Endocrine Methodologies

ome of the methods presently used in endocrinological studies were developed for more practical purposes. In many cultures castration was practiced as a form of punishment. In the Middle and Far East castration was performed to provide servants (eunuchs) for harems; in Italy castrated young boys were trained to be adult sopranos. Castration is presently used to improve the flavor of meat from some domestic animals (e.g., castrated chickens produce capons). These "practical" operations were the forerunners of the gonadectomies of present-day endocrinological studies. The idea that glands contained humors or substances that could act as replacement therapy for a lost function of a gland was first el1lertained seriously by a French physician, Charles Brown-Sequard. He injected himself with extracts from dog, guinea pig, and rabbit testicles and proclaimed that the extracts had remarkable rejuvenating effects. Brown-Sequard even recommended that extracts be obtained from the mature calf to give to men the vigor of horses and other larger animals. It is now believed that these extra cts had only a placebo effect. N evertheless, glandular extraction, purification, and injection into animals have become important methods in elucidating the hormonal role of particular tissues and organs. Although hormones control a large variety of physiological evenls, their basic function is to act at the level of the cell to stimulate cellular physioLogicaL processes inherent in the cell. M eLanocytes, for exampLe, synthesize the pigment meLanin. Melanocyte-stimuLating hormone (MSH) enhances melanin formation by increasing the activity of the rate-limiting enzyme tyrosinase. Cortisol is a hormone produced by adrenal steroidogenic tissue. Corticotropin (ACTI-I) activation of adrenocortical enzyme activity leads to eLevated cortisol biosynthesis. Thus effects of hormones Clre reflected in physioLogical processes in the many cell types present within an organism. This chapter summarizes some of the methods employed by endocrinoLogists to study endocrine glands and the target cells and tissues that they regulate.

nsi

rations

The met hods now emp loyed to study endocrine systems are highly diverse [8,9,29]. lassical in vivo and in vitro me thods include surgical manipulations, tiss ue extract preparation for hormo ne isolation a nd ide ntification , histological methods for the localiza tion of hormones, and numero us hormon e and receptor assay methods. Mod ern enducrin e research utilizes virtually any of the latest molecular, cellular, physiological, behavioral, and gene tic approaches-often in combination with the classical techniques-to further our understanding of hormone synthesis, secretion, physiological roles, and mechanisms of action. The techniques described in what follows will be mentioned in more detail in later chapters.

68

Chapter 4 Endocrine Methodologies

o Endocrinologists

to

bout

orl"lo

Following discovery of a hormone, endocrine studies usua lly focu s on the following investigations (not necessarily in the order delineated):

1. Source. The distribution of the hormone will be determined. T he hormone may be fo und in more tban a single organ, tiss ue, or cellular source. For example, several gastrointestina l bormones are also found within the central nervo us system. 2. Structure determin({/ion and synthesis. D epending on the type of hormon e, the structure will be determin ed by any of a variety of m e thods. I n the case of a peptide hormone, the pri mary (amino acid) sequence will be determined. From this in forma tion the peptid e will then be synthesized and its biological acti vity, compared to th e purified extract, will be determined. This will estab lish the substance and structure as the authen tic hormone. 3. Biosynthesis. T he biosyntbetic pathway of hormone production should be delineated. Knowledge of th e primary stru cture of a peptide hormone can be used to predict the cOITesponding sequence of th e gene that encodes it; the gene can be cloned and the c DNA struc ture corresponding to the hormon e m R N A can be used to predict the prohormone structure and possible e nzymol ogical even ts rel ated to production of the mature horm one.
4. Controlo/secretion. The extrinsic or intrinsic factor(s) regulating the control of hormone secretion must then be determined. E ndogenous stimuli may involve negative or positive feedb ack by other hormones, or the circulating products (oth er hormones, metabolic substra tes). or consequences (blood volume, water and/or electrolyte composition of the blood) of its actions. The nervous system may directly or indirectly regulate hormone secretion from endocrine organs or tissues.

5. Cellular mechanisms of secretion. O nce the first messe nge rs regulating hormone secretion have been determined, it is then necessary to detelmine the nature of the second messengers and structura l elements (ion chann els, cytoplLL<;mic organelles) that participate in the process of hormone release (secretion). 6. Circulation and metabolism. It is importa nt to determine the half-life of the horm one in the systemic circ ulation. Ste roid or peptide hormones may be noncovalently bound to circulating proteins. Fluctuati ons in the levels of the "binding" proteins may affect the total amo unt of hormone present in the blood an d therefore availabl e for hormone action. Th e half-life of the hormon e in the circulation may be affected by degradation or other alterations of the hormone by serum enzymes. The retention time of the horm one in the circulation may also be affected by processes of filtra tion by the kidney. 7. Biological actions and roles. Removal of the hormone fro m the body by one or more methods usuall y will result in physiological effects in the anima l that will predict one or more functi ons for the horm o ne. Administration of the hormone (rep lacement therapy) or related ana log to the ani mal sh ou ld confirm one or more roles for the horm one. Studies on othe r species of animals may suggest add iti on al or alternate roles for the hormone. 8. Mechan isms 0/ Clclio n. Following administration of the hormo ne, in vivo and in vitro ce llular changes in biochemical processes and products shou ld indi cate one or more second messengers involved in hormone acti on. The receptor and signal transd uction mech an isms involved in hormon e action should define the temporal aspects between receptor acti vation and cellular response. Structure-activity st udi es will deter mine the esse nti al features of the hormone required for horm onc action, in the case of a peptide, the "message seq uence." 9. Pathophysiological aspects. The existence of a ho rmone is someti mes glea ned from the symptoms of a human pathophysiological condition. In other cases the discovery of a hormone in experim enta l ani mal s provi des a n ew explanatio n for a previ ously misunderstood clinical condition. In either casc. it is important to study the re lati ons hip be twe en the normal bi ology of a horm one and th e clinical consequ ences of alterations in synthesis. secreti on, and actio ns of that hormone. These studies take place in animal models of h uma n diseases, a nd to the extent that is ethically possible, in h uman clinical popu latio ns.

Hormon R pI c m nlTh r

69

10. Comparative endocrinology. Some bormones are evolutionarily conserved in struc-

ture and/or function in organisms ranging from the primitive to tbe most reccntly evolved. The structures and biological roles of other hormones, however, ha ve clearl y evolved and beco me specialii'.ed for particular adaptations in variolls species. In som e cases, the respon ses to the same hormone molecule are specialized by the types of receptors and signaling mechanisms that al'e present in the respol1sive cells. In o ther variations in the molecular structure of the bormone itself have arisen through gene mutatiol1s, and these can alter and/or expand the biological activities of a particular molecular variant, and even increase the number 01 d iffe rent molecular variants produced within the same org'1nism. Comparative endocrinologists attempl to track the evolution of hormone structure and function, al1c1 determine the adaptational significance of these evolutionary relationships for the physiological ecology of different animal species,

urg'ca

ad

Surgical removal of a putative endocrine gland or ti ssue from <1n animal is followed by subsequent assessment of physiological alterations, A change in functional activity of suspected target organs (i.e., atrophy) suggests an endocrlne function for the extirpated tissue. O ne might also monitor chang s in blood or urinary levels o[ certain meta bolites or electrolytes, Removal of the adrenals, for example, would result in lowercd circula ting levels of adrenal steroids and and a reduction in the plasma I a ' concentration. Transplanting an organ back into the same or a different animal also provides information on the functional role of the organ. In mammals, transplantation of an org<ln from a donor to a host is usually done in genetically related (inbred) strains of animals 50 that the transplant is not rejected by immunological proct:'sses. Endocrine organs can be tran sp lanted to an ectopic (abnormal) site in the animaL usually beneath the kidney capsu le or within the eye, where rapid vascularization often occurs, Removal of the pituitary is referred to as a hY/JophyseclOmy. rlne pituitary target organs of" hypox" animals become atrophic due to the absence of hormonal stimulation. In a fevv examples endocrine ti ssue is normally under a tonic inhibitory controL the tissue then becomes hypertrophic when transplanted to an ectopic site within the animal or when incubated in vltro. Removal of botl1 members of paired (bilateral) target organs (e.g .. adrenal glands or gonads) usually leads to complete loss of dependent tis5ud organ functions [f only one of the pair (unilateral) is removed, the remaining organ usually undergoes compensatory hypertrophy. In otber words, there is an increase in cell size and number in the remaining organ to compensate functionally for the loss of hormone secretion [1'0111 the ablated organ (Fig.. 4. I). In endocrine systems in which one endocrine organ may control secretions by anothcr. the feedback mechanisms that may operate are often studi ed by assess ing the effects of organ removal and hormone replacement on the putative feedback targe t. Other examples of endocrine include pinealectomy (epiphysectomy), adrenalectomy, tl1yroidectomy, anc! thymectomy. The nature of sLich endocrine tissues as the gastrointestinal hormone-secreting cells prevents such (] surgical procedure, Tne pancre<1tic islets are similarly impossible to remove without removing concomitantly the exocrine tissue that comprises most of the mass of the pancreas. lslel ablation, however, can be accomplished by the use of alloxan or streptozotocin, drugs that specifically destroy the insulinsecreting cells of the islets. Cobalt chloride is similarly effective in the elimination of the glucagon-secreting cells of the pancreatic islets. In brain tissucs, localized electrolytlc lesions can be made to e liminate specific brain nuclei that contain cells of interest, and the consequences of the se ablations can be functionally assesscoci .

ce......."",.....t The

The undesirable effects of hormone loss following surgical ablation or loss c!ue to certain di sease states often can be adequately reverse c! by administration of the needed hormone or related analog. At menopaLise. for example, many women experience bone mineral loss (osteoporo sis) clue to declining levels of ovarian estrogens, Estrogen/progesterone

Hts

1 C

71

bovine (ca ttle), ovine (sheep ),porcine (pig), or even equine (horse) origin. Often these foreign proteins are immunologically neutralized within the body follo wi ng ad mi nistration , therefore necessitating a change in the type (source) of the hormone used. teroid hormones a re commonly extracted from the urine of large domestic animals, for exa mple horse ', and us d in th e preparation of hormone replacement therapies, such as the estrogen-containing treatments taken by some postmenopausal women.

