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Pituitary basophils of the Syrian HamsterAn electron microscopic investigation.

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Pituitary Basophils of the Syrian Hamster:
An electron microscopic investigation '
ANDREW DEKKER
Department of Pathology, University of Pittsburgh School o f Medicine,
Pittsburgh, Pennsyhania
ABSTRACT
Histochemical studies have not clearly defined the types or distribution of basophils in the hamster's adenohypophysis. Electron microscopy is used in
these experiments as a more accurate method for identifying types of basophils.
Electron microscopic fields from regional areas of the gland were systematically
photographed, and cell types counted. Hypertrophy of the appropriate basophils was
observed in response to gonadectomy and thyroidectomy. FSH cells are oval, are
adjacent to vascular spaces, have vesiculated endoplasmic reticulum and contain
secretory granules measuring 200 mp maximally. ICSH cells have filigreed cytoplasm,
irregular cell borders, and secretory granules measuring 200 mp maximally. TSH cells
are polyhedral, have sparse endoplasmic reticulum, and secretory granules measuring
100 mp maximally. The three basophilic cell types are randomly distributed throughout the gland.
Histochemical investigation of the m a malian adenohypophysis in conjunction
with appropriate physiologic alteration of
the animal has clearly defined three
basophilic cell types in the rat (Purves
and Griesbach, '54), bat (Herlant, '56) and
dog (Purves and Griesbach, '57). These
usually include two gonadotropes (ICSH
and FSH) and one thyrotrope (TSH)
(Purves, '61 ). Electron microscopic studies have confirmed the presence of these
three basophilic cell types in the rat (Farquhar and Ftmehart, '54a, b) and bat (Herlant, '64).
Histochemical methods have been less
conclusive in defining specific cell types
in the Syrian hamster. Knigge ('57),
using the periodic acid-Schiff (PAS) and
aldehyde fuchsin stains (Halmi, '52),
failed to obtain consistent and precise differentiation of basophils. Serber ('58) and
Thompson ('60), using similar histochemical methods, defined two and three
cell types, respectively. The results of
these two authors also differed in the
staining qualities and the distribution of
the cell types within the gland.
In this study, evidence will be presented to indicate that, after appropriate
endocrine ablation, three basophilic cell
types can be identified by electron microscopy in the hamster's adenohypophysis
and that they are randomly distributed in
the gland.
ANAT. REC., 158: 351-368.
MATERIALS AND METHODS
Animals. Adult male and female Syrian
hamsters (Mesocm'cetus auTatus) were obtained from a local supplier and maintained on Wayne Lab-Blox and tap water
ad libitum. Intact, gonadectomized and
thyroidectomized animals were used in
the study. A thyroid deficient state was
produced by injection of 1.0 mC of Pi
intraperitoneally. The IiS1-treatedanimals
had negligible uptake of 113' tracer doses
and multiple sections through the thyroid
region at autopsy showed necrosis and inflammatory infiltration progressing to fibrosis, changes similar to those described by
Goldberg, Chaikoff, Lindsay, and Feller
('50) in rats treated with Fa'.
Six animals were used in the study of
cell distribution. This group was cornprised of two intact, two 10-day postgonadectorny and two ?-day post-thyroidectomy animals. In addition, the
pituitaries of 36 other intact, gonadectomized and thyroidectomized hamsters
were examined with regard to cell morphology. These glands were obtained at
10, 20, 30, 60 and 90 days after gonadectomy or thyroidectomy.
For comparative purposes, pituitaries
from intact, gonadectornized or thyroidectomized rats were examined with the
electron microscope.
~
'Results were presented in part before the American Society for Experimental Pathology, Atlantic
City, New Jersey, April 14, 1966.
351
352
ANDREW DEKKER
Electron microscqq. The hamsters
were anesthetized with Nembutal and
exsanguinated. After removal of the brain,
the fibrous tissue surrounding the pituitary
was cut with iris scissors. The intermediate and posterior lobes were separated and
the isolated anterior lobe was removed.
The total time for this operation was approximately three minutes.
