THE DISTRIBUTION OF CORTICOTROPIN IN THE

by

Guy Rochefort

THESIS

submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of

Master of Science

The Allan Memorial Institute of Psychiatr,y McGill University, Montreal, ~ebec.

August 2lst, 1956. ACKNOWLEDGE2ŒNTS

Thie investigation was supported by grant& to Dr. R.A. Cleghorn from the Federal-Provincial Mental Health Research Fund and from the Foundations1 Fund for Research in Psychiatry, New Haven.

I wish to express my very sincere appreciation to Dr. M. Saffran for his constant interest, encouragement and able direction.

The beef and hog pituitaries were obtained through the generous cooperation of Canada Packers Limited (Montreal).

The assistance and encouragement given to me by ~ wife and all the members of the Allan Memorial Institute Research Lab- oratories is gratefully acknowledged.

M& sincere thanks to the members of the Laboratory staff who assisted in reading and criticizing the manuscript. TABLE OF CONTENTS Page

INTRODUCTION AND HISTOLOGICAL •••••••••••••••••••••••••••••••••••••••• 1 I. The Problem of Corticotropin Elaboration •••••••••••••••••••••••• 2 II. Concentrations of ACTH in Normal Pituitaries •••••••••••••••••••• 9

1. Anterior Lobe ••••••••••••••••••••••••••••••••••••••••••• 9 2. Posterior Lobe •••••••••••••••••••••••••••••••••••••••••• 9 MATERIALS AND METHODS ••••••••••••••••••••••••••••••••·•••••••••••••••11 1. Sources of Pituitary Tissues ••••••••••••••••••••••••••••••••••• 11

2. Treatment of Tissues •••••••••••••••••••••••••••••••••••••••••••11 a) Rat anterior lobes ·•·••••••••••••••••••••••••••••••••••11 b) Rat poeterior lobes ••••••••••••••••••••••••••••••••••••12 c) Beef and hog anterior lobes ••••••••••••••••••••••••••••12 d) Extraction procedure •••••••••••••••••••••••••••••••••••13

e) Assay procedure ••••••••••••••••••••••••••••••••••••••••13 RESULTS ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••14 1. Concentration of ACTH in rat ••••••••••••••••• 14 2. Distribution of ACTH in rat anterior pituitary ••••••••••••••••••15 3. Distribution of ACTH in beef and hog anterior pituitaries ••••••• 19 4. ACTH in rat, beef and human posterior pituitaries ••••••••••••••• 21 DISCUSSION AND CONCLUSIONS •••••••••••••••·•·•••••••••••••••••••••••••23 1. Concentration of ACTH in rat anterior pituitary •••••••••••••••••23 2. Distribution of ACTH in rat anterior pituitar,y •••••••••••••••••• 24

3. Distribution of ACTH in bee! and hog anterior pituitaries ••••••• 25

4. ACTH in rat, beef and human posterior pituitaries ••••••••••••••• 30

SUMMAR.Y ••••••••••••• • •••••••••••••••••••••••••••••••••••••••••••••••• 32

BIBLIOGRAPHY •••·••••••••••••••••••••••••••••••••••••••••••••••••••••·33

APPENDIX •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••37 INTRODUCTION AND HISTORICAL

Six tropbic have been extract.ed from the anterior lobe of the pituit.ary gland. The secretion and storage of t.heae hormones takes place only in the aeidophilic and basophilie cells of the anterior lobe; the chromophobe cella are believed to be in- active in these respects. It has been established that somatotrophin (STH- growth ) and (lactogenic hormone) are aecreted by the acidophils (1-11), while thyrotropin (TSH- tbyroid stimulating hormone) and the two gonadotropins (FSH- follicle stimulating hormone and LH- ) are secreted by the basophils (13-22). I. The Problem of Corticotropin Elaboration: The problem of acidophilic or basophilie elaboration ot corti- cot.ropin (ACTH) bas, however, resist.ed solution, notwithst&nding the application of ~y methode by numerous investigators. Initially, the basophile were held responsible, due to observations on the adenohypo- physes of Addisonian patients, where the most. characteristic changea are the diminution in number and the regressive changes in the basophile. (23,24,25). However, conflicting evidence soon appeared. Bailiff (26), in

1938 noted in the hypersecreting hypophyses of rate 8Xp08ed to severe cold, a prompt and severe degranulation of the acidophils while the basopbils were only increased in size and vacuolated. At the same tiae, on pathological grounds, Haymaker and Anderson (27) confirlled

Cushing '• contention (28) that adrenocortical tumeurs may be tound accgmpanying the acidopbilic adenomas of acromegaly. Smelser (29), in 1944, therefore attempted to find the solution experimentally. He prepared extracts of the zones in beet pituitaries which Smith (4) bad previously shown contaiRed rather discrete zones of baaophilic and acidophilic tissues and injected them into young immature rats. Judging by the weight gain of the adrenals of the

animale (Moon as~ for ACTH-30) 1 he concluded that the basophilie area contained aix to eight times more ACTH than the acidophilic area.

HoweTer 1 in the same year 1 Heinbecker and Rolf( (31) attributed the secretion of ACTH to the acidophile. They feund complete functional

depression and atrophy of the adrenals in hypophysectoaized dogs,

whereas in "puncture doge" 1 in whoa the infundibular stem only is

severed and the basophile degenerate wbile the acidophile are unaltered1 the adrenals remained normal.

