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Ultrastructure of the pars intermedia of pigmented ratsObservations of melanin-like granules within secretory cells.

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Ultrastructure of the Pars lntermedia of Pigmented
Rats: Observations of Melanin4 ke Granules
within Secretory Cells
L. C. SALAND
Department of A n a t o m y , University of N e w Mexico, School of Medicine,
Albuquerque, N e w Mexico 87131
ABSTRACT
The pars intermedia of non-obese pigmented rats of the Zucker
strain is described using transmission electron microscopy. Secretory cells appear
similar to those described elsewhere in albino rodent hypophyses. Cells which
may be equivalent to ACTH cells of non-pigmented mice and rats are observed
near the border of the pars distalis. Melanocytes are observed to surround lobules
of the pars intermedia and to penetrate among secretory cells. In addition, large
granules which appear to be melanin are present within some pars intermedia
cells. Portions of some cells near melanin or melanin-like granules exhibit expanded rough endoplasmic reticulum and Golgi zones containing dense granules.
Observations are discussed in relation to origin of the melanin, and its possible
significance near cells known to produce melanocyte-stimulating hormone (MSH).
The ultrastructure of normal albino rat
pars intermedia of the hypophysis has been
described in detail and reviewed recently
(Howe and Maxwell, '68; Howe, '73). HOWever, the functions of hormones produced
by the pars intermedia in mammals remain obscure. A number of previous investigators have used methods such as
adrenalectomy (Stoeckel et al., '71) and
alterations of dietary sodium (Kobayashi,
'74a,b,c) in attempts to alter the characteristic cytology of normal intermediate lobes.
Few studies have been reported using pigmented mammals for ultrastructural studies of the pars intermedia, which is well
established as a source of melanocytestimulating hormone (MSH) for adaptation to background in lower vertebrates (Etkin, '67; Hadley and Bagnara, '75). This
investigation utilizes rats of the Zucker
strain to examine the ultrastructure of the
pars intermedia. Of particular interest is
the presence of melanosomes which appear to surround or lie within secretory
cells of the gland.
perfused through the heart with saline until all blood was cleared, followed by cold
6% glutaraldehyde in 0.1 M phosphate
buffer. Methods were modified from those
of Baumgarten et al. ('72) for fixation of
the neurointermediate lobe in rats. Perfusion was continued for approximately 15
minutes. Hypophyses were dissected free
and placed in cold buffered fixative for
two hours. Glands were divided into four
pieces, then washed in buffer and postfixed in 1% buffered osmium tetroxide at
room temperature for 1-2 hours.
2. Histochemistry
Four pituitaries from adult male rats
were used for histochemical localization of
acid phosphatase. Animals were decapitated, and glands were fixed for four hours
by immersion in ice-cold 2.5% glutaraldehyde in 0.15 M cacodylate/HCl buffer.
Fifty-micron sections of neurointermediate
lobes were cut on a Sorvall tissue chopper,
and processed for acid phosphatase by the
Barka and Anderson ('62) modification of
the Gomori method. Tissue was handled as
MATERIALS -4ND METHODS
described in detail for anterior pituitary by
Smith and Farquhar ('66). All blocks were
1 . Routine electron microscopy
Five adult male rats of normal weight dehydrated in graded ethanols and em(250-300 g), and of the Zucker geneti- bedded in Epon 812. Thick (1-2 pm) and
cally obese strain (Zucker and Zucker, '61)
Received Aug. 5 , '75. Accepted Mar. 22, '76.
were lightly anesthetized with ether, and
~
ANAT. REC., 186: 69-78.
69
70
L. C. SALAND
thin sections were obtained on a Cambridge-Huxley ultramicrotome using a DuPont diamond knife. Sections were examined in a n Hitachi HS-7S or Philips 200
electron microscope.
OBSERVATIONS
In three of five rats studied initially, the
meninges surrounding the hypophysis appear pigmented upon gross inspection.
Examination of one micrometer ( 1 pm)
sections (fig. 1 ) reveals the presence of
pigmented cells within investing connective tissue surrounding only the pars intermedia. Small dark areas are sometimes observed deep within the intermediate lobe.
Pigment is not observed around the pars
distalis or pars nervosa.
At the light microscopic level, the majority of cells of the pars intermedia exhibit rounded nuclei and indistinct cellular
margins (fig. 1). Relatively few blood vessels are observed within the substance of
the tissue.
