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Ultrastructural and immunocytochemical studies of prolactin-secreting cells (PRL cells) in the anterior pituitary gland of the female musk shrew (Suncus murinus L.)

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THE ANATOMICAL RECORD 220:415-423 (1988)
Ultrastructural and Immunocytochemical Studies of
Prolactin-Secreting Cells (PRL Cells) in the
Anterior Pituitary Gland of the Female Musk Shrew
(Suncus murinus L.)
TOSHIKO ISHIBASHI A N D MASATAKA SHIINO
Department of Anatomy, Wakayama Medical College, SKyubancho, Wakayama-shi, 640,
Japan
ABSTRACT
Prolactin-secreting cells (PRL cells) in the anterior pituitary gland
of the female musk shrew were identified by the protein A-gold procedure combined
with electron microscopy. Their secretory granules were spherical, and showed
various sizes ranging in diameter from 100 nm to 800 nm according to the difference
in physiological conditions of the animals.
In pregnant and lactating animals, the PRL cells exhibited morphologically active
features, i.e., a large prominent Golgi apparatus and a well-developed rough endoplasmic reticulum (RER) consisting of densely packed parallel lamellae. In pregnant
and estrogen-treated animals, the secretory granules of PRL cells significantly
increased in size as compared with those of virgin animals.
The most remarkable ultrastructural change observed in PRL cells of pregnant,
lactating, and estrogen-treated female musk shrews was the occurrence of “intracisternal” granules. They were small and spherical, and were located without limiting
membranes inside the dilated cisternae of the RER. Intracisternal granules, as well
as ordinary secretory granules, were found to be immunoreactive for PRL by the
protein A-gold procedure. These results suggest that PRL cells of the female musk
shrew may possibly utilize a “bypass” route to form hormone-containing granules
under highly activated cellular conditions.
Recently, the musk shrew became available as a new
laboratory animal. There is little information, however,
concerning the morphology of the anterior pituitary cells
of the musk shrew, except for a few light microscope
reports (Naik and Dominic, 1972, 1978). We have been
interested in studies on the anterior pituitary cells of
the musk shrew, since it appears to be a unique animal
that shows different cytological profiles than those of
other laboratory animals. We have recently demonstrated in somatotrophs of the musk shrew’s pituitary
gland the presence of rod-shaped secretory granules (Ishibashi and Shiino, 1988).
In the present study, we have concentrated on some
specific ultrastructural features of the secretory granules in PRL cells of the female musk shrew as seen in
different physiological states, such as during pregnancy
and lactation.
MATERIALS AND METHODS
Ten virgin female (1 month of age), three late-pregnancy, and four early-lactating musk shrews were employed in the present study. They were kept in airconditioned and light-controlled (0600-2000 hr) quarters, and fed Purina laboratory chow and tapwater ad
libitum. For estrogen treatment, the 10 virgin females
were divided into two groups: One group consisted of
seven animals, which received subcutaneous injections
0 1988 ALAN R. LISS, INC.
of 0-estradiol (E2, 20 pg dissolved in 0.05 ml of sesame
oil) once a day for 2 weeks and the other group consisted
of three animals, which served a s vehicle injected controls.
The animals were killed by decapitation after light
anesthesia with ether. The anterior pituitary glands
were removed from the sella turcica and cut in half.
Small pieces of the pituitary glands were fixed in a
solution consisting of 3% paraformaldehyde and 3%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hr
a t room temperature, and then postfixed in 1% osmium
tetroxide dissolved in 0.1 M cacodylate buffer for 1hr a t
0°C. After a routine procedure of dehydration, the tissues were embedded in Spurr’s plastic mixture. For general electron microscope observation, thin sections were
prepared and stained with uranyl acetate and lead citrate. For immunocytochemical observations with the
electron microscope, thin sections were reacted by the
protein A-gold method described by Bendayan and Zollinger (1983). Control immunocytochemical tests were
carried out by substituting normal rabbit serum or phosphate-buffered saline for the specific antiserum. Electron microscope observations were made with a Hitachi
H-300 electron microscope. Antiserum rat prolactin was
Received July 20, 1987; accepted October 9, 1987.
