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The effect of acute starvation on the adrenal cortex of the hamster.

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THE EFFECT O F ACUTE STARVATION ON THE
ADRENAL CORTEX O F THE HANSTKR
TITTEKTY FIGURES
ISTItODUCTION
Statcs of hyper- or liypoaclrenocorticalis~iare reflected by
various cytological and histochemical changes in the parcncliymal cells of the adrenal cortex. The adrenals of almost
all rritrrvinials are, with minor species diff ercnees, histologically
similar (Kouime, '49). The cortical cells of the rat, f o r example, contain sudanophilic lipid droplets which exhibit birefringence and autofluorcscciice ; they contain histochemically
demonstrable cholesterol and its esters and give positive reactioiis with various aldehyde rcag:.cnts such as leucofuchsin
( Rchiff rctigent ) , 2-4-diniti.o~,licnylli~cli"~~iiie
and 2-hydroxy3-naphthoic acid hpdraziclc. After scctions of adrenal glaiids
arc treated with fat solvent^, the lipid droplets arc no longer
colored by an? of these reageiits. Sites which yield positive
reactions to all of these tests, and which are negative after
cstmction, a 1 ~helicvetl to demonstrate steroid hormones
(T)cmpsey, '48; Groep and Deane, '49). These chemical tests
togethcv with the criteria of cell size and the size of the
inc-liv-idual lipid droplets, are currently used to evaluate the
secretion of hormones by the adrenal cortex of the rat (Deane
and Greep, '46 ; Rergiicr aiid ncanc, '48).
The adrenal of t h e hamster is liistochcniically different
froiii that of the rat. Cortical cells of the hamster contain no
sudaiiophilic lipid droplets (as demonstrated after formalin
fixation) and no histocheniically ticnionstrable cholesterol.
---
.>,>a
556
K A R L M. IINIGGE
They exhibit no birefringence. The zona fasciculata and zona
reticularis are intensely stained with aldehyde reagents but
the reactive material is acetone-insoluble. Lipid is present
in the zona fasciculata aiid zona reticularis in the form of
mitochondria and spheroidal, acetone-insoluble bodies to which
the term liposomes has been applied. On the basis of their
histochemical reactions, Knigge ( '54) suggested that the liposonies are in part a lipoprotein complex, the lipid portion
being phospholipid in nature. Liposomes are not present in
the zona glorrierulosa. Cells here contain instead lipid droplets which exhibit properties suggesting a free (lion-protein
bound) phospholipid. The hamster adrenal is unique in possessing such droplets in the zona glornerulosa, since they
are not present riorrnally in the remainder of the cortex and
liecause lipid with such histochemical propertics has not
been described in the adrenals of other species.
I n view of these differences between the rat and hamster
adrenal cortices, it is of interest to establish the cytological
and histochemical criteria of accelerated or depressed secretory activity applicable to the hamster adrenal. The response
of' the adrenals of hamsters subjected to stressful conditions
has not been extensivclly investigated. Rlpert ( 'SO) observed
a reduction in the Schiff-positive material after administration of corticotropin. Leathem and Stauber ('52) found ail
increase in alkaline phosphatase activity and the appearance
of sudanophilic lipid and cholesterol in the cortex of hamsters
infected 4 weeks or longer with JAshmania donovani. Agate
( '52) likewise ohserved sudanophilic lipid and large amounts
of cholcsterol in the cortex of hamsters hearing implants of
mouse stii-conia 180.
The present work was undertalreii to extend observations
oil the respoiisc of the hamstel. a d ~ e n a undei.
l
various espcrimental circmristances. Since izcu te inanition is considered by
some investigators (Tepperman et al., '-13 ; Selye, '50 ; Sayers,
'50) to evoke a demand for increased cortical hormone, this
<tressor was einploycd to produce a state of hyperactivity
of the adrenals.
STABVATIOS AND ADRESA4L CORTEX
557
MATERIAL AND METHODS
Twenty-four littermate pairs of young adult male hamsters,
ranging in weight from 70 to 85,gm, were used. One animal
of each pair served as a control while the other was deprived
of all nutrient for 8 to 10 days, until it lost 35 to 40% of its
initial body weight.
At autopsy, the adrenals were removed, freed of fat and
weighed by diff ereiice in separate glass-stoppered bottles of
fixing fluid. For general histological study adrenals were
fixed in Bouin's fluid, sectioned at 5 p and stained with hcmatoxylin and eosin or by the Masson ('28) procedure. Glands
fixed in Regaud's fluid (two days) were post-chromated in
3% potassium dichromate (two days), embedded in celloidin
and paraffin and sectioned at 2 or 3 p. Sections of glands of
both control and experimental animals were mounted on the
same slide and stained with iron hematoxylin or Altmann's
acid fuchsin f o r mitochondria. The Golgi apparatus was
demonstrated by the silver nitrate method of Aoyama ('29)
after fixation in cadmium chloride-formalin. Frozen sections
of adrenals fixed in 10% formalin were stained with Sudan
black R, Sudan IV, the Schultz ('24) reaction for cholesterol,
leucofuchsin (Schiff reaction) and with 2-hydroxy-3-naphthoic
acid hydrazide ; other sections were mounted unstained and
examined with the polarizing microscope for birefringence.