A tew years later1 in 1948, D'Angelo, Gordon and Charipper (32) investigated the effects of chronic and acute starvation on guinea

pigs. The adrenal cortices of the starved animala were bypertropbied1 indicating an increased output of ACTH. The ll&in cytological changes

seen in the anterior lobes included a loss of granulation in the acid- ophil cella which decreased in aize and number. The Golgi apparatus

was small and compact. Most of the basophile were enlarged and were

in various stages of degranulation and vacuoli~tion. They possessed abundant mitochondria and the Gol&i apparatus was hypertrophied. The

sigos of heightened secretory activity in the basophile were inter- preted by the authors to be due to an "augmentation of adrenotropin

(ACTH) secretion" by the basophil cella. In the same year Giroud and Martinet (33) in France implanted small blocks of fresh fr

These claims were soon counterbalanced by the work of Finerty

and Briseno-Castrejon (34), who adrenalectomized rats unilaterally and found a marked increaee in the percentage of acidophilic cella and an accentuation of this increase when the animale were subjected to cold stress. These changes were to them indications of the secret- ion of ACTH by the acidophile. In 1951, Halmi and Bogdanove (35), convinced tbat the probleœ of ACTH secretion could not be aolved by cytological meana, decided that a comparieon of the histological picture and the hormonal con- tent of the gland atforded a more promiaing approach to the problem. They therefore used thyroidectomized rats, in whom'- a rapidly pro- gressing hyperplasia and vacuolation of delta 1 cella, degranulation and disappearance of beta cella and an almost complete absence of acid- ophils resulted. The ACTH content of the experimental glands, compared to normals by the adrenal ascorbic acid depletion method, bad decreased but very slightly. Halmi and Bogdanove therefore coneluded that his- tological changes in the acidophils or beta basophile were indepehdent of the ACTH of the gland. Similar experimente, with very similar re- sults, on castrated and estrogen treated rats led them to the same incoaclusive answer wi.th regard to the delta basophile (37). However, wi.th somewhat similar method.s, Tuchmann-Duplessia (3S, 39) concluded from her experimenta that the basopbils secrete ACTH.

After bilateral adrenaleet~, sbe attributed the reduced aize, lesa of granulation and the atrophied Golgi apparatua of the basophil cella

1 delta and beta cella of the anterior pituitary are subclasses of the basophil cella: Ha.l.mi (36), Purves and Griesbach (16). - 4 -

to an increased excretory activity of these cella; at the same time, assayed by the increase in weight in the adrenals of hypophysect6mized. rats (Simpson "repair test") (40), the ACTK content of pituitaries of these animals had appreciably decreased. Corticosteroid administrat- ion-deoxycorticosterone, cortisone, 11-desoxy-17 hydroxycorticosterone-

produced the following changes: increase in the size, number, granulat-

ion and vacuolization of the basophils, increased ACTH content of the glands, and atrophy of the . Treatœent with estrogens gave opposite resulta: first the basophils showed increased activity as evidenced by increase in number and size, followed rapidly by a ' marked degranulation to result in chramophobic cella. stimultaneously, the adrenal cortex was extremely stimulated and the ACTH potency of the pituitaries fell markedly. These facts led to the conclusion that

ACTH was elaborated in, stored in and secreted by the anterior pit- uitary basophile. Marshall (41), assuming that ACTH might occur in the pituitary gland and in certain hormon• preparations as a potentially antigenic protein, applied to the problem the immunochemical method of Coons and al. (42) by which it is possible 11 to localize foreign antigens in tissues by using fluorescent antibody sQlutions as specifie histo-

chemical reagents11 • Marshall injected purified hog ACTH into adrenal- ectomized rabbits (the animals reacted to the injected hormone as to an inert foreign protein), bled the animals after they had shawn strongly positive precipitin tests and fracti onated the serum for gamma-globulin. The globulin was then conjugated with fluorescein and purified by being freed of all other precipitins. Fresh, thin, slices of hog pituitary were then incubated with the fluorescent globulin solution. Using the ultraviolet the fluorescent - 5 ~ cells were charted and photographed. The same slices were then cleared and stained by the azan method. It was found that the fluorescent cells in every instance were basophile. Moreover, the fluorescent globulin was capable of precipitating with a highly potent hog !CTH preparation. The resulting precipitate assayed by the Sayers test, contained most of the hormonal activity. Marshall therefore aoncluded that the basophil cella secrete ACTH.

Convinced that histological, cytological, bistochemical and other similar techniques were only indirect approaches to the problem, Herlant (43,44,45) sought the answer in a more direct fasbion. He isolated the intracellular partiales of sheep, ~og and beef anterior pituitaries by the differentia! centrifugation methods of Claude (46) and Hogeboam, Schneider and Pallade (47), and characterized them by their reactions. His resulte indicated that the acidophilic granular fraction was the most potent in ACTH activity, as measured by the Thorn eosinopenia test (4S) in intact and hypophysectomized rats. In the same year, 1953, two other papers vere published which lent support to the acidophil theory. Soulairac and bis colleagues (49), studying the action of exogenous ACTH on the behaviour of the sexually mature rat and its effect on the pituitaries of these aniaals, concluded that the lack of morphological changes in the acidophil• was a direct argument in favour of an acidophilic secretion of ACTH: the injection of exogenous hormone campletely supressed the secretion of ACTH and the accompanying cytological changes. Desclaux et al. (50) implanted blocks of acidophilic and basophilie beef anterior pituitary tissue on the left adrenals of adult male r ats. The two types of .. 6 -

tissue were equally effective in causing an increase in weigbt of the treated adrenals; however, only the acidophilic tissue was potent in causing a loss of sudanophilia in the and the f&siculata of the recipient glands. These findings were taken to indicate that the acidophile contained ACTH.

