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Effect of adrenocorticotropic hormone (ACTH) on the anterior pituitary of the adrenalectomized young male rat.

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E F F E C T OF ADRENOCORTICOTROPIC HORMONE (ACTH)
ON THE ANTERIOR PITUITARY O F T H E ADRENALECTOMIZED YOUNG MALE RAT
A L E X E I A. K O N E F F
Division of Anatomy, Medical School, and Institute of Experimental Biology,
Owivemity of California, Berkeley
ONE PLATE (ELEVEN FIGURES)
I n a previous communication (Koneff, '44) it was reported that
injection of adrenocorticotropic hormone into normal young male rats
caused diminution in weight of the hypophysis, definite decrease in
size of basophils accompanied by loss of granular material, and changes
in the nucleus and cell organelles. These alterations were interpreted
as indicative of retarded or depressed functional activity of the basophils. No significant changes were observed in the acidophils.
Recent biological studies conducted here have indicated that the
effect of ACTH in adrenalectomized rats is different from that in
normal males.2 Namely, it does not retard the body weight increase
in the absence of the adrenals, causes no decrease in thymus or thyroid
weight (Simpson, Li, Reinhardt and Evans, '43) and does not alter
histologically the testis in the adrenalectomized animal. It was of
interest, therefore, to see whether the morphological appearance of
the anterior pituitary would also differ in normal and adrenalectomized
ACTH-treated rats.
N A T E R I A L A N D METHODS
As in the previous investigation of the effect of ACTH on the anterior hypophysis, young male rats were used. The rats were adrenalectomized at 26 days of age and injections of pure ACTH (Li, Evans and
Simpson, '43) were begun immediately. One milligram doses (divided
into three injections) were given daily intraperitoneally for 30 days.
Aided by grants from the Rockefeller Foundation, the Josiah Macy, J r . Foundation and
General Mills, Inc. I wish to express my thanks to Prof. Herbert M. Evans and Dr. Miriam E.
Simpson for their assistance and f o r tho use of the facilities of the Institute of Experimental
Biology.
"he
report from the Institute concerning the biological effects of the ACTH in young
adrenalectomized rats will be published separately.
163
164
ALEXEI A. K O N E F F
All adrenalectomized rats were supported by 1%sodium chloride in
their drinking water. Only animals in which adrenalectomy was complete were included in this analysis (completeness determined by careful search of site of operation at autopsy and section of any suspicious
fragments of tissue).
Three groups of male rats were studied:
Group 1consisted of nine rats which were adrenalectomized, treated
with ACTH and sacrificed on the fifty-sixth day of life.
Group 2 consisted of ten rats which were adrenalectomized, untreated
and sacrificed on the fifty-sixth day of life.
Group 3 consisted of ten normal rats of 56 days of age. The pituitaries of these animals had also been used in the previous investigation
as controls for normal males injected with ACTH (Koneff, '44).
HISTOLOGICAL AND CYTOLOGICAL METHODS
For general histological study, pituitaries from four rats in group 1
and 2, and six in group 3, were fixed in Zenker-formol and after celloidin
embedding, sectioned at 4 micra. The modified Mallory-Azan technique
was used as a staining method (Koneff, '38).
Five pituitaries from rats in group 1,six glands from rats in group 2
and four in group 3 were treated according to the Altmann-Masson
method and the Nassonov-Kolatschev technique (as adapted by Severinghaus, '32) for the purpose of demonstrating mitochondria and the
Golgi apparatus respectively.
RESULTS
ABterior pituitary of adrelzalectomized coantrol rats
Both acidophils and basophils in the pituitaries of adrenalectomized
rats showed changes when contrasted with the respective types of
chromophil cells in the normal gland. Even preliminary examination
with moderate magnification revealed changes in the acidophils. Although no cell counts were made there was an obvious decrease in the
number. Their size was significantly diminished (see table, M, vs. M2).
The granular material in a large proportion of these cells was markedly
reduced. It should be noted in connection with the changes in size and
the degranulation of acidophils that many cells, ordinarily called
chromophobes, were present which could be identified as acidophilic
chromophobes on the basis of the Golgi apparatus and other morphological characteristics. The basophils in the pituitaries of adrenalectomized rats did not show such pronounced alterations as those noted
165
PITUITARY, ADRENOCORTICOTROPIC HORMONE
in the acidophils. The majority of the basophils closely approximated
the normal in structural characteristics. There were no changes in
Golgi apparatus and mitochondria which would indicate changed f unctional activity. It should be emphasized however, that the average
size of the basophils was significantly reduced (see table, M, vs. h&).
TABLE 1
Effect of ACTH on the size of acidophils and basophils of adrenalectomized rats.
~
OELG SIZFi I N P U N I M E T R I C U N I T S 1
EXPERIMENTAL
CONDITIONS
GROUP
NO.