Electron microscopy
1. General description
The ultrastructure of cells of the pars
intermedia of pigmented rats appears similar to that described for albinos (DISCUSSION), and will only be reported briefly
here. The major cell type appears secretory
in nature, while “stellate-shaped’ cells are
occasionally observed. Another rare occurrence is the appearance of an angular cell
with small, dense granules along its outer
limiting membrane (fig. 2 ) . The latter cell
type is found bordering areas near the pars
distalis, and may be equivalent to adrenocorticotrophic (ACTH) cells observed elsewhere (DISCUSSION). The cytoplasm of the
major secretory cell type is virtually filled
with round or ovoid membrane-bound granules of varied electron densities (fig. 3-6).
Golgi complexes containing very dense material and closely associated dense granules are observed in some cells (figs. 3 , 5).
Some randomly scattered cells also exhibit
partially dilated areas of rough endoplasmic reticulum (figs. 3 , 5). Careful searching did not reveal extrusion of whole granules from any portions of pars intermedia
secretory cells, although occasional cells
show “lining up” of granules along the
plasma membrane (fig. 5). I n addition,
portions of small unmyelinated nerve fi-
bers are observed among secretory cells.
Most are found to contain clear vesicles,
although occasional dense-cored vesicles
are seen (fig. 2 ) .
2. Melanin-like granules
The most striking feature of intermediate lobes examined here is the presence
of cells containing melanin granules and
occasional premelanosomes, both around
the borders of gland lobules and penetrating into the interstices of densely packed
secretory cells (figs. 3, 4, 6A). Portions of
melanin-containing cells are also observed
in contact with the basal lamina of blood
vessels. Melanosomes and premelanosomes
are observed in cells surrounded by extracellular space (fig. 4 ) , and in some instances, areas of the melanocytes are in
direct contact with pars intermedia secretory cells (figs. 3 , 6A). I n addition, occasional secretory cells are found to contain
melanin-like granules either singly or in
small groups (figs. 5, 6B). I n some cases,
a cell containing the granules or a n immediate neighbor exhibits a large Golgi complex containing dense material, and/or
some expansion of rough endoplasmic reticulum (figs. 3, 5). Incubation of neurointermediate lobes for acid phosphatase reveals occasional areas of reaction product
in the Golgi zone and some lysosomes. Positive areas are also found in membranes
surrounding melanosomes within melanocytes, as well as in outer limiting membranes of melanin-like granules in pars
intermedia secretory cells (figs. 6A,B).
DISCUSSION
Glandular cells of the pars intermedia
appear to show little variability in their
morphology among mammalian species
studied. Albino rat and mouse intermediate
lobes contain a secretory cell type similar
in appearance to those of pigmented rats
studied here (Howe and Maxwell, ’68;
Howe, ’73; Moriarty et al., ’75). Recently,
cells stated to contain ACTH have been
described in the albino rodent pars intermedia (Stoeckel et al., ’73). Similar cells
have been observed here in pigmented rats.
I n addition, some investigators have found
a lipotropic substance in the mammalian
pars intermedia (Moon et al., ’73), while
others postulate a hormonal relationship
between the intermediate lobe and aldo-
MELANIN IN RAT PARS INTERMEDIA
sterone production (Kobayashi, '74a,b,c).
Nevertheless, functions of the mammalian
pars intermedia and overall regulation of
its hormonal synthesis and release remain
unclear. In lower vertebrates, such as amphibians, MSH clearly regulates movement
of pigment in melanocytes, thereby controlling background adaptive coloration
(Etkin, '67; Hadley and Bagnara, '75).
The presence of pigment around the
hypophysis in non-albino rats was reported over 35 years ago. Benjamin ('35)
noted that pigment observed around the
rat pars intermedia and pars tuberalis appeared to be extracellular, although Romeis
('40) stated that pigment granules also appeared to be among glandular cells of the
intermediate lobe. Pigment accumulation
in the meninges, particularly around the
hypophysis, has also been observed in some
prosimian primates (H. M. Murray, personal communication). In the present investigation, granules which appear to be
melanin are present within pars intermedia secretory cells. In addition, it is confirmed that melanocytes are only found
around the pars intermedia. The reason
for their presence there and not around
the pars distalis or pars nervosa is not
clear. It is tempting to suggest that melanocytes are present close to a source of hormone related to their function, but the morphology alone is insufficient to confirm or
deny such speculation. Granules which appear to be melanin within pars intermedia
cells are presumed to have migrated from
the numerous melanocytes surrounding the
entire gland. Movement of pigment granules from melanocytes to neighboring cells
is well documented in cells of mammalian
skin (Mottaz and Zelickson, '69). Cells
containing "donated" melanosomes show
acid phosphatase activity within lysosomes
which have incorporated melanin (Mishima, '66; Wolff, '73). Larger melanosomes
of dark skin appear to be donated singly
to keratinocytes (Wolff, '73). Granules
found in the pars intermedia which appear
similar to melanin show some acid hydrolase activity along their outer surfaces, and
may therefore be similar to melanin granules which are donated to neighboring epithelial cells of skin. It appears that cells
of the intermediate lobe are capable of accepting, and perhaps degrading, the melanin-like granules. Large, complex second-
71
ary lysosomes containing melanin granules
were not observed here, as they have been
in epidermal cells (Wolff, '73).