Osmiophilic lipid was demonstrated by fixation in formolZenker-osmic acid. Adrenals fixed in chilled 80% alcohol were
used to demonstrate alkaline phosphatase activity (Gomori,
'39) ; sections were incubated with sodium glycerophosphate,
fructose diphosphate and yeast nucleic acid for one and three
hours at pH 9.2. Eighty per cent alcohol was found to be the
fixative of choice for the demonstration of esterases in the
hamster adrenal cortex ; alpha-naphthyl acetate and naphthyl-AS acetate were used as substrates according to the
method of Goniori ( ' 3 2 ) . With each substrate, sections were
incubated at room temperature for approximately one hour.
The area of 200 nuclei in cells of both the zona glomerulosa and outer portion of the zona fasciculata were measured
froiii Rouiii-fixed glands of three control a i d three starved
hamsters. In the case of the zoiia gloinerulosa, nuclear outlilies were traced from camera lucida images ( X 1300) of
e v e ~ ygloiiiei.ulosa1 cell nucleus in a continuous segment of
the cortex uiitil 200 nuclei were drawn. These drawings were
enlarged an additional 1 2 diameters arid the areas measured
with a plaiiiineter. Siiice nuclei in the zoiia fasciculata a r e
more unifoimily spherical, their diaineters were measured in
order to calculate nuclear area. Under oil immersion, using
an ocular iiiicroincter, the diameter of every parenchymal
cell nucleus in randomly selected fields of the outer portion
of the zona fasciculata was recorded until 200 nicasureincnts
\\.ere obtained.
OBSERVATIONS
l’hc iaorinal crt7reizd c o r t e x
Since the hanister cortex is less well lriio\vn than the cortices
of animals commonly used in the laboratory, a brief desci*ipof it is giveii. T i i addition, ohservations on the Crolgi a p ~ ~ a ratus and the esterase eiizyiiics, a s yet undescri1)cd f o r this
species, a r e presented also.
Zosu. In routiiie prepai-atioiis of IZouin-fixed
glands, this outer baiid of cells occupics a very small 1)oriioii
of the width of the cortex a i d is frequently incoiisl)icnous.
The coiistitucnt cells are, ho\vevei-, uiiique in that tliey coiilaiii
lipid di*opletsn.hich are osmiopliilic (fig. I) but whicEi, after
fixation in 10% foimaliii, cxhihit 110 sutlariophilia, bi i*eFriiigeiice arid coiitaiii no histocheinically denionstrablc cliolestcrol.
Vacuoles, left by solution of the osniiophilic dr.oplcts, awe
rather poori;r preserved after Bouiii fixatioii, but H I T clcai.ly
delineated after fixation in Kegaud’s fluid (figs. 17, 38). IVitli
the latier fixative, mitochondria a r e dcnionstrated in tEi(. wlls
of the zona gionierxlosa as small granules uniformly tlistrihuted throughout the cytoplasm. T h e n stained with either
iron heiiiatoxylin or Altmanii’s acid fuchsin, mitochondria in
these cells a r e coloi-ed less iiiteiiselp than a r e t h o w of the
zona fasciculata and zona reticularis (fig. 1i ) .
The Uolgi apparatus of glomerulosnl cells is a dense argentophilic, justaiiuclear. cap with few radial cstciisioris and
no apparent polarity with respect to tlie vascular channels
in thih zone (fig. 9). In addition to these hlnckciied juxtanuclear caps, there arc‘ present in the cvtoplasm occasional
small argentophilic xi-mules. Thcse frcqnently lie in rows
encircling the nucleus or the vacuoles which cmhinecl the
osmiophilic lipid (fig. 9).
‘rhc zona glomcrulosti esliibits no 01’ only faint alkaliric
pho,sphatase activity after three hours’ iiicuhation with sodium glycerophosphate, fructose diphosphate or yeast iiucleic
acid (fig. 7). Estcimc wtivity, ho\vever, is more intense in
this zone than in tlie r.cinaiiidcr of the cortex. After iiicubatioii with naphthyl-AS acetate f o r periods up to one hour, the
c a p w l c oxhihits no erizyiiic’ activity, tlie cytoplasm of cells
in capsular nodules aiid i i t the eoiia glomerulosa is filled with
fairly coarse, carniine rcd granules, the iiuclci being faint or
negative (fig. 8).
Z o m j’asciculata a w l , ~ o t wr.cticulnris.
previously mentioned, cclls of these zones do not contain lipid droplets. Thus
the zoiia fasciculata and zoiia rcticularis are sudanophobic,
lack bircfriiigcnce and coiitain no cholesterol when examined
in formulin-fixed glands. Thcsc zones, however, stain mi th
leincof’uchsiii aiid 2-liydr.osv-3-nay)ht2loic acid hydrazide (figs.
19, 20), \vliich are reagents for tlie demonstration of aldehydes. Cyells of the xoiia fasciculata and zona rcticularis are
filled with nii tochondria aiid liposomes which exhibit an intciise affinity for iron hematovylin or acid fuchsin after fixation in Regaud’s fluid. When sections a r c stained with these
dyes arid differentiated to a point -cvhere the mitochondria
of the zona fasciculata are clearly outlined, the mitochondria
of the zoiiit glomerulosa are faint or unstained; this results
in a demarcation between thc zona glomerulosa and the remainder of the c o r t a which is not appreciatcid from sections
prepared, by other methods (figs. 4, 17).