Before the advent of modern molecular biological techniques, the structures of most protein hormones were identified by painstaking purification of the source tissue , followed by meticulous biochemical analysis of the hormone's amino acid sequence, A purified ho rm one was chemicaJly identified , and then subjected to a simple analysis of the percentage of carbon , hydrogen, oxygen, nitrogen , sulfur, or other atoms present, thus providing an e mpirical formula. Further analysis of the protein then indicated the number and nature of amino acids present (an amino acid analysis). This was followed by a determination of the primary sequence (exact amino acid sequence) of the peptide or protein under study. T his is nol easy if a large protein hormone is being studied. Nevertheless, one might obtain information on the , -terminal or C-terminal sequences of these large protein hormones. In the more recent past, hormone structures have been determine d by sc reening of cD A libraries, cloning cDNAs representing candidate hormone genes, and det rmining th e predicted amino acid sequence of the putative hormone. In some cases, the gene encoding the hormone was identified using forward genetic methods (from phenotype to genotype); these approaches generally involve the genetic analysis of mutant mouse strains that exhibit phenotypic evidence for disruption of a gene of interest. For example, the gene encoding the adipocyte-derived hormone, leptin, was "positionally cloned" by analyzing genomic markers and deducing the genomic location of the heritable D NA sequence linked to the ob sc phenotype in the mutant (ob/ob) mouse [41]. With the sequencing of the human, mouse, and other animal genomes, it has also become possible to screen sequence data banks and identify putative hormone and receptor nucleotide and amino acid sequences based on the presence of characteristic structural motifs. All of the foregoing methods have similarly been used to characterize hormone receptors and their corresponding genes. From the primary hormone structure, the secondary (a-helical or j3-pleated sheet) and tertiary (folding, intrachain bonding) structures may often be implied from the distribution of basic or acidic amino acids or the presence of sulfhydryl (-SH) groups within the protein. Some peptide hormones, such as insulin, possess a quaternary structure, that is, the hormone is made up of two peptide chains folded together into a three-dimensional structure (conformation). A number of the larger peptide hormones, tor example, follicle-stimulating hormone (FSH ), luteinizing hormone (LH ), and thyroid-stimulating hormone (TS H ), are composed of two chains, the so-called a and j3 subunits of their structures. Chemical analysis may indicate that proteins are modified through sulfation or conjugation to carbohydrate moieties. The pituitary hormones, FSH , LH , and TSH, are examples of glycoproreins (Chap. 5). Determination of steroid or other hormone structures requires different chemical and physical methods of analysis. After determination of the putative hormone structure, it is necessary to synthesize the proposed structure and demonstrate that the na tural and synthetic structures are identical with respect to chemical, physical, and biological characteristics. One can then synthesize related structural analogs of the hormone to dete rmine the structural basis for the biological activity of the hormone. Table 4.1 provides the abbreviations used to symbouze the amino acids present within the primary structure of a peptide hormone. (See, e.g., Figs 5.8 and 1l.S.)

5 The early endocrine studies were, as expected, anatomical and purely descriptive in nature. The light microscope was used to determine the histological nature of the endocrine glands. Many early observations were made on these tissues even before their endocrine role was suspected. Much of this work was accomplished by the great Ge rman , Italian, and other

72

Chapter 4

ldocrln..:: Mothodologles
TABLE 4.
Abbreviations used for amino acids

:ystein'c As partic acid G lutamic aci.d Phenylalaninc G lycine H istadin c Isoleucine Lysine Leucine Methionine Asparagine Proline G lutamine Argi nine Serine 1111'eOn ille Valine Tryptophan Tyrosine

cys asp glu phe gly his ile Iys leu met asn

A C

D E
F G H I K

pro
gIn

arg
SCI'

thr val trp tyr

L M N P Q R S T
V

E uropean cytologists of the nin eteenth century. Although light microscopic me thods are still important, the electro n microscope is now the tool in the investigation of cellular function at the ultras tructural level. The scanning electron microscope bridges the gap be tween light and electron microscopy. Gross observations of endocrine tissue provide only general details of anatomicallocalization , organ size, vascularization , and innervation. Severe alterations from the normal in organ size may provid e clues to an und erlying pathophysiology. The thyroid gland, for example, may become enlarged (goitrous) und er certain conditions of hyperstimulation (see Fi g. 13.11). At the histological level, one is ab le to discern the cytological characteristics of endocrine tissues. T he cells may be hypertrophic (enlarged) or atrophic (diminished) depending on whether they are hyperactive or hypoactive, respectively. Hypertrophic cells contain an abund ance of endoplasmic reticulum and Golgi bodies, as these organelles function in many cellular synthetic processes. A tro phic cells, on th e other hand , lack this synthetic machin e ry and co ntain a much di minished cytoplasmic mass. E ndocrine cell hypertrophy is usually accompanied by hyperplasia . an increase in cell number. Histological stains are available to provide further information on chemical components of cells. H e matoxylin and eosin are two popular dyes for staining cells for routine histological observation. H ematoxy lin , a basic dye, interacts with acidic components of the cell, such as the phosphoric acid of DNA and RNA These components are said to be basophilic or to exhibit basophilia. On the other hand, eosin, an acidic dye, interacts with th e basic components of the cell, which are then sa id to be acidophilic or to exhibit acidophilia. Used in combination, hematoxylin and eosin usually stain the nucleus and cytoplasm blue and pink , respectively. B y the use of a battery of histological stains, one can characterize each cell present in the pituitary. With hematoxylin and eosin , it is only possible to separate the basophils from the acidophils and chromophobic (nonstaining) cells (chromophobes). Other histological stains are needed to further differentiate the several types of basophils. Cer tain stains can be used to demonstrate th e pres ence of specific organic constituents, such as glycoproteins, in gonadotrophs of the pituitary, or glycogen in hepatocytes [29].

Hem
i mm unological methods have been used for many years to characterize and analyze the expression of hormones in endocrine cells. "nley are also used to characterize the specific distribution of receptors, cell signaling molecules, transcription factors, and other proteins that mediate

01

Cdl C tol qlCal Studt

73

Fix

Section

Incubate with reagents

Incubate tissue wittl primary antibody Target

4-.

'\0.

r "'y cr

A
0

A '(

., Principle )f immunohistofluores:ence methods for the nicroscopic detection of l protein of interest in ndividual cells.

Wasil , mount on microscope slide, view under fluorescent microscope

hormone actions in target tissues. Immunohistochemistry refers to the use of spccific antibodies to detect and label specific antigens (molecular targ ts of immune mediators) , for example hormones, in a tissue of interest. This term is often used intercha ngeably with immunocytochemistry, which is a narrower and perhaps more accurate description of those techniques that allow identification of a particular antigen at the level of indi vidual cells, or even within specific subcellul ar compartments (Fig. 4.2). These powerful procedures make usc of antibodies that specifically recognize and bind to a specific epitope (portion of a particular molecule), thereby allowing one to visualize and identify which cells in a tissue contain that specific mol eule. E ither polycfonal or monoclonal an tibodies can be used in immunohistochemical procedures. Po lyclon al antisera are generated by immunizing anim als with a suspension containing the target molecule, and subsequently obtaining the hyperimmune serum from those animals that mount a robust and specific immune reaction to the antigen. The polyclonal antise rum, as its name implies. contains several different clonal strai ns of immunoglobulins that may contrib ute to varying degrees to th total antibody titer. Monoclonal antibodies are produced by immunizing a mouse with a specific antigen, obtaining spleen cells from the anim al, and fusing these ells with chemically selectable myeloma cells to produce hybridomas. E ach hybridoma is capable of expanding to produce a homogeneous clona l line of ce lls that produce chemically and immunologically homogeneous antibodies. Most importantly, the selected clonal cell line produces large quantities of a single antibody species that recognizes and binds to a single antigenic determinant. Irrespective of the type of antibody utilized, the success of any immunohistochemical approach depends upon the specificity and the affini ty of the antibody for a particular antigen.] n a version of immunohistochemistry referred to as immunohistoflllorescence, antibodies to pe ptide or protein hormon es are conjugated to a flu orescent dye and used to identify cells tha t produce the molecule of interest. The tissue to be examined is first treated with a fixative, such as glutaraldehyde, and sectioned into thin slices. After the tissue is placed on a microscope slide, a solution of the antibody conjugate is added to the tiss ue, and the excess solution is then rinsed from the slide several times. Microscopic observations usually revea l that the fluorescent antibody is bound only in those cells that produce the hormone, the antigen (see Fig. 10.8). Specificity is determined by prior incubation with a "blocking peptide", a synthetic peptide that is identical to the portion of the target hormone or receptor that is kno wn to be bound by the antibodies. The

74

Chapter 4

Ildocnne Methodoloql

blocking peptide interacts with the antibody and thus prevents it from binding the endogenous antigen. TIle absence of fluorescent labeling of tissue following preincubation with the blocking peptide confirms the specificity of the antibody for the intended antige n target. In immunoenzyme histochemistry, an antibody to a hormone is conjugated to an enzyme, such as peroxidase.The antibody-enzyme conjugate is then allowed to interact with the tissue slice. When substrate for the enzyme is added, the conjugated enzyme catalyzes very localized reactions that yield a color or opaque product in the vicinity of the antigen (the hormone). By this immunoperoxidase method and use of the electron microscope, it is even possible to localize the site of the enzym e activity to the secretory vesicle s/granules of a cell, which provides strong evidence that these organelles contain the hormone under investigation. In double-labeling immunocytochemical procedures, two antibodies can be used to simultaneously examine the expression of two antigens in a single tissue section. TIle two antibodies possess specificity for the two different antigens, respectively, and they are linked to two different labels for differential visualization under the microscope. The utility of these procedures is far-ranging, and includes the ability to characterize the expression of specific hormone receptors to specific subsets of potential target cells in a complex tissue, such as the brain. Immunohistochemical methods have also proven to be useful in functional studies of hormone secretion and action. The activation of a neurohormone-secreting cell, for example, is often accompanied by the expression of immediate early genes , such as those encoding the proto-oncogene c-Fos [11]. With immunocytochemical methods, cells that express this gene product can be identified as those that are activated under certain experimental conditions. For example, brain cells that are activated by physical or be havioral stress have been "mapped " by means of c-Fos immunocytochemical methods. Similarly, some hormone actions are mediated by intracellular messengers that become phosphorylated upon activation; antibodies can be developed that specifically recognize the phosphorylated form of signaling molecule, and thus they can be used to immunocytochemically map the cell groups in which hormone treatments activate that particular signaling pathway.

