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Response of the pelican adrenal to various stimuli.

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RESPONSE O F THE PELICAN ADRENAL
TO VARIOUS STIMULI1
F. A. HARTMAN, R. A. KNOUFF AKD G . A. HOWARD
Departments of An.atomy and Physiology, The Ohio State University, Columbrrn
NINE FIQUEES
INTRODUCTION
Since it has been shown that the adrenal of the Brown
Pelican (Pelecarczcs occidental&) is especially suitable for
histological and cytological study (Knouff and Hartman, '51)
we have attempted t o determine the effects of various endocrine preparations and other stimuli on the adrenal of this
species.
All experiments were conducted with young birds since
the collection of a considerable series of live adult birds was
impracticable. The effects of adrenocorticotrophic hormone
(ACTH), cortisone, testosterone, desoxycorticosterone acetate
(DCA), stilbestrol and formaldehyde injections as well as
inanition and exposure to cold, were studied.
MATERIALS AND METHODS
Seventy-eight birds were employed in the study, three being adult and the remainder being young taken from the nest.
The age of the birds was unknown except that the young
birds were but a few weeks old when taken. After keeping
the young birds for two or three weeks they ranged in weight
from 1100 to 3600gm, half of them being between 2000 and
2700 gm.
IAided by a grant from the Comly Fund of the Ohio State University.
We are very much indebted to Mr. Richard Archbold of the Archbold Biological
Station, Florida, for his personal assistance in the collection of material and for
the use of the facilities of the Archbold Biological Station.
469
470
F. A. HARTMAN, R. A. KNOUFF AND G. A. HOWARD
All birds were kept in roofed out-door cages and fed fresh
fish twice daily. All injections were subcutaneous. At the
conclusion of each experiment the bird was killed by decapitation and the adrenals were removed immediately and pieces
fixed in 10% formalin or Helly’s fluid and in some instances
Aoyama’s fixative. After washing the Helly ’s material, one
piece was treated with 2% osmium tetroxide for 6 to 12 hours
while another piece was mordanted for a few days with 3%
potassium vichromate.
The Schultz reaction was graded 1, 2 and 3 relative to
normal designated as 3.
OBSERVATIONS
The pelican adrenal possesses a greater proportion of interrenal tissue than is present in any other bird which we
have examined. Likewise the cells of this tissue are arranged
in a manner that suggests the three zones of the mammalian
cortex. No other bird shows this so clearly. The interrenal
cells are organized into cords consisting of a single row of
tall columnar cells radially arranged with their broader ends
forming the surface facing the blood channels and their opposite, narrower, nucleated ends converging to form the solid
central cores of the cords. This cellular pattern bestows upon
the interrenal cells a polarity not found in the adrenal of any
species with which we are familiar. The adrenal of the young
pelican does not show the zonation quite so well as does the
adult bird. Otherwise it seems to differ little from it.
ACTH
We assumed that ACTH injection would be one of the best
means of stimulating activity in the adrenal. This has been
confirmed by our observations. Changes after different intervals following single injections and after daily injections
over periods up to 1 2 days have been noted.
L i p i d changes. The unstimulated gland contained osmiophilic lipid in all zones. After injection of ACTH some of
We are indebted t o Dr. E. B. Hays of the Armour Laboratories f o r the
ACTH.
PELICAN ADRENAL RESPONSE TO STIMULI
471
the lipid of the glomerulosa failed to be stained by osmium
tetroxide. This lipid was sudanophilic and Schultz positive.
We have called it non-osmiophilic lipid. There was no evidence of it in the glomerulosa of the normal gland nor in
-
CONTROL
-
A C TH
5mg.,2hr.before
A C T H
I
I
5mg.,5 hr.bcfore
A C T H
5 mg,,16 hr,before
AC TH
l O m g daily for10 d a y s
fired 6hr.afterlast ini.
-
COLD
5 hT.at5OC.
f i x e d immediolely
INANITION
4doys
m B
I
Fig. 1 Chart showing responses of the different adrenal zones to the birds’
esposure to cold, t.o inanition and to ACTH inject.ions.
472
F . A. HARTMAN, R. A. K N O U F P A S D G . A. HOWARD
other zones except the outer fasciculata at any time. Several
hours after the injection of ACTH there m-as general loss of
lipid throughout the gland.
Three birds were given a single injection (5mg) of ACTH.
The glands from the first were fixed two hours later, those
from the second 5 hours later and those from the third 16
hours later. All showed significant changes in the glomerulosa (fig. 1).
TWOhours after injection, osmiophilic lipid had almost
disappeared from the glomerulosa except in those regions
where blood vessels, ganglia or gonadal tissue were present
on the outer surface of the capsule. Here it was merely reduced somewhat. Most of the cells which did not show 0smiophilic lipid contained droplets of sudanophilic lipid. The
latter type of lipid did not completely replace the osmiophilic
lipid because the total lipid content as shown by Sudan orange
was less than normal.