The differentiation between mitochondria aiid other similarly stained bodies is often difficult and arbitrary. I n the
560
KARL M. K S I G G E
outer portion of the zoiia fasciculata, mitochondria are identified as m a l l granules or short rods which are of a uniform
size and which are frequently arranged in pairs or short
chains (figs. I-a, 1-1); 15). I n addition to mitochondria, there
Fig. 1 1)rawings of cortical cells from the outer portion of
lata, made from glaiids fixed in Regaud's fluid and stained with
('ells Q :tiid b are from n normal anim:il; cells r :md t7 arc from
Legend: I,, liposomes; M, mito(*hondria; G, negatire image
paratus; V, vacuoles.
the zona fascicuiron Ireimtoxylin.
a starred :inimal.
of the Golgi ap-
are similarly stained bodies which are larger, randomly distizihuted among tlic mitochondria, and more variable in number from cell to cell. These larger bodies, OF liposomes, are
frequently encircled by a clear halo-like area (figs. 1-a, 1-b;
1 5 ) . From the mid-portion of the zoria fasciculata to tho
STARVATION AND ADR,ENAL CORTEX
561
cortico-medullary boundary, there occurs a progressive decrease in the number of mitochondria accompanied by an
increase in number of liposomes which may attain considerable size, become irregular in shape and lose some of their
affinity for iron hematoxylin.
Because the cytoplasm of cells of the zona fasciculata is
filled with darkly staining mitochondria and liposomes. any
area from which they are absent is particularly apparent.
Such clear areas represent the negative images of the Golgi
apparatus and, after Regaud fixation, are in the form of
clear, vacuolar or canalicular-like spaces surrounding the
nucleus (figs. 1-a, 1-h; 15). Extensions of these perinuclear
clear areas may extend into the cytoplasm for some distance.
The impressions of the Golgi apparatus gained from the
negative images seen in It-egaud-fixed adrenals are borne out
and amplified when this organelle is blackened with silver
by the Aoyama method. The “typical” Golgi apparatus of
the zoria fasciculata, as described by Reese and Moon (’38)
f o r the rat, consists of a compact, pyramidal-shaped juxtanuclear cap. This type of Golgi apparatus is present in only
relatively few of the cells of the zona fasciculata of the hamster’s adrenal. The most frequent pattern is a perinuclear
distribution of small spheroidal or irregularly shaped bodies
which lie either unconnected to one another at some distance
from the nucleus or closely applied to the nuclear membrane
(figs. 11, 12). Vesicular spaces are occasionally seen within
the substance of the Golgi material, appearing as a series of
small vacuoles or as clear vesicles within the individual
blackened Golgi bodies (figs. 11, 12).
Alkaline phosphatase activity is present throughout the
zona fasciculata arid zona reticularis, with the outermost
portion of the zona fasciculata being somewhat variable in
the amount of enzyme activity present. Of the substrates
used t o demonstrate alkaline phosphatase activity, fructose
diphosphate and yeast nucleic acid yield a somewhat greater
deposition of cobalt sulfide than does sodium glycerophosphate (fig. 7). Moderate esterase activity is present uni-
fo 1*1rilythrong11out t lic m ~ i af a sci cwlata aiid zoiia r e ticnlari s.
\\7tli ii:i1)litliyl-AS acetate (iig. H), cwiyiiie activity is restricted to the cytoplasm of the parenchymal cells.
-- -
‘ Stniitl:ritl c1eri:ition =L.
\
\: ‘(12
- 1
!I%e adreiials are riot altered appreciably in appearaiice
1 1 ~ 7;icutc st;ir.vntion.
The paircd actrciial \wight of starved
iiiiiniiils i h 11.1 irig; tlic .ivciglit of the adreiials of control
aiiiiiinls is 14.2 irig (table 1). The difference in weight is
+yiific;int \vhen csaitiiiic.cI by tlic Student ( ‘ t ” test ( t = 7.45:
at t h e 1%) level, vulucs of ‘ ( t ” greater than 2.7 arc considered
significaiit ).
Sire Zwr. s i w . The distri1)ntiori of the cross-sectional areas
of nuclei of glomerulosal cells is shown in figure 2. The
gixpli confirms the subjectiw ol)sci*vationthat nuclei in this
zoiie cxhibit it widc rmige of sizw arid slinpc~s. Alcutestar\-ation results in a decrease in the area of gloiiwrulosal cell
trucdlci iis reflcc+ctZ 11;v a shift to the left in their distribution
(‘I1 I’Vt’ (fig. a).
The distribution of nuclear areas of cells of the outer fascaiculata is showii in tigiire 3. Tlieii- range of distribution is
STARVATION A N D ADRENAL CORTEX
563
less than that exhibited by nuclei of glomerulosal cells. The
maximum concentration of nuclear areas f o r the normal
adrenal is around 35 p2, from which a mean diameter of 6.6 p
NUCLEAR
AREA,^^
Fig. 2 Distribution curve of the nuclear areas of cells of the zona glomerulosa
in normal and starred hamsters. Each curve represents the distribution of 600
nuclear areas. A shift of the distribution curve t o the left indicates a reduction
in the size of glonierulosal cell nuclei of starved hamsters.
-CONTROL
-----STARVED
7 160
V
3 120
8
a
# 80-
z Z
40-
- --.