In 1954 Kallman and Gordon (51) implanted pellets o! cortis- one and desoxycorticosterone acetate near the anterior pituitary. Ac- cording to the Sayers theory of partial regalation of ACTH release by the blood levels o! circulating corticoids, the resulting hormonal field should inhibit the release of the ACTH from the anterior pit- uitary. The authors observed only slight and variable alterations in the acidophile but striking and extensive hypertrophy and vacuolation in the basophile. They interpreted these changes as consistent with the hypothesis that the basophiJiis the cellular source of synthesis ot adrenocorticotropin (ACTH).

Knigge (52), in 1956, ad.renalectomized rats and studied the cytology and hormonal content of their anterior pituitaries. Staining to differentiate between the beta and delta basophile, he noted that though there soon occurred a progressive decrease in the relative number of beta cells, the per cent of delta cella did not alter and that these delta cells slowly hypertrophied and vacuolated. These signe of increased delta activity, added to the tripled ACTH content, as measured by the adrenal ascorbic acid depletion technique, indicated to him that the delta-basophile are the cellular source of ACTH. Lastly, to render an already indistinct picture even more ambiguous, Hess and Brown (5.3) were unable, by differentia! centri!ugat- - 7- ion, to separate from beef pituitary hamogenates any cellular traction, microscopically defined, containing most of the ACTH, assayed by the Sayers adrenal ascorbic acid depletion method. It is evident, from the above review, that the problem of cellular elaboration of ACTH bas not yet been solved, since a nearly equal number of conclusions have favoured the acidophils as the baso- phils (Table I and II).

TABLE I

Acidophil cell elaboration of ACTH Ret. No. Year Investigators Method

26 19.38 Bailiff Cytology; cold stress in rats 27 19.38 Haymaker, Anderson Clinicopathological data . .31 1944 Heinbecker and Rolf ot hypophysectamized and puncture dogs .34 1949 Finerty, Briseno- Histology; hemiadrenalectamy Castrejon and cold stress in rats 4.3-45 1952 Herlant Isolation of cellular components of sheep, hog and be et

49 ~953 Soulairac et al. Exogenous ACTH and histo~ogy of rat glands 50 195.3 Desclaux et al. Tissue implantation of beer glands in rats. TABLE II

Basophil cell elaboration of ACTH Ref. No. Ye'll" Investigators Kethod

23-25 1921-38 Shumaker and Firor Clinicopathological data Crooke and Russel on Addiaonians Severinghana 29 1944 Smelser ACTH assays on separated parts of bee! pituitaries .32 1948 D1 Angelo et al • Cytology of pituitaries of chronic and acutely starved guinea pigs .33 1948 Giroud and Martinet Tissue implantation of bee! glands into rats•

.38-.39 1950-52 Tuchmann-Duplessis Cytology and ACTH assays of rat pituitaries

4l 1951 Marshall Immunochemi.cal method on hog tissue -51 1954 Kallmann and Gordon Cytology; steroid pellet implantation in rats 52 1956 Knigge Cytology and ACTH assays of rat pituitaries

The work reported in this thesis is an attempt to obtain data towards a detinite solution to the problem of the cellular source ot ACTH. These tacts were not obtained through the atudy of the distri- bution of ACTH in the rat anterior pituitar,y. However, the direct extraction. and assay of the acidophilic and basophilie areaa of freah beet and hog anterior pituitaries has provided a valuable basis for further experimentation towards the solution of this moot probla. - 9-

II. CONCENTRATIONS OF ACTH IN NORMAL PITUITARIES: I. Anterior lobe:

Reliable estimations of ACTH in normal pituitaries awaited the development of specifie and accurate methods of extraction and assay. Astwood, (54) in 1952, reviewed the available literature but most of the values he cites are from personal cgmmunications.

Burns et ,!!. (55) had., in 1948, published levels of ACTH in rat, hog and human pituitaries. The pituitaries were lyopbilized and ground to homeogeneous powders. A known weight of the powders was suc- cessively extracted three times with 0.9% NaCl made alkaline to 0.01 N with NaOB. The combined extracts were assayed for ACTH against a known standard by the adrenal ascorbic acid depletion method. Their value of a total 70 to 100 mU. per rat pituitary was con!irmed in 1954 by Sydnor and Sayers (56) who extracted the fresh or frozen glands with 0.1 N HCl. Later Sa!!ran and Schall.y (57), applied their in Vitro bioass~ to 0.01 N HCl extracts, and obtained average values of 300 to 400 mU. per rat pituitary. In this thesis the glacial acetic ex- traction procedure of Payne, Raben and Astwood· (58) was modified for use with the Saffran and Schally in vitro assay procedure. The values obtained were considerably higher than those reported preTiously.