416
T. ISHIBASHI AND M. SHIINO
TABLE 1. The size of secretory granules in PRL cells of the
female musk shrew
Group
Virgin
Pregnant
Laciating
E2-injecteda
No. of
animals
Diameter of granules,
nm (means f SE)
3
3
313.0
467.2
341.0
382.0
4
7
& 22.7
& 30.4*
f 23.1
If: 21.6*
=E2:0-estradiol.
* P i0.01 as compared with values of the virgin animals.
a gift from Dr. A.F. Parlow a t the Pituitary Hormone
and Antisera Center, Los Angeles, and also from Dr.
Katsumi Wakabayashi a t the Gunma Institute of Endocrinology, Gunma, Japan. Protein A-gold complex was
obtained from Janssen Products, Belgium. Experimental animals used in the present study were kindly provided by Dr. M. Miyajima of the Experimental Animal
Center in our institute.
For measuring the size of secretory granules, 80-120
PRL cells were selected at random from tissue sections
of each group (virgin, late-pregnancy, early-lactating,
and Ez-injected), and photographs taken at a magnification of 5,000 were enlarged to ~10,000.The diameters
(the long axis) of 25 secretory granules from each cell
were measured on the photographs. Significant differences in granule diameters were determined by one-way
analysis of variance, followed by the least-significance
test.
RESULTS
Prolactin-secreting cells of the anterior pituitary gland
of the female musk shrew were identified by the protein
‘1
virgin
A-gold procedure combined with electron microscopy.
Their secretory granules were spherical, and showed
various sizes ranging in diameter from 100 nm to 800
nm, owing to the difference in physiological conditions
of the animals as summarized in Table 1. The distribution of granule size of the PRL cells in each physiological
state is shown in Figure 1.
In pregnant animals, PRL cells contained secretory
granules with the largest average diameter, as shown
in Table 1. Some of the PRL cells contained extremely
large, mature granules (maximum 800 nm), which were
fully distributed throughout the cytoplasm. Most of them
revealed a large, prominent Golgi region associated with
numerous immature granules and a well-developed
rough endoplasmic reticulum (RER) consisting of densely
packed parallel lamellae (Fig. 2). Extruded granular
material was frequently observed in the intercellular
spaces and, like the peripherally located secretory granules, they were found to be immunoreactive by the protein A-gold procedure (Fig. 3). Numerous cytoplasmic
processes forming complicated interdigitating structures filled the spaces between adjacent PRL cells (Fig.
4). Occasionally the cytoplasmic processes of several PRL
cells protruded into a large follicle in which irregularly
shaped dense materials were observed. These materials
were also seen to be immunoreactive by the protein Agold procedure.
In lactating animals, PRL cells exhibited a well-developed Golgi zone consisting of stacked cisternae, numerous vesicles, condensing vacuoles, and prominent whorls
of the RER (called Nebenkern) in the cytoplasm. Several
multivesicular bodies were often seen in the Golgi area.
Secretory granules of lactating animals were much
smaller than those of pregnant animals ( P < 0.01). In
general, morphologically enhanced PRL cells of lactat-
30
“I/
30
Pregnant
Lac t a t i n g
30
I
1
t
10
20
30
I0
50
80
10
10
20
30
Diameter (10 nm)
Fig. 1. Frequency distribution histogram of the diameters of prolactin secretory granules in different
physiological conditions of the female musk shrew.
Fig. 2. Typical PRL cells observed in a pregnant animal. Note the
Fig. 3. Immunoreactive PRL secretory granules in a pregnant animal
revealed by the protein A-gold procedure and immunoreactive extruded
prominent Gulgi complex and well-developed RER. x 6,300.
secretory granules seen in the intercellular spaces (arrows). x 30,000.
418
T. ISHIBASHI AND M. SHIINO
Fig. 4. PRL cells of a pregnant animal having numerous cytoplasmic processes (F‘RC). Note irregularly
shaped dense materials between cytoplasmic processes (arrows). x 30,000.
ing animals revealed rather smaller mature secretory
granules.
In E2-injectedanimals, many PRL cells contained numerous larger secretory granules, which were fully distributed in the cytoplasm. The Golgi apparatus and the
RER were well developed but the Nebenkern formations
were not observed. The most remarkable ultrastructural
change in PRL cells of pregnant, lactating, and also the
E2-injected female musk shrews was the occurrence of
“intracisternal” granules. Intracisternal granules were
small and spherical, and were present, without limiting
membranes, inside the dilated cisternae of the RER (Fig.