I
NUCLEAR AREA,
p2
Distribution curve of the nuclear areas of cells
the zona fasciculata of normal and starved hamsters. An
is shown by a shift of the distribution curve to the right.
in a significant increase in the number of large nuclei,
peak a t 55 p2.
Fig. 3
of the outer portion of
increase in nuclear size
Starvation also results
indicated by a second
564
KARL M. KNIGGE
is obtained. The mean nuclear diameters of cells of the zona
fasciculata of the normal rat is 6.8 ~1 (Odenthal, '52), for the
guinea pig 7 . 6 ~(Boguth et al., '51) and for the dog 4 . 5 ~
(Barkay, '42).
Acute starvation of the hamster results in a n increase in
the cross-sectional area of nuclei of cells in the outer fasciculata (fig. 3), with a maximum concentration being around
40 pa. I n addition, there occurs a significant increase in the
number of larger nuclei, particularly those with a mean
nuclear area of 55 p2.
Zovza gZomwuZosn. The constituent cells of the zona glomerulosa exhibit a marked vacuolation, the resulting decrease
in amount of cytoplasm causing this zone to appear somewhat
lighter than normal in Ahson-stained sections. Cell clusters
are smaller and nuclei are more densely packed than in
control glands, suggesting that a decrease in cell size has
occurred also (figs. 4, 5, 17, 18). Greater variation in the
general architecture of the zona glomerulosa occurs after
acute starvation. I n some areas of a gland, the zona glomerulosa is replaced completely by cords of cells of the zona
fasciculata; in other areas, the capsule exhibits a marked
proliferation of cells which extend into and become continuous with irregularly arranged, vacuolated glomerulosal cells
(fig. 6).
After fixation of adrenals in Regaud's fluid, the increased
vacuolation of glomerulosal cells is conspicuous (fig. 18).
Mitochondria in these cells become larger and exhibit a greater
affinity for iron hematoxylin or acid fuchsin; the zona glomerulosa, therefore, appears darker than normal (fig. 18).
The Golgi apparatus of glomerulosal cells undergoes significant changes after acute starvation. Blackened juxtanuclear caps, although still present in some cells, are less
frequent than normal. The majority of cells contain numerous argentophilic Golgi granules which are considerably larger
than those seen in glomerulosal cells of the normal gland
(figs. 9, 10). Thcse argentophilic granules, most frequently
located about the nuclear membrane, may be the only evidence
STARVATION AND ADRENAL CORTEX
565
of Golgi material in the cell or there may be present also a
juxtanuclear cap which is smaller than normal (fig. 10).
Frozen sections of formalin-fixed glands reveal that the
zona glomerulosa remains sudanophobic, free of cholesterol
and exhibits no birefringence ; aldehyde reagents continue
to stain the zona glomerulosa faintly or not a t all. Fixation
of adrenals in formol-Zenker-osmic acid reveals that the
vacuolation seen in Bouin and Regaud-fixed glands is due
entirely to the presence of osmiophilic lipid droplets (figs.
5, 17).
Acute starvation does not alter significantly the alkaline
phosphatase or esterase activity of the zona glomerulosa ;
activity of the former enzyme remains very weak while esterase activity is intense.
Zona fusciculata cuzd xona reticularis. In Bouin-fixed glands
stained by the Masson technique, many cells of the outer
portion of the zona fasciculata show considerable numbers
of large vacuoles in their cytoplasm. Columns of vacuolated
cells are continuous with those of the zona glomerulosa and
frequently the boundary between the two regions is obscure.
Smaller and relatively less conspicuous vacuoles are present
in the cells of the remainder of the zona fasciculata. Although
their nuclei have enlarged (fig. 3 ) , cells of the zona fasciculata appear smaller than those in normal glands. Cells of
the zona rcticularis are noticeably smaller than normal, contain vacuoles in their cytoplasm and possess nuclei which
are generally smaller, darker and more irregular in shape.
The decrease in size of cells of the zona reticularis results in
a widening of the sinusoids of this region.
Iron hematoxylin-stained adrenals of starved hamsters
reveal that mitochondria have increased in size and that many
cells exhibit cytoplasmic vacuolation (fig. 16). There is variation from cell to cell with respect to the degree of vacuolation, size and shape of mitochondria and number of liposomes.
Several cell types present in the outer zona fasciculata are
shown in figures 1-c, 1-d. Frequently the spherical mitochondria are increased to such a size that differentiation between
566
KARL M. HNIGGE
them and liposomes is difficult (fig.16). I n the mid-portion of
the zona fasciculata, almost all cells are markedly vacuolated
with only slightly larger spherical and rod-shaped mitochondria occupying the cytoplasmic spaces between the vacuoles.
When the entire zona fasciculata of a section is considered,
there does not appear to be a significant alteration in the
overall number of liposomes. Clearly delineated negative
images of the Golgi apparatus are seen less frequently than
in the normal gland. The cells of the zona reticularis are
similar to those observed in normal adrenals, although a decrease in their size has occurred. Vacuoles are present in
some cells of the zona reticularis, but they are smaller and
less numerous than the vacuoles in the zona fasciculata.
Acute starvation reduces the total amount of chromophilic
Golgi material in most cells of the zona fasciculata (figs. 13,
14). I n addition, there occurs an increase in the number and
size of the clear vesicles located in the individual Golgi bodies
(fig. 14). I n some cells (fig. 13) there is present cytoplasmic
vacuolation which is similar to that observed in Regaudfixed glands (fig. 16). I n such cells, the Golgi material bears
no constant positional relationship to the vacuoles.