A transitional zone in the inner glomerulosa or outer
fasciculata was deficient in both types of lipid and appeared
to be a locus of intimate association between lipid and mitochondria (fig. 3 ) . I n the glomerulosa the cells contained
large numbers of droplets of uniform size which stained
with Sudan and gave the Schultz reaction but did not reduce
osmium tetroxide. I n the interstices between these droplets
and in close contact with them were mitochondria varying
from granular to rod-like forms. Passing inward from this
region the droplets became smaller while the mitochondria
became larger and began to blacken with osmium tetroxide.
At the corresponding level in the mitochondria1 stained
preparations vacuoles occurred within what appeared to he
mitochondria. More deeply in the fasciculata these structures became larger and deeply osmiophilic. Still further
inward they were even larger with mitochondria forming a
thin rim around the outside. Among these cells were others
containing larger osmiophilic droplets with granular mitochondria between them. The picture suggested that nonosmiophilic lipid x7as absorbed o r incorporated into the sub-
PELICAN ADRENAL RESPOh’SE TO STIMULI
473
stance of the mitochondria and there transformed into osmiophilic lipid.
The above pattern occurred quite consistently in the ACTH
injected birds. These observations were further confirmed
by the fact that Sudan preparations and aniline-fuchsinmethyl green mitochondria1 stains showed the lipid droplets
to be small and the mitochondria enlarged in the areas described above. The intensity of the Schultz reaction paralleled
the intensity of the Sudan orange stain. The Schultz positive
droplets were more numerous in the fasciculata and were
concentrated at the periphery of the cells in the glomerulosa.
The relative estimates of the material in the different zones
are shown in figure 1.
The bird ( 82200gm) killed 5 hours after injection with
ACTH showed a higher than normal lipid content over the
entire gland, and the highest lipid content of any ACTH stimulated gland. The zone of osmiophilic loss was narrower, but
the cells were more heavily filled with non-osmiophilic lipid
droplets than were the cells of the glomerulosa of the “twohour’’ bird. The area of heaviest lipid deposit was in the
inner glomerulosa and outer fasciculata.
The Schultz reaction was spotty. Some cells showed vacuoles which were Schultz negative near the secretory pole.
Relative intensities are shown in figure 1.
Glands from the bird ( 0 1900 grn) killed 16 hours aftcr
the single injection, showed the lowest osmiophilic content
of all ACTH glands. The glomerulosa was virtually free of
osmiophilic lipid even under gonadal tissue, blood vessels
and nerve ganglia. This is the only ACTH gland in which this
was so. The glomerulosa, however, had the highest lipid
content of the gland due to the non-osmiophilic lipid present.
The total lipid content of this zone as determined by the
Sudan stain was considerably higher than normal. The fasciculata, especially its outer part, contained more osmiophilic
and sudanophilic lipid than the reticularis although they
were both markedly reduced from normal. The lipid loss in
the two inner zones was shown in a reduction in the size of
474
F. A. HARTMAX, R. A. ICKOUFF AXD G. A. HOWAKD
the droplets and in an absence of droplets at the distal end
of the cells. The Schultz test for cholesterol showed a normal
reaction in the glomerulosa but little or none in the inner
zones (fig. 1).
The lipid in glands from birds injected daily for two or
more days appeared to return to normal level and then rise
somewhat higher, the increase occurring mainly in the fasciculata and reticularis. Tmo birds ( 81600 and 82150gm)
injected daily for two days with 5 mg ACTH and given 10 mg
of ACTH on the third day, then killed two arid 7 hours after
the last injection, showed an increase in osmiophilic lipid
in the reticularis and fasciculata over the two previously
described birds. The bird killed 7 hours after injection showed
the greater increase, and had an approximately normal lipid
content. These two birds had probably the widest zones of
osniiophilic lipid loss in the glomerulosa and outer fasciculata (fig. 5) although all of the glands from birds “16 hours”
through “4 days” showed a similar loss.
Three birds were given massive doses of ACTH daily. These
were 10 mg for each of the first 10 days, 15 mg on the 11th day
and 20 mg on the 12th day. The adrenals were fixed 6.5 hours
after thc last injection (fig. 1). The zone (glomerulosa) free
of the osmiophilic lipid loss was narrower than in the preceding experiments, which seemed to be caused at least in
part by a n atrophy of the cells of the inner glomerulosa.
Some cells in this area seemed to be shrunken and have a
lessened lipid content while the cells in the reticularis and
fasciculata had a greater than normal lipid content. The
fasciculata as usual had the highest lipid content. The droplets in these cells mere larger and more irregular than normal.