5, and inset). The size of these granules appeared variable, ranging from 100 nm to 400 nm in diameter. Intracisternal granules were demonstrated to be immunoreactive by the protein A-gold procedure, as were peripheral secretory granules, which were distributed in the
cytoplasm (Figs. 6 , 7). The formation of intracisternal
granules was not observed in the virgin animals. Two
types of PRL cells containing intracisternal granules
were distinguished: One type had a prominent Golgi
complex in which immature secretory granules were
actively formed; the other was fully granulated with
larger mature secretory granules, although the cytoplasmic organelles were not prominent. Furthermore,
the formation of intracisternal granules could be divided
into two cases: One case showed intracisternal granules
of a uniform size (about 150-200 nm in diameter) that
were closely aligned in rows within the dilated RER
cisternae, which formed anastomosing tubules; in the
other case smaller and more heterogeneous dense granules occupied the lumen of the irregularly expanded
cisternae (Fig. 8).Lamellae of the RER containing intracisternal granules in their cisternae were frequently
distributed in close proximity to the cell membrane.
However, we observed neither an image of the RER
lamellae fused with the cell membrane to open to extracellular spaces nor intracisternal granules that had been
extruded from the RER lamellae into the cytoplasm or
extracellular spaces. In early-lactacting animals, welldeveloped PRL cells contained intracisternal granules
inside of the innermost cisternae of the Nebenkern (Fig.
9). These granules were also shown to be immunoreactive by the protein A-gold procedure.
Another ultrastructural characteristic observed in
PRL cells of the female musk shrew was the occurrence
of low-density granules among the highly dense secretory granules. (Fig. 10). This phenomenon was often
observed in late-pregnancy and E2-injected animals.
These granules were limited by rough-contoured membranes and their markedly lower density contents were
often spotted with much denser materials (Fig. 11).These
Figs. 5-7. PRL cells containing intracisternal granules in the anteFig. 6. Intracisternal granules (arrowheads) are demonstrated to be
rior pituitary gland of pregnant animals.
immunoreactive by the protein A-gold procedure as are the ordinary
secretory granules (SG). ~ 2 0 , 0 0 0 .
Fig. 5. Small, spherical and dense granules (arrows) are seen in the
dilated cisternae of the RER even while ordinary secretory granules
Fig. 7. Higher magnification of the area enclosed in the rectangle in
are actively forming within the Golgi area (GI. x 12,000. Inset: Higher Figure 6. Arrows indicate lamellae of RER. ~ 6 0 , 0 0 0 .
magnification of the area enclosed in the rectangle. Note ordinary
secretory granules (SG) and intracisternal granules (*) located inside
the dilated cisternae of RER. ~ 6 0 , 0 0 0 .
Figs, 8,9. Various types of intracisternal granules are observed in
pregnant (Fig. 8) and lactating animals (Fig. 9).
large mature secretory granules and small intracisternal granules
(arrows), which are located in irregularly dilated RER lamellae.
x 20,000.
Fig. 8. T w o types of PRL cells in which intracisternal granules are
contained. The PRL cell on the left contains numerous middle-sized
prominent intracisternal granules that exist in rows within the cisternae of RER-like anastomosing tubules; the cell on the right contains
Fig. 9. Note various sizes and densities of intracisternal granules
present inside the innermost cisternae of Nebenkern. X 30,000.
PRL-CELLS OF THE FEMALE MUSK SHREW
Figs. 10-12. Examples of fully granulated PRL cells in the anterior
pituitary glands of Ez-injected animals.
Fig. 10. Low-density granules (arrows) are accumulated together
with ordinary-density secretory granules in the whole cytoplasm. G:
Golgi area. ~20,000.
Fig. 11. Higher magnification of the area enclosed in the rectangle
in Figure 10.Low-density granules (*) often show heterogeneous contents that are spotted with dense materials (arrows). ~40,000.
42 I
Fig. 12. The immunoreactivity of low-density granules (arrows) visualized by the protein A-gold procedure is weak compared with that
of ordinary secretory granules. ~40,000.