The zona fasciculata and zona reticularis remain sudaiiophobic, free of histochemically demonstrable cholesterol and
exhibit no birefringence as demonstrated in f ormalin-fixed
glands. There is also no significant change in the intensitjor distribution of the staining of these zones with leucofuchsin
01- 2-hydroxy-3-naphthoic acid hydrazide. Fixation of adrenals
in formol-Zenker-osmic acid reveals the presence of osmiophilic lipid droplets in the cells of the zona fasciculata and
zona reticularis (fig. 5 ) . These droplets agree in number and
distribution with the vacuoles observed in Bouin and Begaudfixed glands.
Alkaline phosphatase activity of the zona fasciculata and
z m a reticularis is not altered significantly by acute starvation. ITsing naphthyl-A S acetate t o demonstrate esteraw
activity, areas of increased enzyme activity are present occasionally in the outer portion of the zona fasciculata of
some adrenals.
~
absent
present
absent
Birefringence
Osmiophilio lipid
Alkaline phosphatase
intense activity
absent
Cholesterol
lightly stained
Aldehydic material
I
110
significant rhange
increased markedly
absent
absent
moderate activity
moderate activity
absent
absent
absent
no significant change
present in many cells
absent
absent
no significant change
nioderetc t o intense
increased in size
no significant change
granular,
intensely stained
decreased in total amount
of chromophilic material
absent
more darkly
stained
small, granular,
lightly stained
perinuclear
slight decrease
Starved
increased,
maxima a t 40 and 55 p2
ZONA FASOICULATA
absent
dispersed,
granular
juxtanuclear,
c.ap-like
at 35pe
maximum
conceiitration
-
Control
absent
decreased,
maxirns at, 20
and 30p'
maximum
concentrations
at 30 and 4 0 p ?
Starved
decreased
absent
Esterases
ZONA GLOMIRPULOSA
-
-
Control
Sudanophilic lipid
Mitochondria
Golgi apparatus
N u ~ l e n rsizr
Cell size
CONSTITUENT
TABLE 2
Summary of the major histological and histochemical @dings i n adrenocortical cells of normal and acutely starved hamsters
568
KARL M. KNIGGE
DISCUSSIOS
Several of the cytological changes observed in the cortical
cells of starved hamsters indicate that the adrenals may be
in a state of increased secretory activity. Although the cells
of the zona fasciculata decrease in size, their nuclei enlarge,
mitochondria hypertrophy and the Golgi apparatus becomes
vesicular and dispersed. It has been shown that changes in
nuclear size may be an indicator of the secretory status of
cortical cells. Hypertrophy of the adrenals of the guinea pig,
produced by diphtheria toxin, is accompanied by an increase
in nuclear volume (Boguth et al., '51). Conversely, hypophysectomy of the guinea pig or dog results in adrenal atrophy
and a reduction in volume of nuclei in cells of the zona fasciculata (Boguth et al., '51; Barkay, '42). Atrophy of the
rat's adrenals, as a consequence of the administration of
desoxycorticosterone acetate, is likewise accompanied by a
reduction in volume of nuclei in cells of the zona fasciculata
(Odenthal, '52). Mitochondria are considered to be indicators
of adrenal activity; they decrease in size and number in
cortical cells after hypophysectomy (Deane and Greep, '46)
and undergo hypertrophy during conditions which produce a
need f o r increased amounts of cortical hormones. The Golgi
apparatus is considered by many cytologists to be concerned
intimately with the synthesis and elaboration of secretory
products (Bowen, '29 ; Hirsch, '39 ; Worley, '46). According
to these authors, the Golgi apparatus consists of a portion
which is osmiophilic and argentophilic and a chromophobic
portion, or Golgi internum, represented by clear vesicular
areas such as those seen in the Golgi bodies of cells of the
zona fasciculata of the hamster's adrenals. In several types
of glandular cells it has been suggested that the clear vesiclc
or Golgi internum represents the secretory product of the
cell (Gatenby, '31; Worley, '46). Acute starvation of the
hamster results in an increase in the number and size of these
vesicles in the Golgi bodies of the cells of the zona fasciculata.
This may reflect a more rapid synthesis of the product secreted by these cells. It is to be noted, however, that the total
STARVATION A N D ADRENAL CORTEX
569
amount of Golgi material decreased in the zona fascicuiata
cells during starvation. This contrasts with the behavior of
the Golgi apparatus of the adrenals of other species and of
other organs as well, where hypertrophy or increase in the
total mass of chromophilic Golgi material is reported to ensue
after stimulation (Severinghaus, '33 ; Reese and Moon, '38 ;
Kirkman and Severinghaus, '38).
I n spite of the cytological indications of increased secretory
activity, the adrenals of acutely starved hamsters weigh less
than those of littermate controls. Acute starvation of the
guinea pig results in an increase in absolute weight of the
adrenals (D'Angelo et al., '48a), whereas the rat's adrenals
do not alter significantly in weight (Mulinos et al., '42; Ludewig and Chanutin, '46; D'Angelo et al., '48b). I n the case
of the hamster, it appears likely that acute starvation represents a stressful condition in which the adrenals become
hyperactive but are unable to maintain or increase their mass
in the face of complete denial of nutriment to the animal.