The anterior pituitary was suspended
in 0.1 M phosphate-buffered 2% glutaraldehyde solution for ten minutes, following which it was rapidly rinsed three
times in 0.1 M phosphate-buffered 0.2
M sucrose (Sabatini, Bensch, and Barrnett,
' 6 3 ) . For studying cell distribution, half
of the gland was divided by two frontal
cuts into approximately equal anterior,
middle and posterior segments. Pituitaries from other hamsters were not divided into segments but were otherwise
processed similarly.
The pituitary zonal fragments were immersed in a drop of phosphate-buffered
1% osmium tetroxide and minced into
1.0 mm3 cubes. Following fixation for one
hour in osmium tetroxide, they were dehydrated in graded alcohols and propylene
oxide and embedded in Epon 812 (Luft,
'61). Ultrathin sections were mounted on
uncoated copper grids and stained with
2% uranyl acetate.
In the studies on basophil distribution,
sections of all blocks from half the pituitary of each of the six hamsters were
scanned with the electron microscope. On
the average, ten electron microscopic fields
were photographed of each of the blocks
at a standard magnification and the cells
were identified and tabulated. Electron
photomicrographs were also made of the
pituitary cells of these and other animals
at higher magnifications for morphologic
study.
Light microscopy. One micron sections
were cut from all of the Epon-embedded
pituitary blocks, stained with toluidine
blue, and examined by light microscopy.
Several hamster pituitaries were also fixed
in 10% formalin solution, embedded in
paraffin and stained with hematoxylin and
eosin or periodic acid-Schiff stains.
Nomenclature. The nomenclature used
in this paper is that recommended by an
international committee (Van Oordt, '65),
in which the two gonadotropic cells are
designated ICSH and FSH cells and the
thyrotropic cell a TSH cell.
RESULTS
Three basophilic cell types can be readily distinguished in the hamstefs adenohypophysis by electron microscopy. The
criteria for their identification and the frequency of their occurrence are shown in
table 1. The FSH cell is by far the most
numerous basophilic cell type in both the
male and female hamster's adenohypophysis. A description of the fme structure of
the three cell types follows.
FSH ceEZ. The FSH cell in the normal
hamster is approximately 10 I. in diameter,
oval to round in shape and always in association with vascular spaces. The nucleus is usually eccentrically located,
round and contains fmely scattered chromatin with one or more nucleoli. Cytoplasmic secretory granules of varying
density, measuring 200 mv maximally,
are distributed regularly throughout the
cell. These are decreased in number in
actively functioning cells (see fig. 10).
The mitochondria are generally elongated
and the Golgi complex is usually inconspicuous. Electron dense bodies larger
than secretory granules are often seen in
the cytoplasm of this cell t y p . These
most likely represent lysosomes.
TABLE 1
Distinguishing characteristics and frequency of pituitary basophils of normal hamsters
Cell
type
FSHC
Shape
oval
ICSHC
irregular
TSHC
polygonal
ReIation to
vascular
space
Maximal
granule
size
+
mp
-
Character Of
endoplasmlc
reticulum
Frequency
Male
Female
% total cells
200
vesiculated
200
100
48
24
filigreed
9
1
sparse
2
1
353
HAMSTER PITUITARY BASOPHILS
In pituitaries from the castrated male
and female, the FSH cell is hypertrophied
to approximately twice its normal size.
This occurs as early as ten days following
castration. The proportion of cells showing the changes seen in active secretion
(fig. 10) is markedly increased, nonsecretory cell types being encountered
rarely .
FSH cells are not altered in thyroidectomized animals.
ICSH ceZE. The ICSH cell in normal
hamster pituitaries measures up to 15 u
in diameter, is irregular in shape, and is
not located in association with vascular
spaces. The peculiar, filigreed pattern of
its endoplasmic reticulum is distinctive.
The nucleus is irregular, smaller than
that of the FSH cell, and has clumped
chromatin. Multiple nuclei are occasionally seen (see fig. 9). The secretory granules are 200 mu maximally, of equal density and irregularly distributed throughout. The mitochondria are round and have
light matrices. Free and attached ribosomes are present and the Golgi complex
is not prominent.