E ndocrine studies utilize all the mod ern tools of physiology, mol ecular biology, and genetics to understand hormone function . Below are considered those quantitative methods that have prov en to be particularly important in analyzing the synthesis, secre tion, and actions of specific hormones.

. 1

ctivi

The physiological activity of a hormone was originally determined by bioassay. Although some assays have been replaced by more modern methods, in some instances, the only method available is bioassay. In a bioassay, th e activity of a hormone is studied on living cells, tissues, or organs. Physiological responses such as muscle contraction and relaxation or glandular secretion are monitored. TIle assay may be performed in vitro or in vivo (in situ) depending on the assay used. Usually, the tissue or organ selected is naturally responsive to the hormone. These biological preparations are usually responsive to hormones in the nanomolar (10- 9 M) to picomolar (10 - ]2 M) range [25]. Other hormones may also affect these tissues, but usu ally in pharmacological (micromolar, 10- 6 M) doses. The frog skin bioassay for m elanocyte-stimulating hormone (MSH) is simple, specific, and exquisitely se nsitive. M elanin granules within me lanophores of fro g skin disperse in response to the hormone, causing the skin to turn from light green to dark brown (Chap. 8). T his change can be m easured in a number of ways, but one objective method is to monitor changes in light reflecta nce off the surface of th e sk in l25]. TIl e toad bladder, and such epithelial structures as frog skin and the mammalian renal nephron , hav e been used in vitro to study the actions of vasopressin on transport of wa tel' and other components, such as Na + and urea, across these organs. Studies of ion transport in the toad bl add e r have contributed deta iled information on the mechanisms involved in vasopressin action (Chap. 7) . 111e steroid-primed uterus of the rat has been used in vitro to st udy the mechanisms of oxytocin-induced contraction of this organ. Ma mmary ti ssue from the lactating mouse is used in vitro to determine milk-ejecting potency of oxytocin and related analogs. Other bioassays include in vitro adrenal steroidogenesis and secretion in response to ACTH: prostate and

M thodolO

75

seminal vesicle growth in vivo in response to testosterone in the castrated male rode nt ; measurement of iodide uptake by the thyroid after exogenous TSH injections; meas urement of epiphyseal plate (cartilage) growth in the young rat in response to groWLh ho rmone. Many investigators now use primary cell culture systems or immortalized cell lines rather than intact animals or removal of organs from such animals.

Bioassays determine whether a putative hormone or hormone analog possesses biological activity. Structure-activity studies determine the amount of activity, usually com pared with some standard hormone. The minimal effective dose (MED) and maximal activities within the particular assay are usually determined first. Then , intermediate concen tr ations of the hormone between the two extremes (maximum and minimum) can be utilized L o provide a dose-response curve. Usually, the half-maximal activity of the hormone is compared with a similar half-maximal control response (e.g. , to the parent hormone or to an analog) and expressed as a potency ratio. The hormone or hormone analog may possess one or more times greater or lesser activity (potency) than the native hormone (see F ig. 8.18).

io s ys

ut genesis t

eli n I

Although the primary sequence of many hormone receptors is kno wn, how the receptors function cannot be readily discerned from the amino acid sequence. One would like to know the sequence(s) responsible for ligand (hormone) binding and the sequence(s) responsible for signal transduction leading to a cellular response. A mutant receptor can be synthesized fro m the original normal receptor cDNA and the mutated DNA introduced into "immortalized" (usually cancer) cells. For example, single base changes, multiple base changes, additions, or deletions (subtracting amino acids) can be made. Then, a functional response to a hormone by these cells containing mutated DNA can be measured [26]. or example, one can measure changes in levels of a second messenger, such as cAMP, following addition of the hormone to the cells.

Ie Lev s of
The development of the radioimmunoassay (RIA) has allowed the detection of hormones in minute concentrations and with a high degree of specificity [40). RI A has provided greatly increased diagnostic accuracy of pathological states characterized by hormonal excess or deficiency. These assays are routinely employed to de termine the concentration of hormones and other molecules in blood or other body fluids. As with the immunohistochemical methods, the success of RIA procedures is dependent upon the use of antibodies that specifically recognize and bind the hormone with high affinity. B oth polyclonal and monoclonal antisera can be used in an RI A. Th e second important reagent for an RI A is radiolabeled hormone. In most RIA ', the hormone is iodinated or tritiated ; in peptide hormones or in other proteins, the tyrosine moiety incorporates iodide. he typical RI A procedure depends upon the binding of the specific antibody to the radiolabeled antigen (e.g., hormone) with the same kinetics as described for receptor binding to ligand in the previous chapter: *[H] + [Ab] kon
<---?

[H ) [Ab]

koff where *[H] = radiolabeled hormone, [Ab] = antibody, [H ) [Ab] = hormone bound by antibody, and kon and koff are association and dissociation rate constants, respectively. Like the binding of ligand with receptor, this is a reversible reaction in which kon is greater than KoffAt a fixed concentration of *[H] and [Ab], the reaction equilibrates at a maximum Ie el of '"[H) [Ab]. Th is is referred to as the maximum binding, or Bo. The principle of the R[A is that the unlabeled hormone, [H), when it is added to such a reaction mixture , will compele with the radiolabeled hormone, *[H], for binding to the antibody, [Ab]. [H J + *[HJ + [Ab]

kO'l
<---?

[H) [Ab] + *[H] [Ab]

koff

76

Chapter 4

ldocrme Mcthodologle
Maximum Standard Standard Standard Standard Unknown Unknown etc. y Binding 1 2 3 4 X

A

Antibody ' Hormone Hormone Unknown

+ +

+ + +

+ + ++

+ + +++

+ +

+ +

n
.J

+ +

++++

E I
()

Unknown X

0..

3 Schematic repre sentation of a typical RIA p rocedure with hypothetical standard c urve.

EI (;

<lJ

Unknown Y

!-

Log [Hormone]

Since the a mount of ra diolabeled hormone and antibody is fixed , then increasing leve ls of unl abe led horm one will increasingly bind to th e antibOdy, and displace labeled hormone from doing so. Thus. the higher the concentration of [Ii ] in the reaction solution, th e more the lHl [A b] and the less the [Ab] will be formed. T he R I A is therefore consid e red a compelitive inhibition assay. rn practice, the R IA is executed by adding reagents to a series of test lubes. as depicted in Fig. 4.3. An initial set of tubes constitutes the standard curve, in whieh increasing sta ndard (known) amounts of unlabeled hormone are added to the fixed amoun t of ra diolabeled hormone and antibody. Remaining tubes contain the same amounts of ': '[11 ] and [Ab] as do tubes in the standard curve , but in place of a standard amount of hormone, a given amo unt of the fiuid containing unknown amounts of the hormone (e.g .. serum) is add ed. T he re action is allowe d to proceed to equilibrium. In some RIA procedures, a second antibody is then added that recognizes and binds to the first antibody, creating a larger protein complex that is more easily separate d by th e final ste p, which is ce ntrifugation to separate ':' [H] [Ab] from unbound *[H]. The amount of "'[ H ] [Ab] in the pe ll et followi ng centrifugation is dete rmined by monitoring the radioactivity emitted by the tubes in a gamma radiation counter, and plotted versus the standard amounts of [H] added to yield a sta nda rd curvilinear plot (Fig. 4.2). The amount of radioactivity from " [H] [AbJ in the sample (unknown) tubes is then used to extrapolate the value for the unknown from the standard curve. In addition to protein hormones, cyclic nucleotides and many other nonprotein substances are also measured by these RI A procedures with remarkable sensitivity, precision , an d specificity. The non-proteinaceous substances are, however, usually conjugated to an antigenic carrier protein to produce antibodies for their assay.

idcly Us
llle a vai labil ity of monoclonal antibodies was a major e nabling step in the deve lopment of ne \ve r im munoassays. many of which can be perform ed without the use of radiolabeled reagen ts. Immu/1ometric assays are those which utilize chemically labeled , monoclonal antibodies to directl y measure hormone leve ls in a non-isotopic procedure. Th ese assays usually utilize two diffe re nt monoclonal anti bodies th a t recognize two separate d epitopes on the same target ho rmone molecule. The fir st antibody is attached to a solid phase (e.g., the surface of a we ll in a test p late) and it is used to "capture " the hormone by binding to its orrespond ing epit ope. A second "signal" antibody is chemically attached to a fiuorescent , chemiluminesccnt, or enzymatic label. W hen this signal antibody is subsequently added to th e incuhation, it a ttaches to a second epitope on the captured hormon e. The ch emical signal

An I

01 Harmon Acuor

77

bstrate

Primary Antibody 2 Antigen)

provided by the signal antibody can then be read as a direct re flection of the amount of bound hormone . Figure 4.4 depicts the principle of this type of immunometric assay. which is sometimes referred to as a "sandwich immunoassay." Many other variations of immunometric assays have been developed, and their ease of use and sensitivity h as prompted their widespread use in research and in medical diagnostics. Many of the home pregnancy tests, for example, make use of all immunometric assay principle to detect the earl. pregnancy associated hormone, human chorionic gonadotropin (h CG ), in urine.

Capture Assay "Sandwich"

al si of Horm.one Action
A,Ubody ,

oJ

or

Principle of typical immunometric ,andwich assay."

