
different species, ranging from mammals to the arthropods, possess the
ability to produce both hormones and other substances that affect and
control the release of hormones from endocrine glands.
Thus, it appears that a substance may be classified as a hormone even
though (a) it is secreted by the nervous system, (b) it does not act at
a site distant from its point of origin, and (c) it is not transported by
the vascular system. Once these original restrictions are lifted, it appears
that there exist a large number of naturally occurring substances, some
times called tissue hormones, that frequently are disregarded in the
study of the endocrine system. These are such substances as renin, hista
mine, heparin, and serotonin. Obviously many more data of a physiolog
ical nature are needed before one can attempt to classify such compounds
and the "ductless glands" from which they come. But it must be remem
bered that classifications are simply aids to an understanding of
knowledge. Definitions must continue to remain flexible and open to
revision as new information is made available. In the light of new
findings and the need for definitions broad enough to encompass them,
a hormone might now be defined as a physiological organic substance
that is secreted by living cells in relatively restricted areas of the
organism and that diffuses or is transported to a site in the same organ
ism, where it brings about an adjustment that tends to integrate the
component parts and actions of the organism.
Recently a new subdivision of endocrinology made its appearance; it
is growing with amazing rapidity. This is neuroendocrinology, which
concerns itself with (a) the interaction of the nervous system with the
endocrine system, (b) the secretion of hormones or neurohormones by
4 1. INTRODUCTION
the nervous system, and (c) the influence of hormones on the nervous
system. In addition, the classical approach to the study of endocrinology
has been amplified in recent years by studies concerned with the bio
genesis, metabolism, and mechanisms of action of the hormones and with
the integrative actions of groups or combinations of hormones. These
new approaches and the kinds of information they yield are described
below.
Nature of the Hormones
Compounds that exhibit hormone action may be found in many
molecular sizes and shapes. However, the hormones may be classified
generally into three groups on the basis of their chemical structures:
amino acids or their derivatives, such as epinephrine, norepinephrine,
acetylcholine, and the iodothyronines; steroids related to cholesterol,
such as testosterone, progesterone, corticosterone, and estrone; and
polypeptides and proteins, with or without an additional carbohydrate
moiety attached, such as corticotropin, melanocyte stimulating hormone,
relaxin, follicle stimulating hormone, insulin, and parathormone.
Many hormones are normally bound to plasma proteins and circulate
with them. This includes both steroid and nonsteroid hormones. In the
case of the estrogens, for instance, a specific complex is formed with
blood protein (estroprotein complex) which appears to be of major
importance in the transport and activity of the hormone. If the binding
of the estrogen is prevented by subtotal hepatectomy, activity is lost.
It has long been known that thyroxine is bound to the a-globulin
fraction of blood and to albumin. Actually, one of the chemical tests for
circulating thyroxine level is based on the determination of protein-
bound iodine (PBI). The protein-bound thyroxine is inactive as such
and must be in the free state before it can act at the cellular level. Dur
ing pregnancy in the human being, the thyroxine level of the blood
increases markedly whereas the basal metabolic rate remains normal.
It is apparent that the hormone is not acting at the cellular level under
these circumstances, either as a result of a decreased rate of diffusion
of the hormone into the tissues, a control of the mechanism responsible
for splitting the thyroid-protein complex, or an increase in thyroid hor
mone catabolism or excretion to keep pace with its increased synthesis.
Hormones also appear to show selectivity for certain tissues and are
bound by these tissues. Thus, insulin is rapidly bound by muscle and by
adipose and mammary tissue, and to a limited extent thyroid stimulating
hormone is bound by the thyroid gland, and the gonadotropins appear
to be trapped by the gonads.
ACTION OF THE HORMONES 5
Several general characteristics seem to apply to all compounds that
have been classed as hormones to date. These compounds (a) act in
trace amounts; (b) are not sources of energy for biological reactions;
(c) regulate rates of chemical reactions, but do not initiate new reac
tions; (d) require a latent period before an effect is apparent; (e) are
rapidly removed from the circulation and metabolized by the tissues,
sometimes within minutes; (f) appear to be active only in tissues that
can respond or are sensitive to their action, i.e., have a specificity of
action dependent in great part on tissue sensitivity.
Many of these characteristics also apply to the vitamins and to
enzymes. However, all the hormones, unlike many vitamins, are pro
duced within the animal and thus cannot be considered akin to the
non energy-supplying dietary requirements of the organism, such as
the vitamins. Hormones differ from the enzymes in that they appear to
be more highly tissue specific, are produ6ed in relatively localized areas
by specialized tissues (compared to many enzymes that are universally
present in almost all tissues), and tend to cause physiological adjust
ments that are observable at the organismic level rather than at the
intracellular level. Recent research however, has provided clues which
suggest that the hormones do work in conjunction with enzymes, acting
as cofactors, to regulate reaction rates within the cell.
Action of the Hormones
The actions of the hormones in the living organism may be classified
under three general headings: morphogenesis, homeostasis, and func
tional integration.
A. MORPHOGENESIS
Morphogenesis encompasses the growth, differentiation, and matura
tion of the organism. Nearly all the hormones (with the possible excep
tion of the posterior pituitary hormones, pressor amines, and neuro-
humors) possess some morphogenetic activity. Somatotropin (STH)
provides an outstanding example of this type of action since it stimulates
the overall growth of the organism. Similarly, thyroxine stimulates
growth, differentiation, and maturation of specific tissues, such as the
nervous system, as well as a general metamorphosis in some animals.
Testosterone, a protein anabolic hormone, stimulates both general protein
synthesis and growth of the male reproductive tract. Estradiol is a
hormone primarily responsible for the growth and development of the
female reproductive tract.