Basophils
Acidophils
Mean
1
~
2
-
(M,) 309f102
-
Adrenaleetomized
on 26th day of
life, supported
with NaCl,
untreated ;
age 56 days
(M,) 1 5 6 2 7 M, vs. M,=0.50
4
6
Normal; age,
56 days
3
i
(Ml) 16128
Adrenalectomized
on 26th day of
life, supported
with NaCl and
treated with
ACTH; age.
56 days
M, VS. M4=2.38
(M,) 278+8
~
(M,)195f5
M, vs. MM,=7.45
M, VS. M,=4.33
-
Area occupied by camera lucida tracings of 100 cells expressed as
' S E (of mean) = S D
i
n
1
S R ; Significance ratio was calculated by the formula:
Mean,
4
- Mean,
+
(S E Mean,)*
(S E Mean,)'.
The difference of the means waa considered statistically sigiiificant if the SR was greater
than 3.0.
I n a small number of basophils, degenerative changes were noted. I n
some of these cells the stainable granular material was not uniformly
distributed throughout the cytoplasm as in normal cells, but was confined to the periphery of the cell body and occurred in coarse, darkly
stained clumps. The perinuclear area in this case was light and somewhat reticular. A few basophils showed vacuolation. I n this case, the
peripheral portion of the cytoplasm was first affected; in more advanced
stages, the vacuolation occupied a considerable portion of the cell
body. It should be noted however, that a few basophils were present,
166
ALEXEI A. KONEFF
which on casual observation, appeared normal, but which possessed
a Golgi apparatus which was moderately hypertrophied and of increased complexity.
The changes just described for the pituitaries of rats adrenalectomized at 26 days of age are essentially the same as those previously
reported from this laboratory (Koneff, Holmes and Reese, '41) in the
glands of males, adrenalectomized at 60 days of age and supported by
sodium chloride for a period of 45 days.
Anterior @tuitary of adrenalectomixed rats treated with ACTH
I n the pituitaries of the injected group, as in the glands of adrenalectomized controls, both chromophil cells were altered from normal.
I n acidophils, the morphological changes including the finer cytological
characteristics, were the same, qualitatively and quantitatively as those
described in acidophils of the adrenalectomized controls, the changes
being essentially a matter of size decrease and degranulation. The
acidophils of the injected rats were slightly smaller even in operated
controls, but this was not significant statistically (see table, M, vs. MI).
On the other hand, the basophils in the pituitaries of the adrenalectomized ACTH treated rats differed in several respects from those
in the glands of the adrenalectomized control animals. Firstly, the
young basophils were increased in number. Many small round cells
were present, possessing a dark, basophilic cytoplasm, large, welldefined nuclei and a basophilic type of Golgi apparatus, which should
unquestionably be considered to represent a crop of young basophils.
The young basophils can usually be observed in normal pituitaries,
as well as in those of adrenalectomized controls, but are less frequent
than in this group; here the percentage of these cells called young
basophils was sometimes as high as 15%.
Careful examination of the pituitaries showed a second variety, constituting 5 t o 7% of all basophils. These cells, already noted in glands
of adrenalectomized controls showed the disappearance of the stainable
granular material in the perinuclear area and the clumping of the
basophilic material at the periphery of the cell. This type of alteration
was far more pronounced in injected than in control adrenalectomixed
rats (fig. 8) and was noted in a larger percentage of cells. I n some of
the basophils of this category, various degrees of nuclear degeneration
and alteration in cell organelles was also present, such as clumping
of mitochondria.
The largest group of basophils, in some glands reaching 35% of all
basophils, showed another type of degeneratire change, involving
PITUITARY, ADRENOCORTICOTROPIC HORMONE
167
vacuolation of the cytoplasm. In so far as it was possible to reconstruct
the succession of events, the alteration probably began with the appearance of a few small vacuoles in the peripheral portion of the cytoplasm,
and proceeded unevenly toward the nucleus. Some of the vacuoles
coalesced, thus forming larger vacuoles filled with homogeneous material, dark or light blue in staining reaction. In many instances, a
secondary vacuole, colorless and sharply outlined, appeared in this
homogeneous material, giving the impression that some substance
previously occupying this vacuole was dissolved in the process of fixation or dehydration. The nucleus and Golgi apparatus were crowded
into the center or pushed to the periphery of the cell body by the
accumulation of vacuoles and both were distorted in shape (figs. 9 and
10). I n the final stages the nucleus was irregular in outline, compact
and deep staining and the cytoplasm was obviously disintegrating
(fig. 11). It will be remembered that only a few of the basophils of
adrenalectomized controls were vacuolated. The number of vacuolated
cells and the extent to which vacuolation has progressed in the basophils of the injected group, explains the slight, average, size increase
of basophils in this group (see table, M, vs. M4).