Mifosis. Birds injected with 5 mg o r more of ACTH and killed
within 5 hours after the last injection showed the greatest
mitotic activity in the interrenal cells, the mitotic figures being
f o r the most part in the glomerulosa and outer fasciculata.
The number of these figures found in these glands was considerably above that found in normal glands. Generally
speaking glands f r o m other birds contained a few mitotic
PELICAN ADRENAL RESPORSE TO STIMULI
475
figures, with the exception of the glands from the “12-day”
birds where only rarely could mitosis be found. It was observed that the cells with osmiophilic lipid and those containing non-osmiophilic lipid both undergo mitosis.
Degemeratiow,. Although some degenerative changes were
present in the glands representing earlier stages, they seemed
to become more marked in the glands from animals which
had been injected over a period of three days or longer. The
changes detected were cells with pyknotic nuclei in the outer
reticularis, fasciculata and glomerulosa ; vacuolization of the
interrenal and chromaffin cells ; and the presence of masses
of lymphocytes in the outer areas of the gland. Four of the
8 glands from birds which had received injections 4 days
or longer possessed nerve ganglia showing evidence of injury
o r degeneration, usually masses of lymphocytes and in one
instance cells with pyknotic nuclei, and vacuoles. The glands
from birds which had been injected for 12 days showed considerable congestion in addition to the degenerative changes
mentioned. There was also a suggestion in the “IZday”
glands that the proportion of interrenal material was reduced.
Our observations indicate that ACTH produces changes very
promptly in the glomerulosa of the pelican adrenal t o be followed a little later by an increase then a decrease in total
lipid of the inner zones. All lipid in the inner zones appears
t o be osmiophilic during this cycle while the proportion of
osmiophilic lipid decreases in the glomerulosa. Cholesterol
reduction as measured by the Schultz test is greatest in the
reticularis and with repeated stimulation may involve the
outer zones. Daily stimulation f o r several days may increase
the total lipid in all zones but does not increase the cholesterol
content.
Mitotic activity was increased by ACTH f o r the most part
in the glomerulosa and outer fasciculata but was changed
little in the remainder of the gland.
Daily injections for three days or longer appeared to increase the degenerative changes.
476
F. A. HARTMAN, R. A. KNOUFF AND G . A. HOWARD
COLD
The peripheral stimulation of cold was tried in three young
pelicans weighing 1650 ( 9 ), 2050 ( 8 ) and 2050 ( 8 ) grams.
They were exposed to a temperature of -5'C.
for 5 hours.
The cloaca1 temperatures fell from 4OoC., 405°C. and 41.0"C.
respectively to 35.7"C., 35.7"C. and 36.5"C. One bird was
killed immediately after removal from the cold and the other
two were killed 6 hrs. and 17 hrs. later having been kept in a
temperature of 27.7"C. during these periods.
Lipid changes. Although the sudanophilic lipid content of
the glomerulosa appeared to be within the normal range,
its distribution was very irregular. Some cells contained little
lipid of either type while others showed heavy deposits of
large irregular non-osmiophilic lipid droplets. There was also
a narrow peripheral zone free from osmiophilic lipid. The
lipid in the two inner zones was slightly heavier than normal
(fig. 1 and fig. 6). The adrenals of the "17-hour'' bird differed from those in the other birds in having somewhat less
lipid.
The Schultz test was much reduced (1+) in the inner fasciculata and reticularis (fig. 1).
A considerable number of mitotic figures were present in
the glomerulosa and outer fasciculata in the last two birds.
There was no clear evidence of degeneration.
Starvation
Six birds (2 ? and 4 8 ) in good condition, weighing from
1200 to 1600gm were starved from 4 to 6 days. Loss of
osmiophilic lipid occurred first in the glomerulosa (fig. 1)
and extended later throughout the gland. However beneath
the gonadal tissue lying on the gland, the loss of this lipid
was slight. The non-osmiophilic lipid in the glomerulosa
tended to decrease with the duration of the starvation (fig. 7).
However, in the outer areas of the glomerulosa there were
moderate to heavy deposits of non-osmiophilic lipid droplets
in areas where osmiophilic lipid was absent.
After 4 days starvation the Schultz reaction was low ( 2 -)
in the outer zones and a mere trace in the reticularis (fig. 1).
PELICAB ADRENAL RESPONSE TO STIMULI
477
By the 6th day there was a further reduction of the Schultz
reaction (1in the outer zones and a trace in the reticularis).
Mitosis increased at first during starvation, being most
prevalent in the glomerulosa and fasciculata, but by the 6th
day it had almost disappeared.
TREATMENT
RESPONSE
SLOMERULOSA FASCICULATA
RETICULARIS
CONTROL
FORMALDEHYDE
fixed 7 hr. after
CORTISONE
10 mg.
f ixed 5 hr. later
CORTISONE
3 m g daily for 6 d a y r
fined 9 5 hr.afrerlostinj.