422
T. ISHIBASHI AND M. SHIINO
low-density granules were revealed to be weakly immu- firmed by the protein A-gold procedure that these intranoreactive by the protein A-gold procedure (Fig. 12) and cisternal granules, as well as the mature secretory
ordinarily seemed to be forming from the Golgi vesicles. granules, were immunoreactive.
Farquhar et al. (1978) stated, in regard to the intracelDISCUSSION
lular transport and packaging of prolactin, that the horWe first identified the PRL cells of the anterior pitu- mone is synthesized on attached ribosomes, segregated
itary gland in the musk shrew by the protein A-gold inside the RER, and rapidly transported into the Golgi
procedure combined with electron microscopy and dem- cisternae to be concentrated, where first the earliestonstrated their ultrastructural features. The PRL cells forming granules are recognized. The results obtained
definitely showed morphologically active features in in the present study indicate that morphologically actipregnant and lactating animals. They were character- vated PRL cells are capable of forming visible and imized by a well-developed Golgi apparatus in which im- munoreactive granules inside RER cisternae without
mature secretory granules were present and proliferated the process of transforming a prehormone to the Golgi
RER consisting of densely packed parallel lamellae. Nu- apparatus and condensing it there. It is worth noting
merous cytoplasmic processes were formed between ad- that some PRL cells observed in a pregnant animal
jacent PRL cells. We often observed that the cytoplasmic contained numerous dense, clear-cut intracisternal granprocesses of PRL cells protruded and surrounded a large ules in slightly dilated cisternae of RER, while displayfollicle in which extruded-granules were stored as dense, ing a prominent Golgi zone that was actively forming
immunoreactive material. It is a very interesting fact secretory granules. That is, the formation of granules in
that secretory products from PRL cells are “pooled” in the Golgi apparatus and in the cisternae of RER was in
the extracellular spaces during pregnancy. Secretory progress at the same time within a PRL cell. Chang and
granules were spherical and they exhibited various sizes Nikitovitch-Winer (1976) stated that PRL cells stimuladepending upon the cellular status of secretion. We care- ted by suckling and lactating rats could possibly release
fully observed changes in granule size according to the a soluble form of hormone stored within the RER cisterphysiological conditions and noted that PRL cells under nae directly onto the cell surface. Hausler et al. (1978)
enhanced cellular activity induced by pregnancy or es- also suggested that a dual mechanism of hormone retrogen treatment generally contained larger secretory lease, i.e., a granular and a nongranular form of release,
granules. PRL cells of lactating animals, which showed might exist in PRL cells of lactating rats.
Recently, the preembedding techniques of the peroximorphologically the most hypertrophied cellular features, had a tendency to contain smaller secretory gran- dase-antiperoxidase method have enabled investigators
ules rather than remarkably large granules as seen in to detect ultrastructural localizations of PRL not only in
pregnant animals. It appears that the secretory gran- secretory granules but in both of the Golgi saccules and
ules of PRL cells in the musk shrew generally increase the cisternae of RER (Tougard et al., 1980; Osamura et
in size under enhanced states of hormone synthesis but al., 1982). Judging from the immunocytochemical and
decrease in size under highly stimulated conditions of morphological findings by means of the technique mensecretion during lactation.
tioned above, Osamura et al. (1982) stated that PRL
It is well known that estrogen stimulates the secretory might diffuse directly from the RER into extraceIlular
activity of PRL cells in the female rat (Zambrano and spaces. We observed, in the present study, that immuDeis, 1970; Shiino and Rennels, 1976; Aumuller et al., noreactive granules existed inside the cisternae of RER
1978). Similarly, in the present study, PRL cells of fe- in PRL cells and that these granule-containing cisternae
male musk shrews were activated by Ez-injection and were often located close to the cell membrane. However,
showed prominent Golgi complexes and a significant we have no morphological evidence that RER lamellae
increase in the size of secretory granules. Although we containing immunoreactive granules can open toward
have not observed the formation of Nebenkern in prolif- cytoplasmic or extracellular space to extrude their granerated RER, which was frequently seen in Ez-injected ules.
rats, we did observe the presence of unusual low-density
The finding of intracisternal granules in PRL cells in
secretory granules characterized by weak immunoreac- the present study suggests that PRL cells of the female
tivity. It seems likely that these low-density granules musk shrew may possibly utilize a “bypass” route to
were the result of an incomplete condensation of secre- form hormone-containing granules. It will be very intertory granules within the Golgi complexes due to accel- esting to investigate how the intracisternal granules
erated secretion. The lack of more dramatic ultra- inside the cisternae are finally altered. Further studies
structural changes of PRL cells in the musk shrew, like will be needed to solve the problem.
those seen in the rat, might be due to a n inappropriate
ACKNOWLEDGMENTS
dose or treatment period of estrogen.