2. Posterior lobe: During the course of investigations in our laboratory with hog posterior lobe powders, they were noted to contain a surprisingly high amount of ACTH. The presence of ACTH in the posterior lobe bad already been described in the literature. As early as 1935, Moszkowska (59) claimed that alkaline extracts of beef posterior lobes, injected into normal guinea pigs, caused adrenal hyperplasia and lipid de- .. 10 -

pletion. In 1952 Mialhe-Voloss (60) repeated this work using the

Pincus (61) adrenal weight maintenance test in hypophysectomized rats and came to the same conclusion. In the next year, Si.Dmonet and his colleagues (62) extracted ACTH !rom the posterior lobes of bogs. Mialhe-Voloss (63,64) tben extracted beef, rat and duck posterior pituitaries and demonstrated their ACTH content by the Sayers ad- renal ascorbic acid depletion method. She claimed at that time that the rat contained almost as much ACTH as did the anterior pituitar,y. Because much of the work in our laboratory involved the use or posterior lobe preparations, it became vital to confirm the validity of these claims and to estimate the values for rat posterior lobe ACTH. Utilizing methods different from those of the other workers, we have confirmed the presence ot ACTH in rat and beef posterior pituitary glands. We have also shown the presence of ACTH in human posterior pituitary tissue. -li-

MATERIALS AND METHODS 1. Source or Pituitary tissues: Rat pituitaries were obtained from animals or the Sprague- Dawley strain, supplied by the Canadian Breeding Laboratories and weil acclimatized to the constant temperature animal roam. All animal. a used were from 140 to 200 grams in body weight. <Àl being removed from its cage, each animal was injected with 0.3 mls of a 1.6% sodium nembutal solution and allowed to tall asleep, to obviate the possible stress of transportation to the laboratory• . While still under the anesthetic, the animals were decapitated with a guillotine. The top of the skull was then rapidly eut away and the brain carefully scooped out, exposing the pituitary. With fine-tipped forceps, the anterior and posterior lobes were separated and removed from the sella turèica. Beef and hog glands were obtained at the slaughter bouse less than an hour after the death of the animale, quick frozen on dry ice and kept in the deep-freeze until used. Human glands were obtained through the cooperation of Dr. Calvin Ezrin, from the Department of Pathology, University of

Toronto. The.y were received frozen on dry ice and kept in the deep- treeze until use.

2. Treatment of tissues: a) Rat anterior lobes: For assays on the whole lobe, the tissues were rapidly weighed, dropped directly into tubes containing a known volume of glacial acetic acid, extracted and assayed as described below. For distribution studies, the right and lett wings of three -12-

anterior lobes vere rapidly frozen on dry ice either as removed from the animal or in a drop of distilled vater. They were then carefully positioned on a freezing microtome and sectioned serially at 50 micra. The slices from the right wings were iœmediately placed into small test tubes containing a known volume of glacial aeetic acid; the slices from the left vings were placed on small tared paper disks. Each tube and disk received three groups of four consecutive slices, equivalent to a tissue thickness of 200 micra. The tubes vere then extracted and assayed for ACTH as described below, while the disks were dried.

and then weighed on an ultramicro balance to obtain dry weights of the sections. Three groups of three glands were thus treated, each group being sectioned in one of three planes: sagittal, horizontal and eoronal.

b) Rat posterior lobe:

Posterior lobes were obtained as described above and four to ten glands were pooled, rapidly weighed, extracted and assayed as described below.

c) Beef and hog anterior lobes: The frozen pituitaries were thawed and freed of their mem- brances. With scalpel and scissors, the glands vere then dissected into several parts. The two postero-lateral wings of the glands were combined, extraeted and assayed as described below. The wings are white, lipoid-like and easily discernible; Smith (4), Smelser (29),

Giroud and Desclaux (65) Giroud and Martinet (33), Desclaux et al. (50) have .found these wings of the gland to be very rich in acidophile ., 1.3 -

and very poor in basophils. The antero~edian portion of the gland, which is a reddish tissue and has been described to be mostly baso- philie in cell population (4,29,65,33,50) was extracted and assayed separately, d) Extraction procedure: The rat anterior pituitar.y slices were ground with sand with lOO microliters of glacial acetic acid. The other tissues, whole glands or part8 thereof, were ground "Wi.th sand with 500 microliters of glacial acetic acid. Then another volume of glacial acetic acid (lOO or 500 pl) was added and the tubes were heated, with stirring, in a water bath kept at 80-85°0 for 30 minutes. Gare was taken to exclude condensed water from the tubes. The tubes were removed from the bath and the rods rinsed with either lOO or 500 microliters of glacial acetic acid. The tubes were then stoppered with Parafilm and stored in the deep-freeze. The day before ass~, each tu~ was thawed and placed in a vacuum dessicator over NaOH pellets at roam tempera- ture. In the morning, the dry residue tram the slices was extracted for two hours with 300 microliters of 0.01 N HCl in the dark am that from the other tissue81 with 1000 microliters. A lOO microliter aliquot was then diluted with Krebs-Ringer-bicarbonate-glucose medium for assay. e) Assar procedure: All preparations were assayed for their ACTH potency by the Saffran and Schally in vitro technique (66) against the u.s.P. Provision&! Standard or against powders standardized against the U.S.P. standard. ·14-

RESULTS

1. Concentration of ACTH in the rat anterior pituitary.

In Table III are given the values of ACTH for freeh rat anterior pituitary tissues, extracted with a modified glacial acetic acid method and assayed with the Saffran and Schally in vitro technique. From. these values, it may be calculated that a 5 to 6 milligram rat anterior pituitary contains more than 1000 mU. of

ACTH. This figure ie considerably higher than the 100 mU. per gland reported by Burns et .!J:. (55) and by Sydnor and Sayers (56) and obtained with the adrenal ascorbic acid depletion aas~ on alkaline-NaCl and 0.1 N HCl extracts respectively. It also is greater than the .300 mU. per gland obtained by Saffran and Sehally (57) with their in vitro technique applied to 0.01 N HCl extraets.