CORTISONE
3 mg. daily for II doyr
I-
c
9mg. 12th day
fixed 6 hr. otter lost ini.
DCA
25 mg.
fixed 10 hr. loter
OCA
5mq.dailyforll d a y s
fixed 7 hr. loter
Fig. 2 Chart showing responses of the different adrenal zones t o injections into
the bird of formaldehyde, cortisone and DCA.
478
F. A. HARTMAX, R. A. KNOUFF AND G. A. HOWARD
Degeneration with congestion and change of cell contour
from loss of cytoplasm were very marked by the 6th day.
Pormaldeh yde
Six birds were injected with formaldehyde ( 5 em3 of 3.6%
formaldehyde) subcutaneously. Only one injection was made
in each instance, 60mg of sodium pentobarbital being injected at the same time to prevent the marked painful symptoms which would otherwise occur. The birds weighed from
1250 to 17OOgm. They were killed two hours to 72 hours
after the injection.
Lipid changes. I n the birds killed a few hours after formaldehyde injection there was an increase in osmiophilic
lipid in the fasciculata and a loss of osmiophilic lipid in the
glomerulosa (fig. 2) which was replaced by moderate amounts
of sudanophilic lipid. After 72 hours there was a marked
loss of both types of lipid over the entire gland (fig. 8).
The Schultz test was run on one bird 7 hrs. after injection
of formaldehyde. There were indications of some loss of
cholesterol, the values being 2- f o r the glomerulosa, 3 for
for the reticularis.
the fasciculata and 2
Degenerative c?z,anges. The glands of all birds frequently
contained eosinophils and lymphocytes as well as moderate
to large numbers of cells with pyknotic nuclei, especially in
the outer zones. These changes were least apparent in the
bird killed at 72 hours, in which some repair had presumably
occurred.
There was little evidence of change from normal in mitosis.
Estrogenic and androgenic substances were administered
to a small series of birds.
+
Btilbestrol
Birds of both sexes were injected with stilbestrol for periods
ranging from two to 11 days with doses from 0.8 mg to 6 mg
daily. The birds were killed two to 8 hours after the last
injection.
Lipid chafiges. The adrenals of male birds were affected
less than those from female birds by such injections. Stilbestrol in small amounts (0.8mg) had little effect on osmio-
PELICAN ADRENAL RESPONSE TO STIMULI
479
philic and sudanophilic lipid while the Schultz reaction was
reduced to 1 to 2 in the glomerulosa, 2 in the fasciculata
and 1- in the reticularis. Three birds received these injections for two to 4 days. A larger dose (2 mg) daily plus 4 mg
on the last of 11 days also caused little change except in the
Schultz reaction which was 1- in the glomerulosa, 1- in
the fasciculata and 0 in the reticularis.
I n the female, a small dose (0.8mg) of stilbestrol caused
in two hours a loss of osmiophilic lipid in a narrow peripheral
zone of the glomerulosa. This was replaced by sudanophilic
lipid. Within 24 hours after the injection, the only effect
remaining was a slight reduction in the over all lipid content
of the gland. This dose given daily for 4 or 5 days produced
a greater over all loss of lipid with the greatest effect in the
glomerulosa. The Schultz reaction was similar to that in the
male for this dosage. A larger daily dose (2 mg) for 10 days
and 4 m g on the 11th day produced the greatest loss. Osmiophilic lipid was much depleted everywhere while the sudanophilic lipid was about normal. There was only a trace response to the Schultz test in the outer zone with no response
in the reticularis. The two birds given this treatment were
alike in adrenal response.
Mitotic activity. There appeared to be no marked effect
on mitosis in response to stilbestrol, although some increase
in the number of mitotic figures may have occurred in the
glomerular and fasciculate zones in birds injected for two
to 4 days.
Degenerative changes. Most often glands treated with stilbestrol showed a larger number of cells with pyknotic nuclei
than were present in normal glands. This was true in both
sexes. The glands from birds treated 11 days were most
affected. I n addition to large numbers of cells with pyknotic
nuclei, there appeared non-lipid containing vacuoles in both
interrenal and chromaffin cells, congestion, and cell disintegration.
+
Testosterom
Daily subcutaneous injections of 5 or 6 mg of testosterone
propionate for periods up to 11 days were made in 4 females
480
F. A. HARTMAN, R. A. KKOUFF AND G. A. HOWARD
and 4 males ranging in weight from 1275 to 2950gm. The
hirds were killed from two to 10 hours after the last injection.
Lipid changes. I n general, there was a small decrease in
osmiophilic lipid and a slightly lower than normal sudanophilic lipid content in the glomerulosa together with slight
increases in the osmiophilic lipid of the fasciculata and reticularis of both sexes.