The pituitaries from castrated animals
show hypertrophy of ICSH cells with accentuation of the filigreed cytoplasmic
pattern, giving a highly bizarre appearance (fig. 11). In many cells, multiple
nuclei are present, resulting in a syncytial appearance. The number of secretory granules is decreased. These morphologic changes require at least twenty
days to appear and hence are seen later
than the changes in the FSH cells after
castration.
No alteration of this cell type is seen
following thyroidectomy.
T S H cell. The TSH cell is polygonal,
has sparse endoplasmic reticulum and
small secretory granules which are 100
mu maximally and regularly distributed.
No relationship of this cell to vascular
spaces is obvious. The nucleus is round,
has a fine chromatin pattern and a single
nucleolus. The mitochondria are round,
smaller than those of the FSH cell, and
have a darker matrix.
In the thyroidectomized animal, the
cell remains polygonal despite marked hypertrophy (see fig. 12). The endoplasmic
reticulum is increased but is not vesiculated or as prominent as that of the FSH
cell.
The TSH cell is not altered in castrated
animals.
Distribution of cells. In table 2 the
results of the pituitary cell counts are
given. The frequency with which these
basophilic cell types appear in the three
regional zones of the adenohypophysis
point to a uniform distribution pattern.
This was confirmed by statistical analysis
of these data.
Light microscopic studies. F S H and
ICSH cells can be identified in 1 LI toluidine
blue-stained sections of normal and castrated hamster pituitaries when their appearance is compared with that seen by
electron microscopy. However, the TSH
cell could not be identified with certainty
in normal pituitaries, although they are
obvious in thyroidectomized animals because of the marked hypertrophy (fig. 3,
7). All three types are seen in blocks from
the three zones. These sections afford examination of large areas of the gland and
confirm a generalized cellular distribution.
TABLE 2
Relative % basophils in pituitary zones of intact, gonadectomized, and thyroidectomized hamsters
Sex
Zone
Intact ( C? 90; p 98) 1
FSHC
d
0
1 Figures
Gonadectomy (10 days)
(0” 133; 9 109)
Thyroidectomy (7days)
(d 270; 561)
FSHC
ICSHC
ICSHC
TSHC
FSHC
4
0
11
96
80
95
Anterior
Middle
Posterior
96
0
76
74
24
15
Anterior
Middle
Posterior
92
93
85
0
4
11
ICSHC TSHC
TSHC
4
0
13
0
5
61
57
82
26
20
0
25
18
2
16
3
6
20
28
21
6
8
96
4
0
69
4
4
85
52
13
2
4
73
44
in parenthesis represent total basophil count per animal.
74
354
ANDREW DEKKER
resemblance of the gonadotropes of these
two species is used as a basis for subclassification of these cell types. It is realized
that this similarity in structure does not
indicate functional identity,
Also a marked morphological difference
between the hamster and rat TSH cells is
noted. The sparse non-vesiculated endoplasmic reticulum and the absence of SOcalled “round bodies” in the hamster TSH
cell, is in marked contrast to the rat cell,
in which these structures are prominent
(Farquhar and Rinehart, ’54b). This difference is of interest since a suggestion
of a chemical difference between these
cells has been reported. Serber (’58) with
the aldehyde fuchsin stain found a reversed staining affinity of the thyrotropic
cells in these two species.
The close correlation between Serber’s
total basophil count in the hamster pituitary obtained with histochemical methods
and this study is shown in table 3. This
agreement is not entirely unexpected,
since the periodic acid-SchiE stain conDISCUSSION
sistently differentiates basophils from
Until recently, electron microscopy of acidophils (Purves, ’61). Unfortunately,
the mammalian pituitary was considered the usual histochemical stains useful in
primarily a tool in the study of the secre- subclassification of basophils in other
tory cycle of protein-producing cells species, are not so in the hamster (Serber,
(Farquhar and Wellings, ’57; Farquhar, ’58; Thompson, ’60).
The results listed in table 3 also con’61). This study has shown that electron
microscopy can also be used for identify- firm the “basophilia” of the male hamster’s
ing individual basophilic cell types in 3 pituitary. This differs from the rat where
species such as the Syrian hamster, in the basophils are in the minority in both
which histochemical methods have not sexes (Serber, ’58).