After acute starvation of the hamster, the zona glomerulosa
exhibits a marked increase in the number of osmiophilic lipid
droplets, hypertrophy of the mitochondria and a dispersed,
granular type of Golgi apparatus. The size of the glomerulosal cells is decreased as well as the cross-sectional area of
their nuclei. The capsule of the adrenal may exhibit areas
of proliferating cells, although this is not a constant observation in the adrenals of all starved hamsters. Hypertrophy
of the mitochondria of glomerulosal cells indicates that they
may be metabolically more active than normal, and in this
respect they differ in their response from the glomerulosal
cells of the rat's adrenals which are reported to become distinctly atrophic following acute starvation (D'Angelo et al.,
'48a). Although mitotic figures are seen infrequently in the
adrenals of either normal or starved hamsters, dispersion of
the Golgi apparatus as granules is considered to occur during
cell division (Kirkman and Severinghaus, '38 ; De Robertis
et al., '48; Dalton, '51). The decrease in cell and nuclear
size may also be a reflection of an increased rate of cell
570
KARL M . XNIGGE
division. The areas of proliferating capsular connective tissue cells, produced by such a relatively moderate stressor
as acute starvation, indicates that, as in the rat, this layer
may serve as a source of new parenchymal cells. I n the rat,
a more intense stimulation, produced by overdosage with
corticotropin, causes a marked proliferation and metamorphosis of capsular connective tissue cells into glandular cells
(Baker, '52).
Of particular interest are the osmiophilic lipid droplets
which are normally present in the cells of the zona glomerulosa and which increase greatly in number in this zone and
also accumulate in cells of the remainder of the cortex during
acute starvation. When adrenals are fixed in 10% formalin,
these lipid droplets pass into solution, probably as a colloidal
phenomenon. They are intensely and rapidly blackened by
the osmic acid of certain fixing fluids ; they are not preserved
in fixatives such as Bouin's fluid which are protein precipitants. These and other properties have led to the suggestion
that the material contained in these droplets is a non-protein
bound phospholipid (Knigge, '54). The accumulation of these
lipid droplets in all cortical cells during acute starvation may
indicate that they contain material which serves as a precursor
of the hormones secreted by the hamster's adrenals. I n the
rat, where cholesterol and its esters are thought to be hormonal precursors, an accumulation of these lipids occurs
during the latter stages of acute starvation and other stressful conditions (Sayers, '50). Sudanophilic lipid and cholesterol are present in the adrenals of hamsters infected with
Leishmania donovani (Leathem and Stauber, '52) and hamsters bearing transplants of mouse sarcoma 180 (Agate, '52).
These lipids, however, do not appear in the hamster's adrenals
following treatment with corticotropin (Alpert, '50), and in
view of the fact that they do not appear after starvation,
it seems probable, as Leathem and Stauber ('52) have suggested, that the presence of sudanophilic lipid and cholesterol
marks an exhaustion of the adrenals and is associated with
degenerative processes in the cortical parenchymal cells.
STARVATION AND ADRENAL CORTEX
571
Other intracellular structures which are of interest are the
liposomes. These spheroidal bodies exhibit staining properties identical to those of mitochondria and are differentiated
from them only on the basis of their greater size. Although
the physiological role of these bodies is not fully understood,
several lines of evidence can be presented which suggest that
liposomes may represent transformed or altered mitochondria.
The observation that mitochondria may alter their morphological appearance has been considered by several cytologists
(Bourne, ’51; De Robertis et al., ,48), although the significance of these changes is not fully understood. Severinghaus
(’33) reported a swelling and enlargement of the mitochondria in thyroid follicle cells of the duck after stimulation by
thyrotropin; in this case, the mitochondria “become sufficiently large to resemble in all respects stainable colloid
droplets. ” Sluiter and van Oordt ( ’46) have described cell
types in the interstitial cells of the testis in which the mitochondria are enlarged and indistinguishable from other “fuchsinophilic granules. ” Bodies which are morphologically similar to the liposomes of the hamster have been described in
the rat (Deane and Greep, ’46) and mouse (Miller, ’50). I n
the rat, Miller (’53) recognizes these large organelles as
atypical lipid inclusions and considers them to be mitochondria. Some of the observations which suggest a relationship
between mitochondria and liposomes in the hamster adrenal
are: (A) Liposomes are not present in cells of the zona glomerulosa. Mitochondria here are small and stain less intensely
than do those of the zona fasciculata. (B) I n the normal
hamster adrenal, the number of liposomes per cell in the zona
fasciculata and zona reticularis increases progressively as
the cortico-medullary border is approached ; this is accompanied by a correspondingly progressive reduction in the
number of discreet mitochondria. Since mitochondria may
reflect thc secretory status of adrenocortical cells, this observation supports the belief of a secretory cycle in which
cells move centripetally through a phase of active secretion
in the zona fasciculata to a zone of senescence and degenera-
572
KARL M. KNIGGE
tion at the cortico-medullary border. Belief in this hypothesis, however, is not shared by all investigators (Greep and
Deane, '49). (C) Hypophysectomy of the hamster results
in an increase in the number and size of liposomes in cells of
the zona fasciculata, accompanied by a decrease, and in some
cells, a complete disappearance of all recognizable mitochondria (Knigge, '54). (D) Acute starvation of the hamster
results in an increase in size of mitochondria, thus making
the differentiation between these bodies and liposomcs difficult in many cells.