I n a female bird given a single injection of 6 m g of testosterone and killed 10 hours later the outer rows of cells of
the glomerulosa showed a reduction in the amount of osmiophilic lipid and the presence of some non-osmiophilic lipid.
I n birds given repeated injections over a period of days
the picture in the glomerulosa was similar to the above with
slight variations while there was some increase in osmiophilic
lipid over the normal in the fasciculata and reticularis.
The Schultz reaction for all of the adrenals, except those
from birds injected f o r 5 days and for 11 days, was small
(1 to 2 +) in the glomerulosa and a trace only in the fasciculata and reticularis. The “5-day” bird and the “11-day’’
birds possessed adrenals that gave a fairly good (2 to 2 +)
Schultz reaction in the glomerulosa, a moderate (1 to 2)
reaction in the fasciculata and somewhat smaller (1- to
1+) reaction in the reticularis. These reactions were quite
spotty.
Mitoses appeared to be normal in number.
Degeneration. The adrenals of birds treated f o r a short
time with testosterone showed few degenerative changes,
while glands from birds which had been injected for 5 days
showed some increase in the number of cells with pyknotic
nuclei, The “11-day” glands contained a still larger number
of cells with pyknotic nuclei, and the chromaffin tissue was
vacuolated. There was also some evidence of vacuolization
in the center of the cords.
+
+
Cortisone
Changes produced by the doses of cortisone which we
employed were variable and not marked.
PELICAN ADRENAL RESPONSE TO STIMULI
481
Cortisone acetate suspensions were administered subcutaneously to 14 birds. Three were given one injection. Two
were injected daily for three days, one for 4 days, 5 for 5
days, one for 6 days and two for 11 days. The amounts of
cortisone per injection ranged from 3 to 10 mg. The adrenals
were fixed a few hours after the last injection.
L i p i d chawges (fig. 2 ) . A single injection of 3 mg and fixation 17 hours later was without effect. An injection of 10mg
in one bird and 15 mg in another produced after 4 and 5 hours,
a subnormal lipid content in the adrenals. There was a zone
in the glomerulosa in which the small amount of lipid present
was sudanophilic but not osmiophilic. The widest zone of
osmiophilic lipid depletion was in that of a bird given 3mg
injections daily for 6 days and killed shortly after the last
injection. This depleted zone extended through the glomerulosa into the outer fasciculata and to some extent into the
reticularis while the inner fasciculata contained more osmiophilic lipid than normal.
Birds given larger doses of cortisone for a period of days
(5 to 9 mg daily for 5 days to 11 days) showed a normal or
higher osmiophilic lipid content in the glomerulosa while
that in the fasciculata and reticularis was somewhat lower.
Only the outer row of cells of the glomerulosa contained
small amounts of non-osmiophilic lipid.
The Schultz reaction with a single dose of 10mg (killed
5 hrs. later) showed a small reduction from normal: glomeruand reticularis, trace. Repeated
losa, 2 ; fasciculata, 2
doses were followed by a rise of cholesterol to normal or
above. For example, 3 mg per day for 5 days showed adrenals
with the following values : glomerulosa, 3 ; fasciculata, 3
and reticularis, 2.
iWitosis. A considerable number of mitoses were seen in
the adreiials of birds given 3mg of cortisone while in the
adrcnals from birds receiving injections daily f o r 5 days
o r longer the number of mitoses was below normal. I n general
the more degenerate glands contained fewer mitoses.
+
+
+
482
F. A. HARTMAN, R. A. KNOUFF AND G . A. HOWARD
Degemratiue changes. Most of the glands from cortisone
treated birds showed some evidence of degeneration. The
birds which received the largest doses, and those injected
over the longest period, showed the greatest changes. Those
given small injections ( 3 mg) over periods up to 5 days were
less affected.
The degenerative changes noted were pyknotic nuclei and
cells with large vacuoles containing material which stained
orange with Masson’s stain.
The adrenals containing the most varied, and perhaps the
most extensive degenerative changes were from a bird which
had received 5 mg of cortisone for 4 days, and 10 mg on the
5th day. These adrenals showed many cells with pyknotic
nuclei occurring mainly in the outer zones, cells staining
orange with Masson, large masses of lymphocytes in the
glomerulosa and fasciculata and great numbers of eosinophiles. These glands also had the highest lipid content both
sudanophilic and osmiophilic of any cortisone gland and
showed few or no mitotic figures.
The glands from birds receiving injections for 11 days
showed evidence of a decrease in the size of the interrenal
cells, and a decrease in the lipid content. The cells were
considerably smaller than those of birds injected with ACTH
for 13 days.
Desoxycorticosterorze
Eleven pelicans were injected with DCA in oil, some being
given single injections, others daily injections over a period
of days.