A number of endocrine methods use the radioactive isotopes of various elements (c.g.. 125L 45Ca, 35S, 32p, 2JNa . 14C, 3H) to determine physiological and biochemical respon ses within a cell. The half-life of a hormone, for example. can be ascertained by radiolabeling the hormone and then determining its distribution, excretion rate, and metabolism within the body of an animal. Iodide [12Sr] is taken up by thyroid cells and incorporated into th yroid hormones (T4 and T3)' Radiometric methods can be used to measure uptake of radioactive iodide atoms by the thyroid. Information obtained often reflects the biosynthetic activity of the thyroid gland, and this method is also used to localize hyperplastic thyroid tiss ues. The radioisotope of carbon [14C] can be incorporated synthetically into the str ucture of a molecule such as glucose. Subsequent metabolism of glucose with the concom ila nt evolution 4C] 02 is a measure of the metabolic activity of the cell. of radioactive carbon dioxide C Insulin, for example, increases release of radioactive CO 2 by stimulating the uptake of radiolabeled glucose into diaphragl11 muscle where it is metabolized. This is also a good example of a bioassay. Tritium, a radioisotope of hydrogen CHJ, is widely used in autoradiography and in enzyme assays. The tritium isotope is generally used as a component of some organic structures, such as an amino acid or nucleotide (e.g. , thymidine). The sodium radioisot o pe [23 Na] is most often used to measure Na + uptake into nerve or muscle cells during studi es on transmembrane potential changes in response to chemical messengers or other stimuli. Radioactive calcium [45 Ca] is often used to measure uptake of this divalent cation during muscle contraction and relaxation, nerve stimulation, or cellular secretion. Moreover, this isotope can localize Ca2+ sequestration within mitochondria or in the sarcoplasmic reticulum. Incorporation of sulfur eSS] into the amino acid cysteine has been particularly productive in the study of neurohypophysial hormone synthesis because neurohypophysial peptides contain a high percentage of this amino acid. Such labeling of newly synthesized neurohypophysial hormones has greatly advanced studies on the movement of these proteins by axoplasmic transport down the neuronal secretory axons (C hap. 7). Radioactive phosphorus 2 p] in the form of phosphate can be used to monitor protein phosphorylation as induced, for example. by hormonal stimulation of cells (see Fig. 3.17). A variety of assays use radioisotopes to determine the concentration of a hormone in an endocrine tissue, in the blood, or in the urine. R adioisotope assays can also be used to determine enzyme activation or inhibition by a hormone. In each of these assays specialized equipment capable of measuring the decay of the isotope is used (e.g., liquid scintillation counters or gamma counters).

adioliq

gu1 lion

The actions of hormones in target cells may be subject to physiological regulation, such as upor down-regulation, and endocrine researchers use a variety of methodologies to investigate how these adjustments occur.ln many cases, it has been shown that alteration in the responsiveness of a tissue to a hormone may occur as a result of alterations in the number or affinity of active receptors that are present in the tissue. Chronic hyperinsuJinemia , for example, can induce a pronounced reduction in plasma membrane insulin receptor number, a phenomenon that results from a high rate of ligand binding and subsequent internalization and degradation of receptors in target cells. The reduced number of insulin receptors, in turn. is associated with a reduction in the responsiveness of the target tissues to insulin. Radioligand binding assays can be used to document changes such as these in receptor number or affinity for ligand.

AnalySIS of Gen Expres Ion In Endocnn Sys

79

H*

HO

Figure 4.6
Radioisotope enzyme assay; tyrosinase assay.

b-r-r I I

HO NH2
Tyrosinase-7

II

COOH
Tyrosinase Assay

Ho-L >-T-?-NH" WOH

H COOH
Dihydroxyphenylala nine C [3H]-Water

[3H]-Tyrosine

grown. A sample of ti ssue/organ [rom the anim al or the ce ll cu lture is th e n place d on a microscope slide, as is done for routine microscopy. T11 the darkroom tbe slid e is now imm erse d in a photographic emulsion and kept in the dark fo r a period of time. Decay of the isoto pe (usually tritium) leads to reduction of the ilve r bromide in t he o verlyi ng ernulsion to silver crystals. Because the beta particle r eleased by the tritium tra ve l o nly a sho rt distance, the silver grains d irectly over the emulsion are prefe renti ally exp osed. since they are closest to the source o[ isotope decay. T he tissue with emu ls ion is then pl aced in deve loper and stained, if desired. By this method, for examp le. one can determin e the incorporatio n of tritium-labeled thymidine into D A [14]. This nucleotide is ge nerally used to de term ine the mitotic activity of cells. After orchidectomy, for example, t her en hanced hypothalamic activation of pituitary gonadotrophs (due to the lack of nega tive feedb Llck in hihiti on by testosterone). This leads to increased incorporation o f rad io labclcd thymid in c into the DNA of these cells, which can be visualized by aut ora diogra phic me thods. The action of tc roid hormones involves int eraction with nuclear DNA. Au toradiogra phy using tritium-labeled steroids provides a method for demon stra ti ng the ce llul ar site of steroid action (Fig. 4.7). R adiolabeled peptid e can be used to de te rmine the topographical localization of hormone recep tors in an organ or tissue (sec Fig. 7.7) . Some hormones mediate their effects through stimulation of RNA synth esi s. This is d c: mo ll Slr<1 te d by the use of tritium-labeled uracil, which is incorporated specifically into RNA rather than D A . Using giant chromosomes of the dipteran fly (midgc), Chironomus, for example, it is possible to localize tritium upta ke to specific Ba lbiani rings ( large "p uffs") of thc chromosomes [2], sites known to be responsible for very active RI\A syn tl1c is (F ig. 4.13 ).

nal sis

GeneE

ression in Endocrine

stems

The processes by which the DNA sequence encoding a pro tein is decoded. leading to the production of that p rotein, are collectively re ferre d to a gen e express ion. Hormones often exert their effects by inducing alterations in the gene ex p re ssion of specific proteins in a target tissue. Moreover, hormone genes themselves are ty pic ally su bject to ph ysi ological

19b. e (a) Heavily labeled cells in the medial p reop tic nucleus of the mouse after inj e ction with eHJestradiol ; x 850. (b) Labeled neurons , at a higher magnification, showing the concentration of silver grain s over the cell nuclei; x 1,360. (c) G uinea pig anterior pituitary ce1ls ; r a dioactivity is re tained in n uclei of c ertain cells after injection of e HJestradiol; x 1,360 . (From Warembourg [36 J, with permission.)

80

Chapter 4 f ldocrtne MethodologIes

Pattern of eHJuridine incorporation into RNA produced by larval salivary chromosomes of a dipteran fly. Note intense labeling of Balbiani rings 1 and 6. (From Beermann [2], with permission.)

regulation. In many endocrine studies, therefore, the analysis of changes in the expression of specific genes provides critically important information about hormon e synthesis and action. There are several potential ways in which gene expression may be regulated, including the stimulation or inhibition of (1) transcription of D NA to RNA, (2) processing of RNA to mRNA, (3) rate of m RNA turnover (conversely, its stability) , (4) translation of mRNA to protein, and/or (5) post-translational processing of the translated protein. Some methods permit assessment of the rate of transcription, while others rely on steady-state mRNA levels as a summary indicator of process es 1- 3. Measurements of mature protein accumulation can give an estimate of the overall rate of synthesis, although these values may be difficult to interpret if the rate of degradation or release of the protein is also regulated independently of synth esis.
p

Irvels a

H ybridization, or annealing, involves pamng of complementary strands of nucleic acids. H ybrids can form between D NA -DNA, DNA-R NA , or RNA-RNA strands. DNA is analyzed on a solid support such as a membrane (Southern hybridization, or Southern blot). The oldest and most common method for the analysis of R NA is the Northern blot. In this procedure, RNA is isolated from a group of cells or a tissue, electrophoretically fractionated by size in an agarose gel , and then transferred to a nylon or nitrocellulose membrane. A radiolabeled cDNA probe corresponding to the m RNA of interest is then hybridized to the R NA on the blot. The bands of hybridization are then detected via exposure of apposed X-ray film , and the magnitude of the specific autoradiographic band thus serves as a valid index of the amount of the specific RNA sequence present in the sample [33]. Another common method for mR NA analysis is the ribonuclease protection assay, in which a radiolabeled RNA probe and the corresponding target mRNA are hybridized in a solution, and then treated with a ribonuclease that digests single-stranded but not double-stranded RNA. The remaining probe- mRNA hybrids are then electrophoresed and autoradiographically quan tiiied [21] (Fig. 12.15). The most methods for measurement of mRNAs include reverse transcriptasepolymerase chain reaction (RT-peR) and real-time polymerase chain reaction (real-time peR ) techniques. Both approaches provide powerful alternatives for the measurement of mRNAs Ihat are expressed in low abundance, such as those that encode many hormone recep tors or intracellular signaling molecules. A reverse transcriptase enzyme is first used to reverse tran scribe the m RNA contained within a sample cDNA, and then the sequence of in terest is amplified (copied thousands of times) by the polymerase chain reaction. The p e R m a kes use of a pair of -20-mer Oligonucleotide primers that are designed to hybridize

An yst of Gene ExPI

Total RNA Reverse Transcriptase

Ion m Endocnn Sys

81

dsDNA

Add Primers A & B Primer A

Denature 93-99" C

3'
Annealing 37-59C 3' dNTPs Taq Polymera se Primer B

Extension 72C 1 kb/min

l
Fi Schematic representation of the principle of the polymerase chain reaction (peR),

Repeatin g Cycles of Denaturation Annealing , ExtenSion

Multiple copies of dsDNA of the same size as the distance between primers A and B

with nucleotide sequences that flank the D NA sequence to be amplified (Fig. 4.9) . The reaction mixture contains the samp le cD ' A_ specific oligonucleotide primer pair, a DNA polymerase enzyme, and deoxynucleotide triphosphate (d TP ) precursors, and is subjected to repeated cycles of heating and cooling in a thermocycler apparatus. During each cycle, the following occurs: (1) heating causes the dou ble-s tranded 0 A to be de nc.t tured, i.e. , rendered single-stranded, (2) the prime rs anneal with the corresponding flanking sequences on each 0 A strand, (3) the primer sequences are e longated by the actions of D A polymerase, which incorporates the dN TPs into the extensions, thus producing (4) two double-stranded D NA molecules that correspond to the original double-stranded D. A. These cycles of denaturing, primer ann e tlling, and extension are repeated to yield two-fold amplification of the peR product, or "amplimer," per peR cycle. Thu s, thirty cycles of p eR would be expected to increase th e amount of DNA corresponding to the sequence between th e two primers by 2 30 or -1 billion ti mes! For semi-quantitative RT-PC R, the amplified product is electrophoresed and either stained or autoradiographically analyzed for the extent of radiolabeled d NTP incorporation. The amount of the amplified product is then considered to be proportional to the amount of the specifi c cDNA sequence originally present in the sa mple. Often the results are normalized to those obtained from the amplification of an mRN A sequence of unvarying abundance, for example a "ho usek eepi ng" gene, as a n internal procedural control. More accurate quantitation of mRNA can now be achieved using real-tim e PCR procedures. Several versions of thi s basic method have been developed , but all use a fluorescencesensitive apparatus that monitors in real-time the progress of a PCR that emits fluorescence signal in proportion to the amplification of product. Often the relative amounts of the target sequence present in a sample are indexed as a function of the num be r of PCR cycles or cycle times (C t ) required to reach a minimum threshold of fluorescence.

82

Chapter 4 ..... ndocrine MelhodolOgies

TiSSUt:-

'.