TABLE III mU. ACTH per mg 95% confidence Index of of fresh tissue limita precision

170 80..,360 o.l6 218 141·.3.38 0.10 2.3.3 1CX>-51.3 0.16 250 147-428 0.11 .3.34 154-724 0.15

TABLE III: Concentrations of ACTH in fresh rat anterior pituitary

tissue. The mean value ie 241 mU. of ACTH per mil- ligram of fresh tissue. - 15 ~

2. Distribution of ACTH in the rat anterior pituit!fY.

The resulta of the etudies on the dietribution of ACTH in the rat anterior pituitary are eummarized in Tables IV, V and VI.

In these etudies, the dry weights obtained with left lobe sections were used as an approximation of the weights of the extracted right lobe sections. The values in the Tables are expressed as the amount of ACTH per 200 micrograms of dried tissue, which is roughly equiTal- ent to one milligram of fresh tissue (67).

Table IV presents the ACTH distribution in the sagittal plane of sectioning. The lateral sections in this plane (sections 2 and

3) are higher in their ACTH content than the other sections, although statistically the differences are not significant. Table V liste the distribution of ACTH in the horizontal and

Table VI in the coronal plane of the gland. Although statistically not significant, the middle area in the horizontal plane and the posterior areas (i.e., remote frgm the posterior lobe) in the coronal plane appear to posse~a higher ACTH content than the other sections. - 16 ...

TABLE IV Section mU. ACTH/200 mcgs 95% limite of Index of of dry tissue confidence precision 1 no detectable activity - 2 135 79 .. 231 0.12

3 151 58-393 0.18 4 1œ 59-198 0.10 5 102 54-193 o.u 6 106 83-135 0.03 7 101 44-231 0.15 8 71 21-245 0.16

9 84 39-178 0.15 10 63 36-110 0.10

11 84 52-170 0.12

TABLE IV: ACTH content of sagittal sections of the rat anterior pituitary. Sagittal sections run from the lateral tip of the gland, vertically, towards the mid-line, as shown in Figure 1. FIGURE 1: Top view of rat ------pituitary illustrating sagittal sectioning of the anterior lobe. Left wing Right wing Weighed Extracted 150 Approx. mU. 125 ACTH per 200 mcgs dry tissue. lOO

75

50

Section Nwnber - 17 -

TABLE V Section mU. ACTH/200 mgs 95% limits of Index of of dey tissue confidence precision 1 101 56-181 0.12

2 80 46-1.36 O.ll

.3 2(ff 104-410 0.09 4 142 105-192 0.05 5 127 87-18.3 o.œ 6 l(ff 65-177 0.09

TABLE V: ACTH content of horizontal aections of the rat anterior pituitary. Horizontal sections run from the top of the gland to the bottom, as shown in Figure 2.

FIGURE 2: Frontal view of rat pituitary illustrating horizontal sectioning of the anterior lobe.

Left wing Right wing Weighed Extracted

Section Number

Approx. mU. ACTH per 200 mcgs dry tissue. -18-

TABLE VI Section mU. ACTH/200 mcgs 95% limits of Index of of dry tissue confidence precision 1 145 106-199 0.05 2 lW 57-197 0.12

3 114 63 ... 207 0.12 4 97 60-156 0.10 5 ll5 43-310 0.22 6 93 52-164 0.11 7 209 132-330 0.09 8 284 143-565 0.12

TABLE VI: ACTH content of coronal sections of the rat anterior pituitary. Coronal sections run vertically from the anterior fac• of the gland to the posterior face, as shown in Figure 3.

FIGURE 3: Top view of rat pituitary illustrating coronal sectioning of the anterior lobe.

Left Wing Right wing Weighed Extracted

Section Number

Approx. mU. ACTH per 200 mcgs dry tissue. - 19 -

3. Distribution of ACTH in bee! and hog anterior pituitaries.

The reaults of the assays on the acidophilic and basophilie areas of bee! anterior pituitary are prasented in Table VII and those on the hog pituitary in Table VIII. It may be seen that in ever.y instance the basophilie areas contained at least four times as much ACTH as the acidopbilic areas. However, in bee! gland 3 and in hog gland 3, the lower 95% confidence limita of the basophilie values overlap with the upper 95% confidence limite of the acidophilic values. An ins- pection of the two tables reveals very poor indices of precision in these two assays: the overlapping of these values is undoubtedly due to the relatively poor assays. - 20-

TABLE VII Index of Gland Tissue mgs mU. ACTH/mg 95% limits precisioa

Acidophilic 46 21 10-42 0.14 1 Basophilie 17 152 62-370 0.19

Acidophilic 36 14 5-41 0.20 2 Basophilie 17 183 84-397 0.17

Acidopbilic 23 15.5 9-27 0.10 3 Basophilie 9.5 133 18-941 0.22

Acidophilic 34.5 35 21-58 o.œ 4 Basophilie 16.3 140 98-200 o.œ

TABLE VII: ACTH content of separated acidophilic and basophilie parts of fresh beer anterior pituitary. The mean

for the acidophilic area content is 21 mU, that of

the basophilie area is 152 mU. (ratio 1:7). ,

.. 2~ ..