Lipid changes (fig. 3 ) . Six birds were given 4 mg of DCA
each either once or daily f o r 4 or 5 days. The birds were
killed and their adrenals fixed from one to 15 hours after the
last injection. These glands were normal in three while the
osmiophilic lipid content was slightly higher in two. In the
remaining one, there was some loss of osmiophilic lipid in the
glomerulosa and reticularis and a greater loss in the fasciculata in which the lipid droplets were smaller and restricted
to the middle portion of the cells.
PELICAN aDRENAL RESPONSE TO STIMULI
483
One bird was injected with 25 mg of DCA and killed 10 hours
later. The adrenal showed a marked reduction of osmiophilic
lipid in the glomerulosa and fasciculata. There was a distinct
line of demarcation between the fasciculata and the reticularis
the latter having a somewhat higher than normal lipid content.
Three birds were injected for 11 days with 5 m g of DCA
daily. They were killed 8 hours after the last injection. All
adrenals had a higher than normal osmiophilic lipid content
in the glomerulosa and outer fasciculata, a normal lipid content in the inner fasciculata, and somewhat higher than normal
lipid content in the reticularis. Cells in the reticularis appeared to be smaller than normal, but it may have been
merely due to loss of lipid.
Another bird was injected daily with 15mg of DCA for 4
days and killed 8.5 hours after the last injection. Its gland
showed a very marked increase of osmiophilic lipid in all
zones, the glomerulosa having the highest content (fig. 9).
I n the glomerulosa and fasciculata the cells were distended
with large lipid droplets.
Non-osmiophilic but Sudan positive lipid was seen in only
one bird of the DCA series and this was in the outer glomerulosa.
A single injection of DCA produced some effect on the Schultz
reaction. For example the injection of 25mg and killing 10
hours later gave adrenals with lower reactions as follows :
peripheral zone, 1; intermediate zone, 2
and central zone,
2 -. The picture was similar in the adrenals of birds injected
daily with 5 m g for 11 days, except that the reaction was
normal (3) in the peripheral zone.
Mitosis. There is no indication that DCA stimulates mitotic
activity in the young pelican, but it may actively depress
mitosis because few or no mitotic figures were found in the
adrenals of birds which had been injected for 5 and 11 days.
Degeslerative chunges. The most marked degenerative
changes were found in the glands from the three birds which
received injections for 11 days. However, most of the DCA
+
484
F. A. HARTMAN,
R. A. KNOUFF AND
o.
A. HOWARD
glands showed a larger number of cells with pyknotic nuclei
than are usually found. I n the “11-day” glands there were
many cells with pyknotic nuclei especially in the glomerulosa
and fasciculata, many acidophilic and orange staining cells,
and vacuole formation in the chromaffin tissue. I n addition
to these changes, two of the glands showed masses of lymphocytes and moderate congestion. There was some evidence
of atrophy in the cells of the fasciculata.
We conclude from these observations that DCA when given
in relatively large amounts inhibits the activity of the interrenal tissue. This leads to a higher osmiophilic lipid content
especially in the glomerulosa. Non-osmiophilic lipid rarely
appears in the gland. Sometimes there may be a subnormal
content of osmiophilic lipid in the fasciculata. Mitosis is
probably depressed and degenerative changes become marked.
DIscussIoIs
Our experiments were conducted with birds from three to
<5 weeks old. This estimate was based on our findings at an
earlier visit to the nests when only eggs were present. It is
difficult to find young pelicans since the nesting season varies
from year to year. I n addition the collection and feeding
of such large birds is expensive. Ekperiments, therefore,
are limited.
This investigation was conducted before the time factors
of ACTH action were known. Thus many variations were tried,
which accounts for the small number of birds on a point.
However with these limitations certain trends are indicated
which prove interesting.
The use of osmium tetroxide in the manner described above,
revealed changes in the lipid which is not detected with Sudan.
The tissue was first fixed in IIelly’s fluid and then osmicated
8 to 1 2 hours or less, the estimated minimum time necessary
to assure complete penetration of tissue. We have found
that long periods of osmium treatment fail to bring out the
differentiation obtained by short exposure. The loss of the
capacity of lipid to reduce osmium tetroxide under the con-
PELICAK ADREFAL RESPOBSE TO STIMULI
485
ditions of the experiments suggests a change in that lipid
and since this change is associated with increased activity of
the gland under stress (cold, starvation) it may be inferred
that it is related to formation or release of hormone. What
these changes may be we are unable to say. The lipid may be
altered by the Helly's solution so that it is no longer osmiophilic or it may be of a type that more readily yields its
reduced osmium tetroxide to solvents.