C A T

In situ hyb ridization.The probe (RNA or DNA) is labeled with a radioactive or n onradioactive d etection system. The DNA probe in this example (5'-CATG-3') is hybridizing with the m essenger RNA associated with the rough endoplasmic retic ulum. After hybridization to ce lls or tissue sections , the RNA or DNA of interest c an b e detected by autoradiograp hy or non-isotopic methods. Advantages o f in situ hybridization include more precise intracellular localization of the RNA or DNA of interest and direct visualization of positive cells in relationship to surrounding cells and tissues (From Lloyd , with permission . Endocrine

..

y h ft' t

d Cell-Specific lion

RNA ExpressionPaltern

e Analyzed

llle nucleotides can also be localized within cells and tissues by in situ hybridization (ISH) [20, 32,38, 39] (Fig. 4.10). This powerful technique used to localize mRNA involved in hormone synthesis is beautifully demonstrated in Fig. 8.5. Most ISH methods utilize radiolabeled cDNA or cR NA probes to hybridize to an mRNA sequence present in the cells of a slide-mounted tissue slice. The location of the hybridized probe is indicated by development of apposed photographic fi lm, or more precisely, by exposure of radiographic emulsion coating the mounted tissue. TIl US. th e identity and location of cells expressin g the mRNA of interest are indicated by the appea ran ce of si lver grains in the overlying emulsion. The number of silver grains that develop over an individual cell is proportional to the number of mRNAs encoding the protein of interest that hybridized with the radiolabeled probe. The ISH method can therefore permit semi-quantj tation of ill RNA expression; the principal advantage of the ISH technique is that it permi ts both quantitation and cellular resolution of mRNA expression patterns- an advantage not shared by total RNA hybridiza tion methods. With in situ hybridization, it is also possible to address qllestions regarding differential gene regulation within different nuclei in a give n brain region or within different cell populati()ns in a specific nucleus. This level of resolution can reve al subtleties of gene regulation that cannot be detected with Northem blots or RN ase protection assay methods. Moreover, it is also possi ble to determine if specific colocal ized gl'nes are coordinatL'ly regula ted in brai n nuclei in which some or all of the neurons display such colocalization. The pri ncipal disadvantages of in situ hybridization methods are that they are tim e- and labor-intensive ilnd th at absolute cannot be ensured [6].
fill. I 11 NA Mic;..oarra b Many horm()nes exert their actions on a particular targe t tissue by simultaneously altering the expression of ma ny different gen es. The developme nt of DNA microarray technologies ha s enabled endocrine researchers to identify hormone-responsive genes among thousands of cand idatc$ in single ex periments. Seve ral ve rsi ons of these technologi es have been devel()pe d. In general. however, either oligonucleo tides or cDNA probes corresponding to large nu mbers o f gen es- in some cases encom passing the great majority of genes in a particular genome - a re cova lently attached to specifi ed sites in a micronized grid on glass chips. The R NA sample is converted to eRN A or eDNA , labeled with a fluorescent marker, and hybridized to the immobilized probes on the chip. A specialized chip reader measures the in tensity of the fl uorescence at each probe positi on on the chip, reflecting th e lev el of the co rres ponding I11RNA . Computer analysis and comparison of the data amon g chips then permits ick ntifica tion of those gen es that are express ed to a different extent in hormonestimulate d versus untrea ted conditions [27].

Hor

Uee

es'

ion

Pathophysiology
4:64-72, 1993.)

cts !J " :pl)rtf'r The techni ques of molecular biology also ena ble endocrine researchers to analyze the mech(l ni sms co ntrolling transcription of genes that encode hormone s, receptors, and proteins that are regu lated by hormone action in target tissues. Ofte n the objective of these studies is to iden tify promoter regions of a particular gene that regulate its transcripti on, and analyze regions withi n the promoter that are targets of specific transcription al regulators. Reporter gene constructs are often used in these studies to obtain a "rea d-out" of promoter activity. Such i\ construct might he made by cloning the putative promoter region of a gene, and fusing it to a common reporter gene, such as the firefly luciferase gene. The promoter-reporter constr uct can be tran sfect ed (chemically in troduced) into cells in culture, and the transcriptional ac tivity of the promoter is monitored by measurement of reporter gene expression. In the case of th e lu cifc rase reporter construct, transcriptional activity is reported by luciferase enzyme activity: this is easily qu antitated by lysing the cells, adding luciferin and the luciferase substrate. ATP. and moni toring the lucife rase-ca ta lyzed luminescence with a luminome ter. E ndocrine research ers often study the fea tures () f a promoter by "bashi ng" it: making truncat ions or spi::cific sequence mutations or del etions in the promoter sequence, and then assessing cbanges to its basal act iv ity and/or responses to putative transcriptional regulators. A scyu ence th at normall y med iates a positive or negative regulation of transcription can thus be identified by th e loss o r gain of transcri ptiona l activi ty in its absence.

PhaIInacolog1C

tho

83

Most cells are excitable, that is, they will respond to stim uli by becoming depolarized or hyperpolarized, which results, for example, in relaxation or contraction . res pectively, of smooth muscle. These potenti al transmembrane changes can be mo nito red b in tracellular or extracellular microelectrodes. The respo nse of the cells to hormonal or oth r stimuli therefore can be detected. Most stimuli de polarize cells, but some chemical messe ngers,for example gamma-aminobutyric acid (GABA ), a central nervous system neurotransmitter, hyperpolarize cells. C hemical messengers can be appli ed to th e surface of a cell by microionlophoresis and resulting transmembrane potential cha nges recorded downstre am by microelectrod e recordings. E lectrophysiological methods have been important in st ud ying electrical properties of the neurosecretory neurons of the neurohypophysis (see Fig. 7.3) and the res pon se of pituitary cells to hormones (see Fig. 6.19).

Pharmaco

Many exogenou s substances (e.g. , drugs) interac t with molecular components of cells, and they therefore can be used to study the physiological activity of cells or the mech a nism by which chemical messengers stimulate or inhibit such activity [10]. For example. a t the le vel of the plasmalemma, cardiac glycosides, such as ouabain, are used to inhib it the Na + j K + pump. Certai n hormone secretions are enhanced or inhibited by ouabain . an d th is suggests that active transport (Na + jK + ATPase) systems may be involved in the secretion mecha ni sm of these hormones. Many agents are inhibitors of in tracellular enzyme activity. Of particular inte rest are the methylxanthines, such as theophylline (from tea) and caffeine (from coffee), which are phosphodiesterase inhibitors. These drugs mimic the actions of many hormones because they elevate intrace llular cAMP levels (see Fig. 3.7). There are a number of metabolic inhibitors that affect util ization of substrates for AT P formation. Iodoacetic acid blocks glycolysis, dinitrophenol (D P) uncouples oxida ti ve phosphorylation, and oligomycin prevents mitochondrial phos phorylation of ADP to ATP. The false substrate, 2-deoxyglucose, can be used to slow down glucose utiliza tion with in cells. TIlese inhibitors provide information on th e degree to which the glycolytic ( Embden-M eyerhof) pathway or the citric acid cycle (Krebs cycle) contributes to cellular function. Colchicine, a plant alkaloid, inhibits microtubule asse mbly by binding with tubulin, the pro te in subunit of these intrace llular filaments. Inhibition of hormone action by colchicine or related micro tubule inhibitors (vincristine and vinblastine) might S L ggest, therefore. that the hormone works through a cellular mechanism in which microtubules a re involved. Insulin secretion is inhibited by colchicine. suggesting that microtubules may fu nction in the secretion of this hormone. Cytochalasin B is a fung al me tabolite that specifica lly inte rferes with microfilament function and is without effect on microtubule integrity. Cytochalasin B is inhibitory to the secretion of a number of hormones, suggesting that these filamentous organelles may be involved in the secretory process of these hormones. At the leve l of the nucleus, actinomycin D inhibits RN A producti o n. P uromycin and cycloheximide, on the other hand, inhibit protein synthesis. Thus inhibition of hormone action by actinomycin D implies that the hormone works through a transcriptional process of RNA synthesls. Inhibition of hormone action by puromycin or cycloheximide, in contrast, is interpreted to implicate a tran slational process of protein synthesis in the action mechanism of the ch e mical messenger. Rather specific cation ionophores have bee n discovered and synthesized. These organic cation transport molecules specifically incorporate certain cations into the ir structure. They cross biological membra nes and carry ions into cells. Ionop hore A23l87 , for examp le, is 'pecific for Ca2+ and , because many hormones activate cells by a calcium ion mechanism, this ionophore is in some systems hormone-mimetic. Valinomycin is an ionophore with relativel y high specificity for K+ transport. Some agents, on the othe r hand , are specific inhibitors of ion transport into cells. Verapamil, a Ca2+ channel antagonist , blocks Cal en try in to cells. Usee! concomitantly with a hormone, verapamil 's inhibitory action may s uggest t ha t a particular hormone action is Ca2+ -dependent. Local anesthe tics. such as procaine and te tracaine, ma y also block the action of some hormones by their antagonism of Ca2+ flux into cells.

R omblii TULE .1.

Some pharmaceutical and other agents used in cell physiology studies

nl

DNA T

hruqu

85

Actinomycin D Alloxan Chlorpromazine Cobalt chloride Colchicine Cycloheximide Cyproterone acetate Cytochalasin B 2-DeoxY-D-glucose Dexamethasone Dinitrophenol Ergot alkaloids (e.g., ergocryptine) lodoacteic acid (or iodoacteamide) [allophore A23 187 Liposomes Local aneslhctics (procaine, tetracaine) Methylxanthines (theophyline, caffein e) Oligomycin Puromycin Saralasin Spironolactone Thiouracil [3H]Thymidine [' H]Uracil Valinomycin. Verapamil

Inhibits RNA syn th esis (transc ri pti on) Destroys beta (insulin-secreting) ce lls of pancrea tic islets Dopamine recepto r antagonist Destroys alpha (gl ucagon- ecrering) cells of pancreatic islets Disrupts microtubules 1nhibits protein synth.:sis Tes tosterone re ce p tor an tagonist Disrupts microfilam cnts Metabolic inhibi to r: inhibits glucose uptake and utilization Synthetic glococorticoid agonist Metabolic inhibitor: uncouples oxida ti ve phosphoryla ti on D opam in e receptor agonist (inhibit prol actin secre tion) M e tabolic inhibito r: inhibits glycol ys is Calcium (CalT ) transport carrier (ionophore) Drug carriers Irthibit ce llul ar uptake of Cal .. Phosp hodies teras c inhibitors (elevate cAMP ) Metabolic inhibitor: inhibits phosphorylation of ADP Lo ATP Inhibits prote in synthesis (translation) Angiotc nsin II rece ptor antagonist Aldosterone receptor anta goni t Inhi bits lilyroid iod ide up ta ke and T.,-TJ synth e is Studies on D A synthesis S tudies on RNA syn th es is Potassium tr a nsport carrier (ionophore) Inhibits cellular uptake of Ca h

proceptive (e.g. , ho pping and darting in females) as we ll as consummatory behaviors (e.g., mounting, intromission and ejaculation by the male, or lordotic behavior of the female). Specific behavioral assay methods a re co nsidere d in subsequent chapters as they are us ed to st udy hormone effects on specific behavi rs. Se nsitive and sophisticatcd behavioral assays hav e been developed to assess the effects of hormones on feeding , drinking, sexual behaviors.