TABLE VIII

Index of Gland Tissue mgs mU. AC TH/mg 95% limits precision

Acidophilic 20.6 68 47-98 o.œ l Basophilie 16.0 344 156-756 0.17

Acidophilic 27.4 49 25-96 0.14 2 Basophilie 33.5 461 194-1092 0.17

Acidophilic 35.3 115 84-157 0.05 3 Basophilie 18.0 417 139-1246 0.24

TABLE VIII: ACTH content of separated acidophilic and basophilie parts of fresh hog anterior pituitary. The mean for the acidophilic part content is 77 mU., that of the basophilie part is 407 mU. (ratio 1:5) ... 22 -

4. Concentrations of ACTH in rat, bee! and human posterior pituitaries.

The ACTH content o:t rat, bee! and human posterior pituitaries are given in Table IX.

The human posterior pituitary was obtained !ive hours post~ortem from a 63 years old male, wi th no known endocrine disorders, who died from broncho-pneumonia :tollowing a transurethral prostatectomy. It is interesting to note that a posterior pituitary gland removed 15 hours post-mortem from a 51 years old female who died of a cerebrovascular accident did not cantain sufficient ACTH to give a valid assay, although the presence of the hormone was indicated by an increase in the for- mation of corticoids in the assay.

TABLE IX

mU. of ACTH per mg 95% confidence Index of Gland of :tresh tissue limite precision Rat 40.7 19-87 0.14 Rat 23.3 10-53 0.16 Rat 26.4 16-44 0.09

Rat 36.2 29-44 0.04

Beef 6.0 3-14 0.17

24.6 15-40 0.10

TABLE IX: ACTH concentrations in rat, beef and hurnan posterior pituitary tissues. -~-

DISCUSSION AND CONCLUSIONS:

1. Concentration of ACT& in the rat anterior pituitary.

The resulta in Table III on the ACTH content of the anterior pituitary of normal male rats are significantly higher than any previously reported (55,56,57). Birmingham and coworkers (68), in this laboratory, have in- Testigated the reasons behind the wide divergences in the values and it has been shown to be due mostly to the extraction methods. Comparative assays on halves of the same pituitaries by the Saffran and Schally in vitro technique yielded such values as 25 mU. ACTH per milligram of fresh tissue when extracted with 0.01 N HCl and 225 mU. ACTH per mil- ligram when extracted with glacial acetic acid. The superiority and efficiency of the glacial acetic method is thus evident; moreover, Birmingham et al. (68) found that the values of the dilute HCl extracts increased with extended storage in the refrigerator. The modified extraction procedure used in this work has cambined the two aethods. The values obtained by this modified method- Table III, mean of 241 mU. ACTH per milligram of fresh tissue- are higher than the ones obtained by the original glacial acetic acid method (68)- mean of 145 mU. ACTH per milligram. The glacial acetic acid-heating treatment probably liberates the hormone from its sites of attachment in the celle; drying the extract under vacuum removes the glacial acetic acid, which interferes with the bioassay, without harming the hormone; and finally, extraction with dilute HCl redissolves the ACTH and also possibly breaks down any further bonds which may have escaped, or which were resistant to, the acetic acid treatment. -24-

The possibility, however, exists that part of the discrepancy between these values and those of Sayers and his school may be due to the different methods of assay. HCl extracts of normal rat anterior pituitaries gave a total of 70 to lOO mU. ACTH by the adrenal ascorbic acid depletion method (55,56) and 300 mU. ACTH by the in vitro procedure (57), while purified preparations, assayed by both methods, gave almoat identical potencies (66,69). It may be that the native ACTH has more corticosteroidogenic activity than ascorbic acid -depleting activity. Moreover, the in vitro technique is based on the known property of ACTH to stimulate the adrenal production of corticosteroids, whereas the basis of the Sayers assay, the depletion of ascorbic acid in the , has not yet been related to the formation of corticosteroids. other arguments, outside of its comparative simplicity, in favour of the in vitro bioassay for ACTH, such as its specificity, precision, reproducibility and small percentage of error, have been discussed by the

&uthors, Saffran and Schally (66). At the very least, it may therefore be said that the in vitro bioassay is the equal of the official adrenal ascorbic acid depletion method, and is to be favoured for its saving of time and expanse. Conclusion: The anterior pituitary of the rat has been shown, by glacial acetic acid-dilute HCl extraction and assay against known standards, to contain more than 1000 mU. of ACTH. This high value necessitates the re-examination of all works in wbich the ACTH content of the anterior pituitary was measured by other methods after various stressful procedures (35,37,38,52,56,70).

2. Studies on the distribution of ACTH in the rat anterior pituitary. - 25 -

The even distribution of the ACTH in the three planes of the rat anterior pituitary, as evidenced by Tables IV, V and VI, was to

be expected from theoretical and practical considerations. On the one hand, the cellular distribution in the rat anterior pituitary is very ill-defined and no large discrete areas of acidophile or basophils exist (65,16). On the other hand, the small aize of the gland and the thickness of sectioning (200 micra) rendered doubtful the sufficiently discrete separation of zones in the gland. Although it was seen that the solution to the problem of cellular elaboration and storage of ACTH could not be gained from these studies, it remained nonetheless important to complete them. The rat anterior pituitary is used extensively in experimental studies and how much and just where the adrenocorticotropic hormone was present had not been previously determined with certainty. Moreover, the development of the methods was important; and their successful application to the quantities of ACTH in normal rat anterior pituitaries has already been discussed.

Conclusions: The rat anterior pituitary, sectioned and assayed in its three planes, sagittal, horizontal and corona!, presents a uniform distribution of ACTH, within the statistical limits of precision of the experimental procedure.