The difference in activity suggested by osmium reduction
in the pelican would indicate distinctions from the mammal
because the glomerulosa of the latter does not respond so
readily. In the pelican stimulation of the adrenal by means
of a single injection of ACTH leads first t o changes in the
glomerulosa which involves a loss of the capacity of the lipid
to reduce osmium tetroxide although the lipid reacting with
Sudan may be unchanged or even increased. There follows
a marked reduction in osmiophilic and total lipid in the fasciculata and reticularis. With daily injections over a period
of days the osmiophilic and sudanophilic lipid rises above
normal in the two inner zones. All of the lipid appears t o
be osmiophilic as well as sudanophilic.
The apparent transition between lipid and mitochondria
in the inner glomerulosa and outer fasciculata reminds one
of the picture reported by R. A. hIiller ('50) in the mouse
adrenal cortex. With hypertrophy there was reduction of
lipid concentration accompanying proliferation of the mitochondria or with atrophy, the reverse, increase in lipid and
reduction in the number of mitochondria.
Cholesterol as indicated by the Schultz reaction was reduced most in the reticularis and less in the fasciculata and
glomerulo sa.
Mitoses increased considerably in the glomerulosa following short periods of daily injection or after a single injection
but were depressed after long periods of daily injection.
Exposure to cold showed moderate stimulation of the
adrenal. Starvation which was a stress of much longer duration, gave evidence of much greater stimulation. Likewise
486
P. A. HARTMAS,
R. A. KNOT-FF AXD G. A . HOWARD
formaldehyde produced a picture of marked stimulation.
However mitoses showed little change from normal. Stilbestrol produced little effect in the males but a decided effect
on the lipid picture in the females but no significant change
in the rate of mitosis. Testosterone produced a small effect
on the adrenals.
Of the two methods employed to produce inhibition of
interrenal activity, the injection of cortisone was less effective. Injections over a period of days was accompanied by
an increase in osmiophilic lipid in the glomerulosa but a
decrease of that in the fasciculata and reticularis with very
little appearance of non-osmiophilic lipid in the glomerulosa.
Xitosis was less frequent and the interrenal cells became
smaller.
DCA when injected in small amounts for short periods had
little effect in some birds. A large dose caused a marked
reduction of osmiophilic lipid in the outer zones by 15 hours.
Injections of DCA daily f o r a relatively long period were followed by a marked increase in osmiophilic lipid in all zones.
This represented the total lipid since in only one instance
did non-osmiophilic lipid appear. Mitosis was depressed and
there “as considerable evidence of atrophy. This however
was not limited to any one zone. All evidence indicated inhibition of interrenal cell activity when DCA was injected
repeatedly f o r several days.
The outstanding feature in this study is that the glomerulosa and to some extent the outer fasciculata appear to be the
most active in the whole gland when there is a response. This
is interesting because the glomerulosa is only about 10% of
the thickness of the gland and the fasciculata only 2076, the
remainder being the r e t i c ~ l a r i s .m7ith
~
prolonged stimulation
the inner zones are affected.
Miller and Riddle ( ’42) reported that the stimulated adrenal
of the pigeon possessed considerably less lipid and cholesterol
than the normal adrenal. The peripheral zone was affected
These figures are based on unpublished measurements from the adrenals of 10
young pelicans.
PELICAN ADRENAL RESPONSE TO STIMULI
487
with OW levels of stimulation while with high levels the whole
gland responded. They noted changes as early as 10 hours
after a single injection of ACTH. We observed changes within
tx-0 hours after injection. They found that the mitochondria
and lipid merct inversely correlated.
A[. R. Miller ( ' 5 2 ) in his study of the lizard Xcnntusiu vigilis
found that ACTH caused marked lipid depletion and a decrease
in interrenal cell size with two injections and hypertrophy
and lipid formation after 4 doses. Cortisone also caused a1iriost complete evacuation of lipid and shrinkage of cells and
thinning of interrenal tissue. DCA likewise caused lipid deplc t ion.
SUMMARY AND CONCLUSIONS
1. A study of the effect of ACTII, cold, starvation, formalcichyde, cortisom, UCA, stilbcstrol and testosterone on the
ndrenal was made in pelicans a few weeks of age.
2. ACTH produced prompt changes in the lipid of the glomerulosa which were followed later in the inner zones. At
first t,her.e was a decrease in osmiophilic lipid in the outer
zone. Daily injection f o r several days sometimes increased
the total lipid (sudanophilic) in all zones but did not increase
the cholesterol content (Schultz reaction). Mitosis was increased in the glonierulosa and outer fasciculata but was
little aff'ected in the rest of the gland. Daily injection f o r
several days increased degeneratirc changes.
3. Exposure to - 5°C. for 5 hours caused irregular effects
on the lipid, some cells being depleted and others containing
more than normal amounts. A narrow peripheral zone was
frcc from osrniophilic lipid. Cholesterol was much reduced.