R ecombinant DNA technologies are now used to cx press recombinant pro te ins in a varie ty of in vitro expression systems. In some cases the purpose of th e recombinant protein ex pres sion is to a nalyze its biological activi ty or its structure- func tion relatio nships in a biologica l co ntext. In other circum s ta nces, larger a mounts of purified pro te in are required fo r bioph ysical studies of its molecular stru ct ure. Express ion and p urificat ion at the larg .st scale, and perhaps posing some of the biggest technical ha ll nges, is req uireJ ror the product ion of hormones for medical and/or commercial pu rposes. H ormones play important Toles in reg ulati ng the activity o t most cells of the body: indeed , some hormon es are necessary for life. Parath orm one and a ldosterone, two hormones that re gula te th e electrolyte composition of the body arc examples. G rO\ til ho rmone (GH) is necessary for growth, and its absence in th e young anim a l r suits in short stature. Fail ure of the pancrea s to produce insulin results in diab tes mell it us. In ul in replac ment th erapy requires obtaining th ese hormones from other an im al so urces, but some indivi duals develop antibodi es to th ese nonhuman sources of the horm one. [n the cas(: of GH.o nly pituitaries obtained from human cadavers or primates can be lIsed , since GH from other animals is ineffective. Recombinant DNA techniques offer the hope tha t a number of hum a n hormones can be synthesized by bacteria in large amounts and at reasonable prices. Recombinant DNA

86

Chapter 4

t:ndocrtnc Melhodolooles
E. coli E. coli

fJ-Gal

Galactosidase

A Chain

t Beta

fJ-Gal

Galactosidase

Insulin A Chai n o:::t::fXX)

1. Partial Purification 2. Cleavage with CNBr 3. Purification of Insulin Chains

B Chain

Insulin B Chain
y

Recombin ant DNA technique for the mic robial p rod uction o f insulin. (From Rig gs et al. [23], with permission. )

cx;x:x;x:o
S S S S

Active Insulin

Photograph of gigantism produced in the mouse by injection of metallothionein-growth hormone fusion genes into the pronucleus of the fertilized egg followed by implantation within the uterus of a recipient m other. Giant mice (left) became almost twice as heavy as littermates (not bearing the GH gene). (Reprinted by permission from Palmiter et al. [18]. Nature, 300 [5893]: 611-l5.1982 by Macmillan Journals, Limited.)

techniques are essentially in vitro extensions of natural phenomena. These methods involve purification or synthesis of genetic material and its insertion into a bacterial host. Several bacterial strains have been constructed that can produce human hormones, and genetically engine ered bacteria now provide important sources of peptide hormon es [23]. Two methods have bee n used to provide the genetic material to be cloned. For shorter peptides (e.g. , insulin , somatostatin), DN A synthesis is used to make a gene of the desired hormone. In the case of the two chains of insulin, the 21-amino-acid A chain and th e 30-amino-acid B chain are made in separate bacterial strains as tails on a larger precursor protein , the enzyme The individual insulin chains are then clipped from the precursor proteins and, after purifica tion of the separate insulin chain s, they are joined by air oxidation (Fi g. 4.11). The method used for large r proteins utilizes mRN A and reverse transcriptase to make a D NA copy that is then inserted into the bacterial genome and cloned. For example, cD NA fo r proinsuJin is inserted into Esch erichia coli cells, which are then grown by fermentation to produce proinsulin. 1l1e connecting peptide is then cleaved enzymatically from proinsulin to produce human insulin.

The functions of hormones and their receptors are now routinely studied using the powerful and remarkably informative approaches afforded by genetic engineering. These methods are commonly used to produce a desired genetic modification in a mouse a nd observe the phenotypic consequences of the genetic alteration. Most often, genetic engineering is used to generate animals in which either a foreign gene is expressed (a transgenic animal), or a specific gene is inactivated (a gene "knockout" animal). I n the early 1980s, endocrine researchers first injected foreign genes into mammalian embryos and found that this transgenesis approach could provide a powerful tool for use in e ndocrine research. Foreign DNA can be introduced into th e mammalian genome by microinjection of DNA molecules of interest into pronuclei of fertilized cells, followed by the implantation of th e eggs into the reproductive tracts of recipient mothers. The integration of the fo reign DN A into one of the host chromosomes at an early stage of e mbryonic deve lopment results in the development of a transgenic animal [1,13,22,34, 35]. In th e earlier experiments. microinjection of th e stru ctural gene for rat growth hormone into the pronuclei of fertili zed mouse eggs resulted in the development of some offspring that grew significantly larger than their litlermates (Fig. 4.12). This methodology has important implications for

EnQln

nn

87

studying the biological effects of growth hormone: as a way to accelerate animal growth, as a model for gigantism , and possibly as a means of corre cting genetic disease. In the years following these original studies, transgenesis has bee n used in my riad ways to study hormo ne and receptor function. Transgene expression can be targeted , such that the trans oene is only active in a desired ceIl or tissue. This can be accomplished by placin g the coding region of lhe transgene under the control of a promoter sequence that is aclive on ly in the desired target cells. In some cases this is done to assess the consequences of overexpression of a hormone or receptor by the specific targeted cell group; in other circumstance:.. the transgenic protein is designed to be functionally inactive and serve as a dominant negative (inhibitory) modulator of its endogenous counterpart in a targeted cell group. Transgenesis is also use d to label cells of a certain phenotype, such that they may be visualized in the living sta te. For example, the promoter sequence for the hypothalamic neurohormone, gonadotropi n-releasing hormone (Gn RH), can be fused to a gene e ncoding the jellyfish green fluorescent p ro tein (GFP) to create transgenic mice in which the transgene, and hence the fluorescen t JFP molecule , are only expressed in Gn RH neurons. This particular animal model has been used to pe rmit visualization of these few ne urons in living brain tissu ' $ fo r electrophysiological experiments [28]. Transgenic technologies have been studied in several commercially important animal species with emphasis on growth enhancement as a strategy to shorten long production cycles. For the production of transgenic Pacific salmon, an "all-salmon" genetic construct consisting of a metal10thionein-f3 promoter fused with the full-length type I GH gene was developed . 'D1e pO nMTGHI DN A was inj ected into coho salmon eggs with extraordina ry result s (Fig. 4.13). On average, the transgenic salmon were more than 11-fold heavier than the nontransgenic controls. Most interes ting was the observation th at the transgenic fish precociously developed a silver body coloration typical of salmon und ergoing the physiologica l preadaptation (smoltification) necessary for the spring migration from freshwater to the marine environment. Gene knockout animals have provided a wealth of vital information about the physiological roles of hormones and their receptors. Indeed , the loss of function in a gen e deletion mutant animal can confirm the essential role of a protein in a specific endocrine process, or even recapitulate a disease state that is believed to arise from an analogous gene mutation in humans. The first step in the gene targe ting procedure is the illtroduction of a disrupted allele of the target gene into cultured e mbryonic stem cells. The targeting construct is fashioned to contain a selectable marker, such as the gene encoding a protein that confers resistance to a cytotoxic drug. O nly those cells in which the homologous recombination event occurs will therefore survive growth in media containing the toxic drug . Using this process, the cells het e ro zygous for the targe ting construct are selected, grown in culture, and the n injected into mouse blastocysts. T hese embryos are then

Nontransgenic (left) and transgenic (right) coho salmon siblings at 14 months old showing size difference and silver appearance of transgenic individuals, indicative of transformation to seawater adaptability. Length of top large fish (fork length), 41.8 cm. (Used by permission from Devlin et al., "Extraordinary salmon growth." Nature 371:209-10,1994.)

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Chapter 4

docnne Methodotoqtes

surgically transfe rred into the uteri of pse udopregnant femal e mice. The resultant progeny are chimeric for the targeted cells. Bree ding of th ese founder animals produces some mice carrying a single copy of the disrupted gene in all cells, and thus, subsequ en t breeding of two heterozygous mice will produce some mice that are homozygous for the inactivated gene sequence. These homozygous "k nockouts," if they are physiologically viable, can then be studied to assess the potential loss of function that accompanies the loss of th e specific ho rm one, receptor or other protein of in teres t. In so me cases, mice carrying a germline mut ation in a vital gene have lethal defects tha t prec lude their use. To circumvent this probl e m, gen e ticists have developed methods that !,c rmit cell-specific gene targeting, in which the gene mutation can be induce d in a specifically targeted subset of cells in the body. The 10x P-C re recombination syste m is one such met hod. This strategy involves the generation of mice be a ring site-specific recombina tion sites, call e d loxP sites, in the intronic sequences that flank an ess ential e xon of the target ge ne. A se co nd line of mice is produced that harbor a transgene construct containing a gen e promoter fused to the Cre recombinase gene; importantly, the promot er is known to be active only in the cells of interest . Mating of the two mouse lines results in the expre ss ion of C re protei n only in the targeted cells, where it acts at the 10xP sites to delete th e exo n of the targeted gene. Thus. the specific gene deletion only occurs in the target ce lls, avoiding the lethal complications of total germline gene deletion. M any strik ing ex am ples of the successful use of thi s system are found among studies of the tissue- sp ec ific actions of insulin. Conditional disruption of th e insulin rece ptor gene in brain. for exa m ple , recently provided unambiguous confirmation th a t insulin acts centrally to regu late feeding behavior and energy homeostasis [4].

Both vert e b rate and invertebrate animals are used as model sys tems for endocrine research. A few examples of vert ebrate models are discussed.