To date, electron microscopic techniques
been decisive (Serber, ’58; Thompson,
’60). Moreover, the finding of two gonado- have, understandably, not allowed a thortropic and one thyrotropic cell types in the ough analysis of the spatial relationship
hamster’s adenohypophysis is of interest of the cell types within the pituitary gland.
and is consistent with the findings in other An attempt, however, at such an analrodents (Farquhar and Rinehart, ’54b; ysis using microdissection techniques has
recently appeared (Rennels, ’63). The
Barnes. ’62).
Although the cell types identified in
TABLE 3
this study are specifically labelled, their
functional specificity is not considered Comparison of cell counts i n intact hamsters,
using light (L.M.) and electron
fully proven. Further studies to elucidate
microscopy ( E . M . )
the functional specificity of these cell
types are required.
Basophils, frequency
NO.
Method
animals
The morphological resemblance of the
Male
Female
used
hamster gonadotropes to those of the rat
% total % total
is striking (Farquhar and Rinehart, ’54a).
L.M.
59
25
17
Also the slow response of the ICSH cell to (Serber, ’ 5 8 )
castration is similar in these rodents
E.M.
59
26
2
(Farquhar and Rinehart, ’54a). The close
Conventional light microscopic pituitary
preparations stained with hematoxylin
and eosin and periodic acid-Schiff stains
are not useful in identifying basophilic
cell types (fig. 2). PAS-positive cells, however, are generally distributed throughout
the gland.
Electron microscopy of the rat pituita?y.
The main findings of Farquhar and
Rinehart (’54a, b ) concerning the rat pituitary basophils are confirmed. FSH and
ICSH cells of the rat and hamster show
a marked similarity in most of their features. This includes the delay of the ICSH
cells of these two rodent species to react
to castration. However, the TSH cell of
the rat, in comparison to the hamster, is
different in structure. The endoplasmic
reticulum of the rat TSH cell is more well
defined and vesiculated. Also, “round
bodies” (Farquhar and Rinehart, ’54b)
present in the endoplasmic reticulum
vesicles of the rat are not seen in the
hamster TSH cells.
HAMSTER PITUITARY BASOPHILS
method devised in this present study of
zonal analsyis has proved fruitful. The
generalized distribution of the three basophilic cell types in the hamster pituitary
gland is an interesting contrast to the
localized distribution in the rat (Purves
and Griesbach, '54; Purves, '61), which is
graphically represented in figure 1. This
generalized distribution of at least some
of the basophils in the hamster was suspected by Thompson ('60) for he showed
the aldehyde fuchsin-positive cells to be
generally distributed.
Although the model of the rat pituitary
shown in figure 1 seems to be generally
accepted (Farquhar and Rinehart, '54a;
Serber, '58; Thompson, '60), there is no
unanimity either as to distribution of the
cells in the gland or their function (Barnnett, et al., '56; Rennels, '63; Herlant, '64).
HAMSTER PITUITARY
HORIZONTAL VIEW
In other mammalian species such as
the bat (Herlant, '64), there is a more
generalized distribution of the pituitary
cell types. The hamster represents another
example of such distribution of pituitary
cells.
ACKNOWLEDGEMENTS
The author is grateful for the help of
Dr. Wang Yen in handling radioactive materials, Dr. L. J. Gerende in performing
the statistical analysis of the data included in this study, and Dr. R. Mark for
constructive criticism of the manuscript.
The technical assistance of Miss S.
Gonsowski and G. Morici is gratefully
acknowledged.
LITERATURE CITED
Barnes, B. G . 1962 Electron microscopic
studies on the secretory cytology of the mouse
anterior pituitary. Endocrinology, 71 : 618628.
Barrnett, R. J., A. J. Ladman, N. J. McAllaster
and E. R. Siperstein 1956 The localization
of glycoprotein hormones i n the anterior
pituitary gland of rats investigated by differential protein solubilities, histological stains
and bioassays. Endocrinology, 59: 398-418.
Farquhar, M. G. 1961 Origin and fate of
secretory granules in cells of the anterior pituitary gland. Trans. N. Y. Acad. Sci., 23:
346-351.