SUMMARY
Young adult male hamsters were subjected to acute starvation until they lost an average of 36% of their initial body
weight. The adrenals undergo a significant decrease in absolute weight although their gross appearance is not altered
appreciably.
The cells of the zona glomerulosa become smaller and osmiophilic lipid droplets accumulate markedly in their cytoplasm.
The mitochondria of these cells enlarge and stain more intensely than normal ; the Golgi apparatus becomes granular
and dispersed. The cross-sectional area of glomerulosal cell
nuclei decreases. These changes are considered to reflect an
increased activity of the glomerulosal cells and a more rapid
rate of cell devision.
Cells of the zona fasciculata appear smaller than normal
after acute starvation, although the cross-sectional area of
their nuclei becomes larger. Osmiophilic lipid droplets, normally not present in cells of this zone, become especially
numerous in the mid-portion of the zona fasciculata. Mitochondria hypertrophy and the Golgi apparatus becomes more
vesicular although the total amount of chromophilic Golgi
material is reduced somewhat. These cytological changes may
reflect an increased secretory activity, with material contained in the osmiophilic lipid droplets serving as hormonal
precursors.
Sudanophilic lipid, cholesterol or birefringent material do
not appear in the hamster adrenal following acute starvation.
STARVATION A R D A D R E N A L CORTEX.
573
Also, the staining with aldehyde reagents (leucofuchsin and
2-hydroxy-3-naphthoic acid hydrazide) and the activity of
several enzymes (alkaline phosphatase and esterase) do not
alter significantly in the adrenals of starved hamsters.
ACKNOW LEDGMENT
The author wishes to thank Dr. Burton L. Baker of the
Department of Anatomy, University of Michigan, for making
available the facilities of his laboratory where portions of
this work were done.
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ALPERT,M. 1950 Observations on the histophysiology of the adrenal gland of
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BOGUTH,W., H. LANGENWRFF
AND E. TONUTTI 1951 Zellkerngrosse als Indikator der Funktionsbesiehung Hypophyse-Neveniiierenrinde. Physiologische Anpassungsreaktion und toxische L;ision des Rindenorgans durch
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G. 1949 The mammalian adrenal gland. Oxford, Clarendon Press.
1951 Cytology and cell physiology. Oxford, Clarendon Press.
DALTON,
A. J. 1951 Cytoplasmic changes during cell division with reference
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D’AHGELO,S. A., A. s. GORDONAND H. A. CHAMPPER
1948a The effect of
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KARL M. K N I G G E
DE ROBERTIS,
E. D. P., W. W. NOWINSKI
AND F. A. SALZ 1948 General cytology.
W. B. Saunders Company, Philadelphia.
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1952 Microscopic histochemistry. Unii-ersity of Chicago Press.
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1938 A review of the Golgi apparatus.
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J. H., AND L. A. STAUBER1952 Adrenal lipid and alkaline phosphatase in hamsters infected with Leishmania donovani. Endocrinology,
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STARVA‘PIOR’ A N D ADRENAL CORTEX
575
SAYERS,
G . 1950 The adrriial cortex mid homeostasis. Physiol. Rev., 30:
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A. 1924 Eine Methode des mikrochemischcn Cholcstrinnachweises a m
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A. E. 1933 Cytological observatioils on secretion iii normal and
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J. W., AND G. J. VAR OORIYP 1946 Experimental d a t a on cytology and
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PLATE 3
EXPLANATION OF FIGURES
Adrenals for figures 4 to 6 are sectioned a t 4 and stained by the Masson technique, those f o r figures 4 and 5 being fixed in formol-Zenker-osmic acid and that
for figure 6 in Bouin's fluid. Adrenals fixed in 80% alcohol and sectioned a t 5 a
are used for the demonstration of alkaline phosphatase (fig. 7) and f o r esterase
activity (fig. 8). I n figures 7 and 8, the cortico-medullary boundary is a t the
lower border of the photograph.
4 Zona glomerulosa and outer fasciculata, control male. Osmiophilie droplets
are present in the zona glomerulosa but are absent from the remainder of
the cortex. X 600.
5 Zona glomerulosa and outer fasciculata, starved male. Cells of the zona
glomerulosa are in clusters which are smaller than in figure 5. Glomerulosal
cells exhibit a marked accumulation of osmiophilic droplets which are situated
in that portion of the cell bordering a sinusoid. Osmiophilic droplets are
present also in the cells of the zona fasciculata. X 600.
6
Zona glomerulosa and outer fasciculata of a starved male, showing an area
of proliferating capsular connective tissue cells. The more lightly staining
glomerulosal cells are irregularly arranged with some of them in cords which
are continuous with those of the zona fasciculata. X 600.
7
Section of a control adrenal, incubated with yeast nncleic acid for one hour
a t p H 9.2. Alkaline phosphatase activity is present in the capsule, zona
fascieulata and zoua reticularis. The zona glomerulosa is negative. X 250.
8
Section of a control adrenal, incubated with naphthyl-AS acetate for one
hour at pH 7.5. Esterase activity is intense in capsular nodules and in the
zona glomerulosa whereas the zona fasciculata and zona reticularis exhibit
moderate activity. The capsule and all nuclei are negative. x 250.