Th es e most primitive vertebrates, which include the hagfish and lampreys, have not been extensivel y stud ied . T hese eel-shaped animals spend their lives in both marine and freshwater t:nv ironments and therefore most likely possess a complex endocrine system as in other more evolved ("higher") vertebrates.

TIlese ancestors of the bony fish es include sharks, skates, and rays and , although prevalent in numbers and readily available, they have not been the subject of much endocrine research.

These fish represent the largest and most diverse group of vertebrates. They have evolved adaptive features that enable them to survive in a multitude of ecological niches. Many of their speciali zed physiological functions are under endocrine regulation [20]. Migration to and from the sea by some species requires unique roles for some hormones. Prolactin, for example, is essential for survival in some fish transferred from saltwater to freshwater (as happens in salmon migrations). The control of color change in fish is much more complex than in other vertebrates and may, in some species, use a recently discovered melanin-concentrating hormone, whose function may be limited to more recent (teleost) fishes (Chap. 8). Th e corpuscles of Stannius are also unique to certain bony fi shes and produce a hormone, teleocalcin, not found in other vertebrate species. '1l1e urotensins derived from the urophysis of certain species provide another example of hormones whose roles may be restricted to certain bony fishes (Chap. 7).

A mphibia ns incl ude urodeles (salamanders, e.g" "mudpuppies." newts) and anurans (frogs and lo ads), as well as less well-known representatives. 111eir ready availability, their relative ly sma ll size, and the fact that they go through a dramatic metamorphic change from

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larval to ad ult life has made them imp orta nt models for c ndocrin rese arch. O ur und erstanding of the roles of the thyroid gla nd an d its hormon es in e ndocrin e physiolo gy has been greatly influ enced by studies on amphibians (Char . 1.") ). 111e amphibian egg has always been an important mod el for an understa nding of ea rly vertebrat e de e lopme nl. T hese eggs have p rovided evidence for roks of ho rmones (grow t h factors and ste roid hor mones) during the ea rli est devel op mental stages.

Snakes, lizards, turtles, and tort o ises, as we ll as the crocod ilians a nd related specie s, compose this vertebrat e group. T he re has been relatively li llIe research done on these animaL. but important information o n so me uniqu e reproductive an d be ha ' io ral stra tegies under e ndocrine control have been discovered (see Fig. 7.12).

These descendants of th e reptil es includ e a large var ie ty of birds that occupy a grea t d ive rsity of ecological niches. It is not surprising, the re fore , that these a nim als have evolved some unique endocrine strategies. Both wild and domes ticated birds (chicken, quail) are available in substantial num bers for experiment al stud ies. Birds have provid ed uni que mo de ls for unders ta nding the n c uroanatomica l substrates controlling behavior (e.g. bi rd song, co urtship; Chap. 16). Th e role of the kidney in vitamin D hormo ne productio n has been best studied in birds beca u,' e of th e imp orta nt relatio ns hip b tween the hormone and the co ntrol of C a 2 -'- metabolism necess ary for eggs he ll formation (Chap. 9).

T he mouse, rat, hamster, and guinea pig have bee n pa rticularl y importan t anima ls for the adva nceme nt of o ur knowledge of e nd oc rin ology. U se of the mouse has predominated in molecu lar genetics, and the rat ha s perhaps proven most effec tive in the study of e ndocrine physiology. The hamster has proved to be a par ti cul arly useful model for und erstanding th e role of the pineal gland and its horm o ne, melatoni n, in t he control of reproductiv(; cycles (Chap. 20).l11e nude mouse has bee n a use ful mode l for supporting the growth of human tumors. sin ce it lacks an active immune ,ystem neces. ary to reject the transpl an ts. The Bra ttleboro rat lacks the hormone vasopre ssi n and th e refo re is a unique model of " di abetes insipidus" (C hap. 7).

Monkeys, because of their close evolutionary relatedness to humans, have played im portant ro les in und e rstandin g the human endocrine syste m. For example. our kno wledge of the structure a nd function of the corpus lute um of the ovary has been aided by use of prim ate models.

Most endocrin ologists would probably strongly agree tha t studies in animal s have been ess en tial for th e progress of endocrinology. Surgical rem o va l of various rgans has provided k ey insi ghts into th e exis tence and origin of ho rmon es. Most endocrinologi sts wo uld also p robably agree that animals are still absolutely esse n tial fo r basi c studies in endocrinological research. On ly recently, for example, an atrial natriuretic fa clo r (A F ) was isolated from the he art of rats. This a tri al peptide probably plays a pivo tal ro le in th e pathophysiology of several disease states. Ne vertheless, most endocrinol ogists p ro bably woul d agree that not all ani mal s have always been treated humanely and , in many cases, t he numbers of animals used may have been in excess of the actua l numbers needed. 'n le past in d iscre tions are being rem(;d ied a t most or all resea rch centers. Molecular biology has now provided a variety of non anim al model systems wherein the actions of hormones can be studied in vitro using cell cultures r37]. Although these methods will not e ntire ly supple ment studies on living animals, they will provide some alternate, less intrusive, me thods of tudy.

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As in other subdisciplines of biology, endocrin ological research faces some inte resting ethical
C ontroversies regarding the use of embryonic ste m cells or fetal tissues are similar to those of other bioethical debates, but with a novel twist because of the co ntested status of the fe tus and abortion. Fetal tissue transplants hold great hope for m an y pat ients. E xtensive work with anima l models has shown that hum an fetal brain cells transplanted into the substantia nig.ra o f monkeys with exogenously produced Pa rkinson 's dise ase have had beneficial effects. R esearchers think that the time has come for ex panded clinical trials. Cl inica l trials, conducted e ithe r abro ad o r with private funding, have shown that fetal tissue impla nts can significantly improve the symptoms of Parkinson's disease in some individuals. Transpla ntation of feta l brain ce ll s fro m 6- to 8-week-old fetuses into multiple sites on each side of the patient's brain resulted in a n incre ase in brain dopamine productio n a nd . more importantly, symptoms were greatly red uce d for a considerable period of time [31]. EXtJerimenta l evide nce is also strong that feta l isk t cell transplants can restore normal insu lin function in diabetics. A nd fetal thymus and liver tran spla nts may have utility for blood and immune sys tem disorders. M a ny othe r examples could be cited to docllm ent the urgent need of fetal tissues for use in clinical medicine. Cnfortunate ly. " respect for the needs of such patients appe ars to conflict with respect fo r pre natal hu m a n life an d larger soci e tal concerns" [24]. More recently, ma ny of the same bopes and CODce rns ha ve arisen regarding the use of e mbryonic stem cells: 1l1ese cells possess the capacity to be d ifferentiated into any type of ce ll (totip otent), or at least a number of different types of cells (pluri po te nt) . E mbryo nic stem ce ll s are potentially de rivabl e from tho usands of unused, fertilized hum a n eggs stored in the freeze rs of in vitro fertilization clinics. Advances in developmental cell biology have opene d up a new frontie r of biomedical science , in which these ce lls may be induced to differentiate into cells th at may be capa ble of restoring specific tiss ue function in seve ral devast ating disease states. As of the writing of this ed itio n of this te xtbook, the United States Congress is considerin g legislation to permit the use of fe dera l funding for research with embryonic stem ce ll lines beyond the limited number that are currently approved. R ega rdless of th e legislative outcome, concerns o n the part of some citize ns about the use of these cells m akes ill ike ly 1hat the public debate on this issue will continue into the foreseeable future.

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ectop ic transplantation a xoplasm ic tra nspo rt (rlow) atro ph v sand wic h assay immuJl osY Ill pat heeto my re ve rse transcrip tase peR Western blot re port e r const ruct con d itiona l gen..: targeting binding protein immunocytoch e mistry glycoprotein h or mon e an a log io n cha n nc l imm uno histofluoresce nce tra nsfee tl o n gene k nocko ut a nim a l ste m cells

1. Short stature in children may occur as a normal inh erited trai t, or it may result from a clinically identifiable pathology or e ndocrine pat hophysio logy. Growth hormone deficiency (GHD) is an e xample of an endocrin e patllop hys io logy in wh ich the abnorma lly low secre ti on of GH ca n be the appare nt ca use of short stature in ch ildren. How would a clinical e nd ocrinologist de te rmine if serum le ve ls of G H are abnormally low in a ch ild wit h short stature? What m e thod would he or she use to a nalyze GH le ve ls in blood? What re age nts wou ld be required ? How does the method work? 2. E strogen (ER) and progeste rone (Pg R ) re ceptors are prognostic factors in breast cancer. The expre ssion of E Rs a nd Pg Rs in breast cancer cell s is a lso commo nly a n alyzed to g uid e d ec isions regarding th erapeutic op ti ons to pursue. Wha t m eth od wo uld be used to cha ra cterize th e ce ll-spe cific express io n of the ER or PgR proteins in breast tumor tissue secti ons? D escribe the procedure, and list th e specific reagent; and major e qu ipment that wo uld be require d. 3. G e ne targe ti ng methods ha ve bee n use d to assess fun ctio nal co nsequences of hormone rece ptor ge ne ablation, and to thereby clarify th e functional role s o f the targe ted horm one receptor. The targeting of th e receptor gene