Thcrc was an incrcwse in mito& in the outer zones but littlc
evidence of cellular degeneration.
4. lnanition for 4 to 6 days caused a loss of osmiophilic
lipid first in the glornerulosa and later throughout the gland.
The non-osmiophilic lipid also teiidcd to decrease with d u n tioil of inaiiition. Cholesterol bccarrie much rcduced throughout. Mitosis increased at first hut had almost disappeared
by the 6th day. Degeneration ~vasquite marked by the 6th
day.
488
F. A. HARTMAN, I&. A. KXOTTFF AND Q. A. HOWARD
5. Formaldehyde after 72 hours caused a marked loss of
both types of lipid over the entire gland. Mitosis appeared
to he little affected while degeneration was moderate.
6. Stilbestrol caused some reduction in the cholesterol in
the adrenal of the male but little effect otherwise. In the
female, however, this compound caused a reduction of osmiophilic lipid in the glomerulosa, which was replaced by sudanophilic lipid. Repeated injections especially with larger doses
caused depletion of osmiophilic lipid and cholesterol everywhere while the mdaiiophilic lipid appeared to be little affcbctcd. There was no marked effect on mitosis but there was
some indication of degenerative changes.
7. Testosterone produced no great changes in the osmiophilic and sudanophilic lipid. The cholesterol, however, was
much reduced especially in the inner zones. Mitosis appeared
i1ormal and degenerative changes were moderate.
8, Cortisone produced variable but not marked changes
in adrenal lipid. Continued daily injections resulted in a
normal or higher osmiophilic lipid content in the glomeruloxa and tl. somewhat lower amount in the inner zones. Cholesterol was normal or higher. Repeated injections reduced
mitosis and increased the degenerative changes. Eventually
there was decrease in cell size and lipid content.
9. DCA when given in relatively large amounts inhihitcd
the activity of the interrenal tissue. This led to a high osmiophilic lipid content especially in the glomerulosa. Nonosmiophilic lipid rarely appeared. Mitosis appeared depressed and degeneration became marked.
10. Our observations indicate that the glomerulosa and to
some extent the outer fasciculata are the most responsive
portions of the pelican adrenal when the bird is stimulated.
LTTERATCRH CrCTED
KKOIW*, R. A., AND F. A. HABTMAN1951 A microscopic study of the adrnlal
of the Brown Polican. Anat. Bee., 1/13: 161-188.
MILLRR,M. R. 1952 Tho normal histology and experimental alteration of the
adrenal of the viviprroiis lizard, dYu&u8ia i ! i g i h . Anat. Rcc., 218:
309-323.
PELICAN ADRENAL RESPONSE TO STIMULI
489
MILLEK,JL A. 1950 Cytological phenomena associated with experimciital alterations of secretory activity in t h o adrenal cortex of mice. Am. J. Anat.,
86: 4 0 5 4 3 7 .
MILLER,R. L4., AND 0. RIDDLE 1942 The cytology of tlre adrenal cortex of normal
pigeons and in experimentally induced atrophy and hypertrophy. Am.
J. Anst., 71: 311-341.
PLATE 1
ElPLANATION OP FIGURES
Utiretoochrd })~iotoniicrogr;ipIisof scctioiis of the adre~ialsof tlic Iirowii P(~Iie;iii,
3
Two Eiorirs a f t e r injectinn of ACTH dinwing disappe:traiice of niucli of the
osmiopliilie lipid from t h r glomerulosit and transition between niitoc1iond~i:i
:ind lipid droplets
(?A ).
Vacuoles cont;iiiied sudaiiopli
fixation followrd by osiiiiurn totroxidc. Jf:isson stain. X 960.
1
TTiistimulated gland. I’rocedurc a s above. X 3 00.
5
Two hours a f t e r the last of three injections of 9 C T l l in tlirce clays, showing
markrd drpletion o f osniioplijlie lipid iii the glowerulosa. Procecluw as abox e.
x 300.
490
491
PIATE 2
EXPLASATION OF FIQCRRS
Cnrctouclicd ~~liotoiiiisrogru~lir
of scctioiis of the adrenal8 of thc Brown Pslican.
0 After expowre to -5'0.
7
for 5 bourn. Procedure a8 rboro. X 100
After 5 dnyr inmition. Procedure a8 above. X 100.
8 After (72 hrs.) injcc+tion of fom~ildaliydosliowhg loss of lipid. Prowduru
as above. X 100.
0 DCA, 15 m g daily for 4 days, Killed 8 hre. &tor tlic! bet injection. Promdure
us aborc. X 100.
492
P E I J C A N Al)I<lCKATJ l<lCSL’OXW TO STIMULI
F. k. IIARTMAN, It. A . IZNOUBF AXD G. A. H O W A R D
493
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