576
TARYATION AXD ADRENAL CORTEX
K A R L 31. KNJGGR
'I'LATF, 1
PLATE 2
R X P L l N - 4 T I O N OF’ FIGURES
Adre~ialsfor figures 9 t o 14 are f i ~ din eadriiiiim-eliloride forrri:iIin, prepared
by the Aoy:inia method f o r the demonstration of the Golgi apparatus and sectioned
st, 4 p. Adremls f o r figures 15 a i d 16 are fixed i n Regaud’s fluid, sectioned a t 3 E”
and st:iincd with iron Ecmatoxylin t o drmonstrate mitochondria.
9
Zona glonierulosa, control male. The Golgi apparatus is a compact, juxta
nuclear cap without rai1i:il cxtrnsions. Suclci a r e unstained and 1 acnoles
are presriit i n some erlls. x 1200.
10 Zona glomerulosa, starled m:ik. Many cells slrow :I dispers:il of tlic Golgi
apparatus i n the f o r m of granules which lie most frequently ncar the nuclear
mcmbrane. Other cells contain, in addition t o the granules, juxtanuclear caps
wliieh are sm:iller t h a n norni:rl. V a e ~ o l c sarc more nurnwous than in figure
9 and litive co:ileseed i n niany plac~ls. X 1200.
1.1. Uolgi :ippcir:itus, fascicnlatn crlls of a control mile. Golgi hodics are distrjbutcd i n ii peiiiiuclcni pattein. Individual Golgi bodies contain srn:ill, i f
a n y , clironiophobic centn b. X 1500.
12
Clolgi apparatus, f
icwl:itx cell of :I control m:ilc. Tlic Golgi matcrinl i b
closely applied to tlw iiuclear r n v i n l ) i : i i w . Tlit. :irrow indirates a groiip of
wcuoliir-like spaces, nliicli arc infrcquently seen in the riorinal g l m d :ind
niay be the ctiromophohic component of thc Golgi apparatus. X 1500.
13
(lolgi apparatus, fascicul:itn cclla of x st:irued m:ilc. The t o t n l amount of
nrg(wtop1iilie Golgi matc,ri:il is less than that in tlic ~iorm;rIgland (figs. 11
:iiiil 1 2 ) . The Golgi m:iterial which is dispersed i n the cptopltism is not
rc%l:ited positionally to the vacnoles which arenmulate iii ninny cells. X 1500.
1.4 Crolgi npliaratus, fascicnlata crlls of ii st:irved male. Tho viicuolar-like clear
sl):iccs ill tlic Golgi liodies :irc 1:irgcr and more numcrous than in control
glands. The total nniormt of Golgi m:iteri:il j s d s o diminished. X 1500.
1.5 Outer portion of the zon:~f:iscicnl:it:1. control ninlc. The eells :we filled nitli
granular and short roil-like niitocliondri:~ mid some liposomes (1,). The
neg:itiw image of the Golgi appar:itiis i s intlicatcd at (1. x 1900.
1 (i Outer portion of tlic zon:i f:isciculatn, starred male. Mitochondria are enliirgcd :ind onc wll iii this field exhibits n ~nnrlted cytop1:~niriic r:ieuolation
nhilc otlirr wlls Iia\ c ft>nvi \ acnoles. X 1900.
578
STARVATION AND ADRENAL CORTEX
KARL M. XNIQQE
PLATE 2
.2dreuals for figuiw 1 7 :ind 18 a l e flyer1 in Reg:iud’s fluid, sectioned at R fi
:uid stained with iron 1iem:ctosylin. Tliosc f o r figiircs 19 and 20 arc fixed in
10% fornrnliii, sc~ctioiiedo i i tlic f reczing ~ n i c r o t o ~ nand
c stained with leiicofuchsin
(fig. 19) :ilicl ? - l i y d r o s ~ ~ - : ~ - i i : i ~ ~ lacid
i t l i o Iiyclrnxitle
i~~
(fig. 20).
17 Zoiiii gloiiicrulosa and outer fascieulatn, control malc. Yitochoiidria of the
m i i n gloinclulosa iit:iiii lightly, v:iii~iiig :I slinip ilrlinention ljetxycen this z011t:
:ind the zoiiii fascivuhtn irliicli st;iins iiitc.iisrlp. X 700.
18 Zoim glonrcrulosa and o a t w f : i s c i d : i t n , st:Irved male. Crll elustrrs in the
zoiin g1oiueriilosa are smaller t h a n iioriii:il, lint the m i l e as a ~ v l i o l c stains
darker tlian i n figurc 17. C:-toplasinic rneuolatioii is increased markedly.
X 700.
1.9
(‘ontrtrl inalc. IAcwrwfuchsin,witlioiit prior treatment mith mercorie clrloridc,
staiiis the zoiia f:cscicul:it:i :ind zonii rrticnl:1ris biit the zonn glomcrulosa is
faint or iiegntilr. X 180.
20
(’oiitrol mnlc. Tlie ~lslilrel-Seligm:rnrwc-tiou i s intcllse in tlic zona f : i s c i d a t a
ant1 zoii:i reticiilnris. The zoiia gloiiivni1oq:i is g c i i ~ ~ ~ :neg:iti\
i I l ~ c., hiit soine
gloiiirrulo~nlcell5 wliicli lie iiiiinc~1i:itcl~
1~c~iic:itli
the cnlisiilc rnay 1)r st:iincd
also.
x
180.
580
PLATE 3
TARVATION AND ADRENAL CORTEX
KARIr X. KNIGGE
581
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