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is pres um ably accompanied by a loss of the receptor protein. In stud ies such as th ese, it is incumbent upon the inves tigator to demonstrate that th e binding. o f hormone to the targeted receptor is indeed climinated, or at least significant ly diminished, in targe t tissues of the "ge ne knocko ut" animals. a. Describe a receptor bin ding d'. :dy me th od that could be used to measure th e number a nd affinity of recc:ptors in the wild-type (no rmal ) anima ls and those in th e rece ptor gene knocko ut animals. What reagents would be required? How would the rece ptor binding assay proced ure be performed? b. Assume that yo u have pe rformed the assay me th ods described in (a) , on tissues from th e wild-type a nd (homozygo us) receptor ge ne knock out anima ls, as we ll as from a hete rozygous group of animals in which the receptor numbers may only be partially reduced, but not eliminated by th e di srup ti on of on ly one al lele of the receptor ge ne. For eac h of the three ana lyses, drClw approxim at ions of th e saturation binding curves that yo u wo uld expect to derive from th e bi ndin g assays. Include total, specific, a nd nonspecific binding curves for eac h. How would th ey differ amo ng the th ree groups'! Additionally, draw schematic represe ntations of th e Scatchard plots of each se t of data on th e same graph. 1low wo uld they differ? H ow woul d they be simil ar? 4. Th e decline in es trogcn leve ls in mcnopausal wome n is associated with increased ri sk of excess ive bone demi neralizat ion , a co nditi on common ly known as osteopo rosis. Many investigators are atte mpting to und ers ta nd where and how estroge n normally acts to protec t th e ske le ton from bon e de min e ra liza tion , with th e ho pe of developing new dru g th e rapies for patien ts wi th osteoporosis. a. There are kno wn to be two isoforms of the estrogen receptor, ERa: and th at are encoded by separa te genes. Which molecular methods could you use to determine the relative amounts of the respective receptor isoform mRNAs that are expressed in bone ce ll s'! Wh ich method would yo u use to asce rtain which specific types of bone ce lls, e.g. osteoblasts or ostcoclasts, express the mR NAs encoding or b. Consid e r how you might investigate the effects of es troge n decline on bone mineral izatio n in an exper im e ntal a nim al. Wh a t surgica l me thod mi ght be use d to induce a loss of circ ul ating estrogen in a female rat? c. Suppose you have avai lable to you both a specific ERlX ago nist a nd a specific agonist. You wish to determine if one or the other, or bot h, of the E R isoforms is more impo rta nt in med iating the effects o f estrogen on bone de nsity. Usin g th e experimenta l anim al model in (b), above , design an experi ment in whic h you could use these new dru gs to assess th e re lative in volvement of the two recepto rs in mediat ing estroge n's protective effects on the skele ton . 5. In sulin activates L11e insulin recep tor (TR ) in hepa tic ce lls, promptin g it to beevlJ1 e phosph orylated on key tyrosi ne residues. TIle rece ptor, in tuUl, is a tyrosine kinase tha t phosph orylates downstream signaling molecules, such as th e in su lin rece ptor substrate I (1 R. 1) protein, the reby ac tivating this irllraceUular messe nger. What method

might yo u use to assess th e degree of IR and IRS I ac ti va ti on in cult ur of hepati c ce lls? For your expe rime nts.. yo u have avai lable to you a pa nel of monoclonal antibodies that recogni7.e tota l fR, phosphotyrosin e-l R. total IRS l, and phosphoty rosine- fRS l. res pectively. Describe the mcthod that you would usc. 6. nabo lic steroid abuse has bce n anecdotally linked to a va riety of pa thophysiologics and beha vio ral di sordc rs, but their molecular actions in the brain rc:main poorly un derstood. It is likel y th at these and roge ns, lik e other steroid ho rm on es, C)(;[t their actions in the brain by simult aneo usly altering. the expression of many different ge nes, and in seve ral diHerent partS of the brain. a. What method cou ld be used to simultaneo usly ide ntify genes whose expression levels arc alte red by in the brains of experimental an imal s'! Describe th e c.- perime ntal method and technologies that mi ght be used. b. If specific testosterone-respon sive genes were identifi ed by th is method , then what methods could be used to specifically identify which brain ce ll populations exhibit testosterone-induced alterat iow. in the expression of thesc genes'!

[I] Aguzzi. A , et al. 1994. Tra nsge nic and knockout mice Mode ls of ncurological di sease. Brain Pathof. 4:3- 20. [2J Heermann , W 1973. Di rect changes in th e pattern of Balbiani ring pu ffing in ChironOI17I1S: Effects of sugar trea tm en t Chromosotlw 4:297-3 26. [31 Black. J 1Y89. Drugs from emascul ated hormones: The principl e or syntropic a ntago nism . In Vitro Cell. Develop.
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(41 I3runing 1. C , et al. 2000 . Role of brai n ins ulin recep tor
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[5] Burgoyne, R. 0 .. and A . Morga n. 1993. Regulated exocytosi s. Biochem. J 293:305-16. [6] Ca mp, P.. e t al 1992. (ie ne express ion of peptiJergic signa ls: rreets of sle. ro id s. Nr>uropro tocols 1:67-76 . [7] Chi rguin , J M . 1990. Molecul ar biology for nonm o lecu la r biologists. Diabetes Cale 13:188- 97. [8] Davis, J R. E. 1996. Molecular biology techniques in endocri nology. Clin. L nriocrino l. 45: 1.25-33. [9] Fukamizu.A 1993.Transgenic animals in endocrinological invc:stiga tion.i. L ndocrinol. In vest. 16:46 1-73. [101 G ilm an, A G., L. S. G oodman , and A Gilman . 1990.
Go odman and G ilman's the pharmacological basis of th erapelltin . . ew Yo rk: Macmillan.

[1 '1J Hoffman, G. ., M. S. SmitiJ . and J G. Vcrbalis. 1993. c-Fos and related immeJia te t!arly ge ne products as markers of activ ity in neuroendocrinc syste ms. h ont. NeL/roendocrinol. 14:'173- 2 [3.

[12] H olzwa rth , M. A , 1. R. Sl adek , Jr. , and K. M. Knigge.

1976. Monosodi um glutamate induced lesions of th e arcuate nucleus. Anal. Ret. 186:197- 205 . [13] Kudlow, 1. E. 2002. Transgenic a nilJ1 allll odels of pitu itary function . In : J D. Baxte r. S. Melmed , and M. 1. C W, eds. G enetics in endocrinology. Modern Endocrin ology Se ries ( L Ma rtini, serlC:s cd.).- Philadelphia : Lippincott Willi ams & Wilkins, pp. 27-36.

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[14] Lebl o nd, C. P. 199 1. Time dimensi o n In cell biology. A radi oa utographic sur vey of the dynamic fe atures of celis, ce ll co mponents, and ex trace llula r matrix. Protoplasm a 160:5-38. ("15) Mc('ube, J T. , et al. 191\6. Front. N eu roendocrino/. 9:149-67 . [16] Mi llar. S. E. , et al. 1989. Vacc ination with Cl sy nthetic zona pe llucid:! peptide produces 10Jl>!-term con trace pti o n in female mice. Science 246:93 5-8. [17) N!u mby, M. L , et al. 1985. Mo noclonal antib o dies as pro bes oj stru cture, fun ction and isoenzy me forms of th e type II regulatory subunit o f cycl ic A MP -depe ndent protein kin ase . Pharma co l. Ther. 21';::'\67-87 . [18] Palmiter, R . D , e t 81. 1982. D ramatic growth o f mice that develop from eggs microinjected with m e lall otio ne ingrowth horm o ne fu sio n genes. N ature 300:611-15. (1 9) Pa rmentier, M .. e t a1. 1989 . Molecular clo nin g of the tllyro tropin re ce ptor. Scien ce 246 :1620-2. [20] Powe rs, D . A. 1989. Fi sh as mod e l sys te ms. Scien ce 246: 352-8. [2 11 P re digc r, E. A. 200 1. De te cti o n a nd quantitati on o f m RNAs uSing ribo nu clease protec tion assays. M ethods 1'.101 Bioi. 160 :495 - 505. [22) R eve ntos, J , and F M un e lL 1997. Tran sgen ic a nim al models in re productive e ndocrine research. E/.//: J Endocrin o!. 136:566-80. [23] Ri ggs, A. D. et aL 1980. Synthesi s. clo nin g. and expressio n o f hormon e genes in E lch erichw coli. Rec. Prog. H o m1. Res. 36:26 1- 76. (24) Robertson, 1. A . 1988. R ight s, sy mbolism. and public policy in feta l ti ss ue t ran spl a n ts. Hastings Crr. Rpt. 5-12. [25) Sh iz u me. K ., A. B. Le r n e r. a nd T. B. F it z patrick. 1954. In vit ro bioa ss ay lor m e la nocyte stimula ting h ormone. Endocrinology 54:5 53- 60 . [26] Simonsen , f l., and H. F. Lodish . 1994. Cloning by function: express io n cloni ng III mam malian ce ll s. Trends Phurnlil co l. S ci. 15::137-41. [27] Soulet, D . and S. Ri ves t. 2002. Pe rspec tiv e: how to mak e microana v, se ri a l a nal ys is of gene exp ression,

and proteomic re le vant to da y-t o -day endocrine problems and phys io logical sys te ms. Endocrin o logy 143: 1995-200 l. [28J Spergel, D. J.. et al. 2001. Using reporter genes to label. selecte d ne ur ona l popul a ti o ns in tran sge nic mice for gene promo ter, anat o mical. and ph ys io logical st udi es. Prog. NeL/mniol. 63( 6)673-86. [29] Spice r. S. S. 1993. Ad vantages of hist ochemis try fo r the stud y of cell b io logy. Hisro chem J 26 :531-47. [30] Theoharides, T. C , and W W Douglas. 1978 . Secre tion in mast cells induced by calcium e ntr apped within phospholipid vesicles. S cien ce 201:1143-5. (31) Th ompso n, L. 1992. Fetal trans plants sho w promise. Scien ce 257 :868-70. [32] Tong, Y . et al. 1990. Glucoco rti coid regulati o n of proopiomelanocortin mRN A levels in rat arcu ate nuc le us. Mol. Cell. Newosci. 1 :78- 83 . [331 Tra yhurn. P. 1996. N orthern blotting. Proc. Nurr. S oc. 55583- 9. [34) Wa ll , R. J. , D. E. Ke rr. and K. R. B ondioli. 1997. Transgenic dairy ca ttl e: Genetic enginee ring on a .large sca le. J Dmry S ci. 80:2213-24. [35] Wall , R. 5. , e t al. 1997. Tra nsge nic anim a l technology. 1. Androl. 11\:23 6-9. [36 J Ware mb o urg. M. 1976 . Detecti o n of diffusible su bstances. 1. de Mi croscopie et de Biologie Cellulaire 27:277-RO. [371 Weiss, J., and J L. Jameson. 1993. P e rifused pituitary cells as a model fo r stuciies of gonado tropin bi osy nth e sis and secre ti o n. TE M 4:265-70. [38) Wilcox , J N. 1993. Fund a me nt a ls of ill si tu hybridi za ti o n. 1. f-fi stochem. Cy rochem. 41: 1725-33. [39] Woodruff,T. K. 1998. CelluJar localizatio n of mRN A an d protein: in situ hy bridiza ti on histochemistry and in situ ligand binding. Methods Cell Bioi 57:333-5 1. [40] Yalow, R . S. 1978. R adioi mmunoassay: A probe for the fin e st ructure of bi Ologic systems. Science 200: 1236-45. [41] Z h a ng, Ye t al. 1994. Posi tional clol1ln g of the mous e obese gene a nd its hum an ho mologue. NalLire 372:425-32.