The occurrrence of intracisternal granules in PRL cells
The authors wish to express their thanks to Dr. Edof pregnant, lactating, and E2-injected animals was one ward G. Rennels for his kind advice on preparing the
of the most characteristic ultrastructural features in the manuscript. The authors also wish to thank Dr. A.F.
musk shrew. The formation of intracisternal granules Parlow, of the Pituitary Hormones and Antisera Center,
in the anterior pituitary cells is well known in thyroid- Harbor-UCLA Medical Center, Los Angeles, and the
ectomy cells of the rat and other species, and a few cases National Hormone and Pituitary Program, University
of intracisternaI granules in PRL cells of lactating rab- of Maryland School of Medicine, Baltimore, for their
bits have been illustrated by Young et al. (1967). How- generous gifts of antigens and antisera.
This study was supported by a Grant for Scientific
ever, the intracisternal granules observed in PRL cells
of the musk shrew are more numerous and prominent Research from the Ministrv of Education. Science and
than those of Young et al. (1967). Additionally, we con- Culture, Japan, No. 62570016.
PRL-CELLS OF THE FEMALE MUSK SHREW
LITERATURE CITED
Aumuller, G., R. Wagner, and K.J. Graf (1978) Fine structure of rat
prolactin cells after treatment with a long acting depot contraceptive. Acta Endocrinol., 89:251-262.
Bendayan, M., and M. Zollinger (1983) Ultrastructural localization of
antigenic sites on osmium-fixed tissues applying the protein Agold technique. J. Histochem. Cytochem., 31:lOl-109.
Chang, N.G., and M.B. Nikitovitch-Winer (1976) Correlation between
suckling induced changes in the ultrastructure of mammotrophs
and prolactin release. Cell Tissue Res., 166:399-406.
Farquhar, M.G., J.J. Reid, and L.W. Daniel1 (1978) Intracellular transport and packaging of prolactin: A quantitative electron microscope autoradiographic study of mammotrophs dissociated from rat
pituitaries. Endocrinology, 102:296-311.
Hausler, A,, H.P. Rohr, P. Marbach, and E. Fluckiger (1978) Changes
in prolactin secretion in lactating rats assessed by correlative morphometric and biochemical methods. J. Ultrastruct. Res., 64:74-84.
Ishibashi, T., and M. Shiino (1988) Unique features of secretory granules observed in the pituitary growth hormone secreting (GH) cells
of the musk shrew (Suncus rnurinus L).Cell Tissue Res. 251:111116.
423
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shrew, Suncus murinus L. (Insectivora), with special reference to
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Naik, D.R., and C.J. Dominic (1978)Functional significance of the cells
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(Suncus murinus L.). J. Endocrinol., 79.9-102.
Osamura, R.Y., N. Komatsu, S. Izumi, S. Yoshimura, and K. Watanabe
(1982) Ultrastructural localization of prolactin in the rat anterior
pituitary glands by preembedding peroxidase-labeled antibody
method Observations in normal, castrated, or estrogen-stimulated
specimens. J. Histochem. Cytochem., 30:919-925.
Shiino, M., and E.G. Rennels (1976) Recovery of rat prolactin cells
following cessation of estrogen treatment. Anat. Rec., 185:31-48.
Tougard C., R. Picart, and A. Tixier-Vidal (1980) Electronmicroscopic
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Young, B.A., C.L. Foster, and E. Cameron (1967) Ultrastructural
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Zambrano, D., and R.P. Deis (1970) The adenohypophysis of female
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prolactin, shrew, anterior, cells, immunocytochemical, ultrastructure, suncus, secreting, pituitary, gland, female, musk, prl, studies, murinus
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