I n some apparently normal basophils or in those revealing only the
initial stages of vacuolation, there was evidence for a considerable
degree of heightened activity. This was manifested by one or more
of the following characteristics, increased cell size, various degrees of
granular depletion, prominent mitochondria or hypertrophied Golgi
apparatus (figs. 6 and 10). It appeared as though these basophils were
making an effort to compensate for the function lost by the degenerating cells.
The chromophobes in the pituitaries of adrenalectomized ACTH
treated rats were numerically increased and, as in adrenalectomized
controls, this was due t o degranulation of acidophils as far as could be
determined from general as well as specific staining methods.
DISCUSSION
I n an attempt to evaluate the above mentioned findings in the glands
of adrenalectomized ACTH-treated rats, one must consider whether
the changes are directly related to the adrenalectomy. I f so, are they
related to the general physiological condition of the adrenalectomized
animal, or are they secondary to alteration of other endocrine organs,
and finally are the effects due in part or entirely to action of the hormone on the parenchyma of the pituitary?
I68
ALEXEI A. KONEFF
It seems improbable that the effect of the ACTH on the anterior
pituitary of adrenalectomized rats was mediated through the testes
or thyroids inasmuch as no deviation from normal was found in these
organs. I n fact, it is strange to have such marked alterations in the
pituitary not reflected in changes in thyroid or testis but it is possible
that regardless of the degeneration of some basophils, sufficient numbers of these cells remain to assure adequate support of these other
endocrines.
The body weight change of adrenalectomized ACTH-treated rats and
their controls was approximately the same when maintained on salt
in the drinking water.
It has been previously reported from this laboratory (Koneff, Holmes
and Reese, '41) that the pituitary changes resulting from the adrenalectomy of 60-day-old male could be prevented t o a large degree by the
sodium chloride therapy. It should be emphasized, however, that sodium
chloride did not maintain the anterior pituitary in its completely normal
state. Both chromophil cells were never quite as large in the glands of
animals maintained on salt as in those of normal control rats of the same
age. The acidophils showed greater than normal d e g r a d a t i o n and
reversion to the chromophobe state, while in a few basophils the initial
stages of degeneration or signs of heightened functional activity were
observed. The same deviation from the normal morphology of the
anterior pituitary was also detected in the adrenalectomized controls
as well as in the ACTH-treated animals of the current series. Therefore, only the severer degenerative changes involving the cytoplasm,
the nucleus, and the cell organelles characterizing adrenalectomized
ACTH-injected rats are to be considered as effects of the hormonal
injections. In other words, one can readily see that chronic injection
of ACTH into young adrenalectomized male rats does not modify the
characteristic nature of the pituitary acidophils of this animal. One
can also see that, contrary t o this lack of response on the part of the
acidophils, the basophils in the ACTH-treated rat's pituitary showed
constant and conspicuous degenerative changes.
The experiments with ACTH-injected, normal male rats (Koneff,
'44) showed that the prolonged injections do not alter appreciably the
acidophils but cause a significant size decrease of the basophils, marked
diminution in granular content of the cytoplasm, reduction in size of
the Golgi apparatus and number of mitochondria in these cells. These
alterations were interpreted as being indicative of a depressed o r
retarded functional activity of the basophils. There was no evidence
that this effect of ACTH on the pituitary cells was mediated through
PITUITARY, ADRENOCORTICOTROPIC HORMONE
169
testes or thyroid and the possibility was considered that detectable
changes were due to a direct effect of the hormonal injections on the
anterior pituitary.
I n the present study of the effect of ACTH injections on the pituitaries of adrenalectomized rats, there also was no significant effect on
the acidophils while the other type of chromophil cells -the basophils
- showed considerable alteration. Here, in adrenalectomized rats
again, there was no evidence of the mediation of the effect of hormonal
injections through testes or thyroids, and, moreover, in the present experiment, the possible influence of the highly stimulated adrenal cortex
on the pituitary was eliminated. Therefore, the present study seems t o
corroborate the previously expressed suggestion that the effect of the
ACTH might be considered as a direct one on the parenchyma of the
pituitary. One might even postulate that the basophils are participating
in the production of the ACTH inasmuch as they are the only chromophi1 cells of the pituitary that manifested appreciable changes following the hormonal injections. This assumption is in accord with the
recent findings of Smelser ( '44), who demonstrated greater concentration of adrenotropic hormone in extracts of tissue taken from the
eentral basophil-rich zone of the anterior pituitary of a beef, than in
the peripheral acidophil-rich portion of the gland.
CONCLUSIONS
1. Changes were observed in both types of chromophil cells of the
pituitary of adrenalectomized male rats, maintained on sodium chloride
in drinking water and injected with ACTH.