Farquhar, M. G . , and J. F. Rinehart 1954a
Electron microscopic studies of the anterior
pituitary gland of castrate rats. Endocrinology, 54: 516-541.
1954b Cytological alterations in the
anterior pituitary gland following thyroidectomy: a n electron microscopic study. Endocrinology, 55: 857-876.
Farquhar, M. G., and S . R. Wellings 1957
Electron
microscopic evidence suggesting
secretory granule formation within the Golgi
apparatus. J. Biophys. Biochem. Cytol., 3:
319-322.
Goldberg, R. C., I. L. Chaikoff, S. Lindsay and
D. D. Feller 1950 Histopathological changes
induced in the normal thyroid and other
tissues of the rat by internal radiation with
various doses of radioactive iodine. Endocrinology, 46: 72-90.
Halmi, N. S. 1952 Differentiation of two types
of basophils in the adenohypophys,is of the
rat and mouse. Stain Techn., 27: 61-64.
Herlant, M. 1956 Corrklations hypophysogenitales chez la femelle de la Chauve-Souris,
Myotis myotis (Borkhausen). Arch. Biol., 67:
89-180.
1964 The cells of the adenohypophysis
and their functional significance. In: International Review of Cytology. Academic Press,
New York, 17: 29S382.
-
RAT PITUITARY (AFTER PURVES)
HORIZONTAL VIEW
SHC
ICSHC
Fig. 1 Diagramatic representation of the
hamster and rat anterior pituitary contrasting
the generalized distribution of the cells in the
hamster with the regional distribution in the
rat (Purves, '61). The intermediate and posterior lobes are shown i n the diagram of the
rat pituitary.
355
356
ANDREW DEKKER
Knigge, K. M. 1957 Influence of cold exposure upon the endocrine glands of the
hamster, with a n apparent dichotomy between
morphological and functional response of the
thyroid. Anat. Rec., 127: 75-95.
Luft, J. H. 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem.
Cytol., 9: 409-414.
Purves, H. D. 1961 Morphology of the hypophysis related to its function. In: Sex and
Internal Secretion. Young, W. C. and G. W.
Corner, eds. Williams and Wilkins, Baltimore,
Md. 3rd Ed., pp. 161-239.
Purves, H. D., and W. E. Griesbach 1954
The site of follicle stimulating and luteinizing
hormone production in the r a t pituitary. Endocrinology, 55: 785-793.
1957 A study on the cytology of the
adenohypophysis of the dog. J. Endocr., 14:
36 1-3 70.
Rennels, E. G . 1963 Gonadotrophic cells of the
rat hypophysis. In: Cytology of the Adeno-
hypophysis. Benoit, J., and C. Da Lage, eds.
Colloq. Internat. Centre Nat. Rech. Sci., (Paris),
128: 201-205.
Sabatini, D. D., K. Bensch and R. J. Barrnett
1963 Cytochemistry and electron microscopy:
the preservation of cellular ultrastructure and
enzymatic activity by aldehyde fixation. J.
Cell. Biol., 17: 19-58.
Serber, B. J. 1958 A cytological study of the
anterior pituitary gland of the normal, gonadectomized, and thyroid deficient hamster
(Mesocricetus auratus). Anat. Rec., 131:
173-192.
Thompson, R. J. 1960 Cytology of the hypophysis i n the adrenalectomized golden
hamster (Mesocricetus auratus). Am. J. Anat.,
106: 55-71.
Van Oordt, P. G . W. J. 1965 Nomenclature
of the hormone-producing
cells in the
adenohypophysis. Gen. Comp. Endocr., 5 :
131-134.
PLATE 1
EXPLANATION OF FIGURES
Figure 2 is a photomicrograph of paraffin-embedded hamster pituitary fixed in 10% formalin and stained with hematoxylin and eosin.
Figures 3-7 are photomicrographs of 1 p thick sections of Epon-embedded Syrian hamster pituitaries stained with toluidine blue. Figures 2-7
were photographed a t the same magnification. X 250.
Intact male. Note nondescript nature of cells.
Intact male. The vacuolated cells, one of which is indicated on the
left (arrow), are FSH cells. The polygonal cell with the clear
cytoplasm on the right (arrow) probably represents a TSH cell.