3. Studies on the distribution of ACTH in beef and hog anterior pituitaries. The extraction method, developed in the work on the rat anterior pituitary, was then applied to separated parts of beef and hog anterior pituitaries. The results are summarized in Tables VII and VIII. These glands were chosen for several reasons: their availability, their wide-spread use, their size and their histological structure. Ref- erence has been made to the cellular zones in these glands: the antero- " 26 - median portion is very rich in basophile and the posterolateral wings are very rich in acidophils (4,29,33,50,65). However, it must be noted that these zones are not pure acidophile or basophile: they both contain chromophobes and a variable contamination of the other stainable cells (65). Nevertheless, it could be argued that if one of these zones contained a much higher amount of ACTH, then presumably the ho~one was stored in the prevalent cell type of that zone, As may be seen in Table VII, the basophilie areas in every instance possessed much more ACTH than the acidophilic areas.

This method of pituitary zone extraction and assay is not novel. Smith (4) first used it to attribute to the acidophile the elaboration of somatotropin. Smelser (29) used it with sucess to allocate thyro- tropin and the gonadotropins to the basophils, and somatotropin to the acidophile. He also, by the same method, assigned the elaboration of

ACTH to the basophils and noted a ratio of 6:1 and 8:1 between the ACTH activities of the basophilie areas and the acidophilic areas of beef pituitaries. The results in Table VI were obtained with methods much more specifie and accurate than Smelser's. Yet it must be noted that the ratio between the mean ACTH content of the basophilie areas and that of the acidophilic areas is 7:1, a figure identical to that of Smelser. Similar resulta were obtained with hog glands, as may be seen in Table VIII, although the ratio is lower, about 5:1. However, these resulta must not be interpreted as mere corro- bation of previous data. The demonstrated specificity and accuracy of the extraction and assay methode are in themselves strong arguments to allocate to the basophile at least the storage of ACTH. No other ex- planation is possible. It is true that the resulta do not pinpoint the - 27 -

secretion of the hormone to either the beta or delta basophile. This problem cannot be solved by such means alone.

On the other hand, histological and/or cytological procedures by themselves will also not solve the problem. Any type of treatment of the animal, designed specifically to alter the ACTH secreting cell, (either by obliging the pituitary to release rapidly and constantly its ACTH, thereby stimulating new hormone secretion, or by preventing the release or secretion of new hormone), affects both the acidophile and basophile. This renders evaluation of cytological changes very ambi- guous (32,34,38). The reasons behind this apparent interdependance remain unexplained. Halmi recognized this pitfall and strove to bypass it by combining the histological and cytological data with direct ACTH measurements on the pituitaries of the experimental animale. However, Halmi was not successful in establishing the cellular origin of ACTH. Firstly, he did not do cytology and ACTH assay on the same gland. Secondly, the HCl extraction method he used was inefficient, as has been shown in this thesis. Thirdly, he allowed a thirty day lag between the experimental thyroidectamy or castration and the assays for ACTH content, during which time the pituitary stores returned to very near normal. In fact, Sydnor and Sayers (56) and Gemzell ~ !1,. (71) have shown that within one week after adrenalecto~, the pituitary ACTH levels return to normal. It is reasonable to assume that one month after thyroidecto~ or castration, which are lees stressful than adrenalectomy, the pituitary ACTH stores should have returned to normal. Tuchmann-Duplessis (38,39) independently employed the same principles and more or less the sam.e experimental procedures as Halmi. Although " 28 -

Tuchmann-Duplesis measured the ACTH levels in the pituitaries only nine to ten days after adrenalect~ and administration of corticoids, thyroxine and ACTH, her work is gravely weakened by the unspeoific extraction method and relatively inaocurate method of assay employed. Herlant also recognized the weaknesses of cytological and histological means. To bypass them, he utilized differentia! cent- rifugation of sheep, beef and hog pituitary homogenates to separate the cellular fraction containing the ACTH. Assayed by the eosinopenia, thymus weight decrease and adrenal weight increase methode in hypophy- sectomized rats, Herlant (43,44,45) found that the granular fraction obtained from centrifugation at a acceleration of 3000 X g for one hour, contained most of the ACTH. Herlant claimed that these granules were mostly acidophilic, as shown by their staining reactions. However, Hess and Brown (53) repeated the work of Herlant and assayed the ACTH activity of all the isolated fractions, whose purity was determined with various stains and under the electron microscope. ~ the thymus involution assay, the mitochondria, the basophilie granules and the supernatant showed at least twice the ACTH activity of the acidophilic granules. Assayed by the adrenal ascorbic acid depletion technique, the mitochond- rial fraction was the most potent and the acidophilic granular fraction the least potent in ACTH activity. The inherent difficulty in the interpretation of the resulte of homogenization and differentia! centrifugation is the losa of cellular integrity and the consequent release and mixture which are of the intracellu- lar elements which are then .eseparated only on the basis of their physical properties, such as size, shape and density. These properties may easily be altered by the mechanical treatment and the separated fractions may - 29 - not necessarily correspond to the original possessed by these elements in the intact cells. For example, Catchpole (74) has suggested that the basophilie granules are aggregates of submicroscopic particles and that these aggregates are broken by homogenizing. This ia1 in a sense, sup- ported by the spread by corticotropic activity to all the fractions after the application of these methods (53). In summary, it appears that any method designed to solve the problem of cellular elaboration of ACTH must meet the following re- quirements: (a) the cells of the glands must be kept intact; (b) the histology and cytology of the glands must be carefully studied; (c) the ACTH content of the glands and the separate cellular zones must be determined; (d) the histology and the cytology of the gland must be closely correlated to the ACTH content of the same gland, extracted and assayed by proven specifie and accurate methods. Further work, based on the findings reported in this thesis and incorporating the above principles, is planned on this problem.