2. I n the acidophils, these changes (decrease in number, diminished
size and degranulation) were the same as those found in the glands of
adrenalectomized, uninjected control rats and therefore could not be
regarded as a result of the ACTH treatment.
3. In the basophils, the outstanding change observed in a great number of cells was a degenerative change involving the cytoplasm, the
nucleus and the cell organelles. The second feature was the presence
of a crop of young basophils. Inasmuch as such changes were seen
rarely in the basophils of the adrenalectomized control rats, they are
considered as the direct effect of ACTH injections. This conclusion
seems justifiable in so far as there was no indication that the effect
of ACTH was mediated through the other endocrine glands studiedthe testes or thyroids.
170
ALEXEI A. KONEFF
LITERATURE CITED
KONEFF,ALEXEI A. 1938 Adaptation of the Mallory-Azan staining method to the anterior
pituitary of the rat. Stain Technol., vol. 13, p. 49.
1944 Effect of adrenocorticotropic hormone on the anterior pituitary of the
normal young male rat. Endocrinology, vol. 34, p. 77.
KOWEFP,ALDXI A., R. 0. HOLMESAND J. D. REESE 1941 Prevention of adrenalectomy
changes i n the anterior pituitary of the r at by sodium chloride administration.
Anat. Rec., vol. 79, p. 275.
LI, CROH HAO,H. M. EVANSAND M. E. SIMPSON3943 Adrenocorticotropic hormone. J.
Biol. Chem., vol. 149, p. 413.
SEVERINGHAUS,
AIJRA E. 1932 A cytological technique for the study of the anterior lobe
of the hypophysia. Anat. Rec., vol. 53, p. 1.
SIMPSON,
MIRIAM E., C: H. LI, W. 0. RFJNHARDT A N D H. M. EVANS 1943 Similarity of
response of thymus and lymph nodes to administration of adrenocorticotropic hormone in the rat. Proc. SOC.Exp. Biol. and Med., vol. 54, p. 135.
SMELSER,
GEORGEK. 1944 Differential concentration of hormones in the .central and peripheral zones of the bovine anterior pituitary gland. Endocrinology, vol. 34, p. 39.
PL.4'TE
I
E X P L A S B T I O N OF FIGI'RES
Photographs of f i g u r e s 1, 0 and 3 ivere taken with 4 mui. objective; magiiificatioii X 400.
Mallory-Szan stain.
Photographs of figures 4 to 11 7wre taken with ?-inin. obdective; magnification X COO.
4 and 6 Nassoiio~-k'olatsclierpreparations; 5 , 7 to 11 Mallory-Azaii stain. A key t o figures
4 to 11, slioning position of nucleus (N),Colgi apparatus ( C ) and vacuoles ( V ) is given
cpposite plate 1..
1 Field selected froin a section of the anterior pituitary of a normal male rat. Note general
size and distrihutioii of acidophils. h'otp iioimal granulation of these cells.
2 Field selected from a section of the anterior pituitary of a n adrenalectomized rat s u p
ported hy lye sodium cliloridr in drinking water. Note changes in size and granulation of
the acidophils.
3 Field selected froin a section of the anterior pituitary of a n adrenalectomized-ACTH
trea.ted rat. Note general simi1arit.p of the acidophils in figures 2 and 3. Note presence of
vacuolated basophils.
4 and 5 Basophils of a medium size f r o m pituitary of normal rat. Note the size and shape
of the Golgi apparatus.
6 Basopliil from pituitary of adrenalectomized rat treated with ACTH. S o t e change in
size and shape of the Golgi apparatus; compare with figures 4 and 5 .
7 Basophil of a very small size. This variety of basophil cells was found in the pituitaries
of adrenalectomizrd ACTH-treated rats.
8 Basophil showing clumping and peripheral concentration of the basophilic material in
the cytoplasm.
9, 10 and 11 Basopliils froin the pituitaries of adrenal~ctouiizedACTH-treated rats. Note
a significant increase in cell size and raruolatiou of the cytoplasm. Note pyknotie condition
of the nucleus i n figure 11.
PLATE 1
PITUITARP, ADRENOCOHTICOTKOPIC HORJIOSE
AljFXhI A
IIONBFF
............. 7 r ............
.. ..
1
: *
G .......
..... .G
N
........ N
.! ..
2.........
............J
I2...........
....".....,
-j
.:
G ....... ....@!\
N ........i. .
....
/
,,,
@... ......
;
..
.i. :..
'7
L............
-I L
...........J
................................
1
......-.
'6
A
..
N ........
I
G .....
.....v
.......N
G ......
................................
................................... 1
...... .........v
r..
...........................
1
.... i.. .......
....v
.. -:....
:*
i' . .
N... ..!......
! II
L
................................
172
t
i
-.I
PLATE 1
173
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