The predominance of basophils is apparent. VS, Vascular space.
Ten-day male castrate. The pale hypertrophied cells (arrow) are
FSH cells. All sections of this animal had the same appearance as
shown. This tends to confirm both the frequency and generalized
distribution of this cell type.
Ninety-day female castrate. The pale hypertrophied cells (arrow)
are FSH cells. They appear similar, but paler than those seen in
figure 4.
Intact male. Arrows indicate ICSH cells. Even a t this magnification
the filigreed pattern of cytoplasm and dark, irregular nuclei are seen.
One-week thyroidectomized female. Arrow indicates hypertrophied
TSH cell, Compare its size to TSH cell illustrated in figure 3. Note
that other cells are unaltered.
HAMSTER PITUITARY BASOPHILS
Andrew Dekker
PLATE 1
357
PLATE 2
EXPLANATION OF FIGURE
Figures 8-12 are electron photomicrographs of Syrian hamster pituitaries fixed in 2% glutaraldehyde and 1% osmium tetroxide buffered with
phosphate buffer and embedded in Epon. All sections are stained with 2%
uranyl acetate.
8 Intact male. A n oval FSH cell is contrasted with a polygonal TSH
cell. The larger secretory granules ( s g ) and varying density (arrows)
of the granules of the FSH cells are evident. Also these cells have
more prominent endoplasmic reticulum (er). Acidophils ( Ac) surround the FSH cells. A nucleus ( N ) of a non-granulated cell is indicated. Dense bodies (db) are present in both cell types. X 12,000.
358
HAMSTER PITUITARY BASOPHILS
Andrew Dekker
PLATE 2
359
PLATE 3
EXPLANATION O F FIGURE
9
360
Intact male. The ICSH cell is a large cell with irregularly scattered
granules ( s g ) and filigreed endoplasmic reticulum (er). The secretory granules (sg) measure 200 mp. Note the multiple nuclei ( N ) ,
which may be present in these cells, giving them a syncytial appearance. X 13,000.
HAMSTER PITUITARY BASOPHILS
Andrew Dekker
PLATE 3
361
PLATE 4
EXPLANATION O F FIGURE
10 Ten-day female castrate. A n FSH cell is adjacent to a vascular space
( V S ) . The cell is hypertrophied to approximately twice its normal
size and has vesicular endoplasmic reticulum ( e r ) and decreased
secretory granules ( s g ) . These are of usual sizc, but tend to be
more peripheral in the cell. In addition, bound and free ribosomes
are conspicuous. These changes are thought to be correlated with
increased cell function.
Also frequently seen in these hypertrophied cells, but not illustrated here, are nuclear irregularity and “myelin figures” in the
cytoplasm.
These phenomenon are accentuated in FSH cells of animals which
have been castrated for longer periods of timc.
Acidophilic cells (Ac) surround the FSH cell. A dense body (db)
is indicated. X 9500.
362
HAMSTER PITUITARY BASOPHILS
Andrew Dekker
PLATE 4
363
PLATE 5
EXPLANATION OR FIGURE
11 Ninety-day female castrate. Hypertrophied ICSH cells are present.
These cells respond more tardily to castration than the FSH cells.
The endoplasmic reticulum (er) is markedly dilated and attenuated
and there is marked nuclear irregularity (N). The secretory granules (sg) are arranged along the periphery of the cell. Acidophils
( A c ) are present which have larger secretory granules than the
basophils. A portion of a n FSH cell is indicated. G, Golgi complex
x 9000.
364
FUMSTER PITUITARY BASOPHILS
Andrew Dekker
PLATE 5
365
PLATE 6
EXPLANATION OF FIGURE
12
366
Seven-day thyroidectomized male. The TSH cell shown here is hypertrophied but retains its polygonal shape. The endoplasmic
reticulum ( e r ) is poorly developed. The mitochondria ( M ) are
smaller than those of other basophilic cell types as are the secretory
granules ( s g ) which measure 100 mp. A Golgi complex ( G ) is present. Acidophils (Ac) are indicated. X 11,000.
HAMSTER PITUITARY BASOPHILS
Andrew Dekker
PLATE 6
367
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