~perimentation will bear on two points: first, the ACTH content of separated cellular parts of hog anterior pituitary will be correlated to their cytological picture; second, the variations in the ACTH content of rat pituitaries will be connected to the alterations in the cytology of the same glands, as produced by a major stress, such as adrenalecto~. Conclusions: The ACTH content of separated acidophilic and basophilie cellular zones of fresh beef and hog anterior pituitaries ha3 been determined. In every instance, the basophilie area ACTH content was greater than the acidophilic area content, the mean ratios being 7:1 in beef glands and 5:1 in hog glands. These results are in line with the theory of basophilie cell elaboration of ACTH. - 30-

4. ACTH in rat, bee! and human posterior pituitaries.

The results on the ACTH content of rat, beef and human posterior lobes, summarized in Table IX, confirm the claims of Mialhe-Voloss (60,63,64) who first conclusively demonstrated the presence of the hormone

in p~sterior lobes. The implications of these findings must not be minimized. Mialhe- Voloss (72,73) has demonstrated that the ACTH in the posterior lobe of

rats plays a definite role in the response of the animal to 11 neurotropic11 stresses, such as noise and light. Subjecting the animal to loud noises decreases the ACTH content of the posterior lobe but does not alter that of the anterior lobe. Systemic stresses, on the other hand, such as a histamine injection, deplete the ACTH stores of the anterior lobe but do not affect that of the posterior lobe. However, Mialhe- Voloss extracted acetone powders of the posterior lobes with isotonie saline, which probably does not extract quantitatively the ACTH. Again, the presence of ACTH in the posterior pituitary raises the very important question: is the hormone secreted in the posterior lobe or is it but stored there? If secreted, then the elaboration must necessarily belong to the , since they are the only cells there. The posterior lobe is, however, considered to be a site of hormonal storage and secretion, not a site of hormonal elaboration. If stored, where is the ACTH for.œed and how does it reach the

posterior lobe? It cannot be due to post~ortem diffusion from the anterior lobe, since (in the case of the rat, at least) the time lag between death and separation of the lobes from one another is less than

a minute. If it is due to an in vitro diffusion from the anterior lobe, then we should expect to find present all or some of the other tropic - 31 - hormones, such as the gonadotropine. This has never been shown (63). Mialhe-Voloss (63,64) has also tested the for ACTH activity and did not find any.

Conclusions: The presence of ACTH in the posterior lobe of rat and beef pituitaries has been confirmed. The presence of ACTH in the human posterior pituitary has also been demonstrated. ... 32 -

SUMMARY

1. The anterior pituitary of the rat hae been shown, by glacial acetic-dilute HCl extraction and by bioassay against known standards, to contain more than 1000 mU. of ACTH.

2. The rat anterior pituitary, sectioned and assayed in its three planes, sagittal, horizontal and coronal, presented a uniform distribution of ACTH, within the statistical limita of precision of the assay.

3. The ACTH content of separated acidophilic and basophilie cellular zones of fresh beef and hog anterior pituitaries has been deter- mined. In every instance, the basophilie area ACTH content was greater than the acidophilic area content, the mean ratios being 7:1 in beef glands and 5:1 in hog glands.

4. The presence of ACTH in the posterior lobe of rat and beef pituitaries has been confirmed. The presence of ACTH in the human posterior has al so been demonstrated. - 33 .. BIBLIOGRAPHY

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!PPENDIX

Histology of separated acidopbilic and basophilie cellular zones of bee! anterior pituitaries.

It bas been claimed that bee! and other mammalian pituitaries contain zones of cellular concentration: an antero-median area, very rich in basophile and the postero-lateral wings, very prominent in acidophils. This was confirmed, by histology, in the bee! pituitary. A bee! gland was thawed and dissected in the same manner as for assay (page 12). The separated cellular parts were then fixed in Bouin's fluid for 24 hours, processed, sectioned at 5 micra and stained by the Masson Trichrome method. With this procedure, acido- pbils stain red, basophile purple. Colour photographs (taken by Mr. Charles Hodges, Montreal Neurological Institute) of fields in the slide obtained of the acidophilic and the basophilie zone, are given in Figures lA and lB respectively. The overall preponderance of the red acidophils in Figure lA and of the purplish basophile in Figure lB confirma the literature and gives greater significance to the values presented in Tables VII and VIII.

I wish to express my most sincere thanks to Mr. Gilles Boyer, Bsc. (M.T.), head of the Histology Laboratory at St. Jean de Dieu Hospital (Montreal) tor the preparation ot the slides on the bee! pituitary. ii

FIGURE lA

FIGURE lA: Photomicrograph of a field in the beef anterior pituitary acidophilic area. (Section 5 micra thick, stained with Masson Trichrome. Magnification: X 450.) iii

FIGURE lB

FIGURE lB: Photomicrograph of a field in the beef anterior pituitary basophilie area. An island of acidophile may be recognized by their intense red stain. (Section 5 micra thick, stained

wi th Masson Trichrome. Ma.gnification: X 450.)