Of additional interest is the partially expanded appearance of the rough endoplasmic reticulum and Golgi complex in
some secretory cells containing melaninlike granules, or in cells adjajcent to areas
of melanin. Occasional cells of normal albino rat pars intermedia show such Golgi
and ER profiles (Howe and Maxwell, '68;
Howe, '73), and they appear in great abundance i n reserpinized albino rat partes
intermedias (unpublished observations).
Similar changes are reported in the mouse
pars intermedia after restriction of sodium
in the diet (Kobayashi, '74a,b). Very recently, Moriarty and co-workers ('75) have
shown some expansion of endoplasmic
reticulum and depletion of cytoplasmic
granules in the pars intermedia of rats subjected to neurogenic stress. In dark-adapted
amphibians, similar changes in secretory
cell organelles are thought to indicate synthesis of material after release of cytoplasmic stores (Saland, '68; Castel, '72).
In the case of the pigmented rats studied
here, alterations of organelles may be indicative of local production of hormone or
precursor material, perhaps influenced by
the proximity of melanin granules. Changes
are presumed to be in response to release
of material from secretory cells. Although
"lining up" of granules near the limiting
cell membrane was noted, release G f discrete granules (exocytosis) from secretory
cells has not been observed here, nor has
it been described in the pars intermedia in
other studies (Howe, '73; Moriarty et al.,
'75). Investigations are in progress using
pharmacologic agents to induce release of
intermediate lobe hormones in both albino
and pigmented rodents.
ACKNOWLEDGMENTS
Non-obese Zucker rats were obtained
from the colony maintained in the Animal
Resource Facility at the University of New
Mexico School of Medicine. My thanks to
Ms. Judi DeLongo for excellent technical
assistance.
LITERATURE CITED
Barka, T., and P. J. Anderson 1962 Histochemiical methods for acid phosphatase using hexazonium pararosanillin as coupler. J. Histochein.
Cytochcm., 10: 741-753.
Baumgarten, H. G . , A. Bjorklund, A. F. Holstein
72
L. C. SALAND
and A. Nobin 1972 Organization and ultrastructural identification of the catecholamine
nerve terminals in the neural lobe and pars
intermedia of the rat pituitary. Z. Zellforsch.,
126: 483-517.
Benjamin, J. A. 1935 The occurrence of pigment in the pars intermedia and pars tuberalis
of the hypophysis, and i n the hypophyseal leptomeninges of the rat (domestic and wild). Anat.
Rec., 61: 331-340.
Castel, M. 1972 Ultrastructure of the anuran
pars intermedia following severance of hypothalamic connection. Z. Zellforsch., 131: 545557.
Etkin, W. 1967 Relation of the pars intermedia
to the hypothalamus. In: Neuroendocrinology,
VII. L. Martini and W. F. Ganong, eds. Academic Press, New York, pp. 261-282.
Hadley, M. E., and J. T. Bagnara 1975 Regulation of release and mechanism of action
of MSH. Amer. Zool., 15 (Suppl. 1 ) : 81-104.
Howe, A. 1973 The mammalian pars intermedia: A review of its structure and function.
J. Endocrinology, 59: 385-409.
Howe, A., and D. S. Maxwell 1968 Electron
microscopy of the pars intermedia of the pituitary gland in the rat. Gen. Comp. Endocrinology,
11: 169-185.
Kobayashi, Y. 1974a Quantitative and electron
microscopic studies on the pars intermedia
of the hypophysis. I. Dietary effects of brown
rice on the kidney, adrenal and pituitary of
C57BL/6 mouse. J. Elec. Micro., 23: 107-115.
-__ 1974b Quantitative and electron microscopic studies on the pars intermedia of the
hypophysis. 11. Alterations of the pars intermedia and the adrenal zona glomerulosa of
albino mice following sodium restriction. Annotatione Zoologicae Japonenses, 47: 221-231.
1974c Quantitative and electron microscopic studies on the pars intermedia of the
hypophysis. 111. Effect of short-term administration of a sodium deficient diet o n the pars
intermedia of mice. Cell Tiss. Res., 154: 321327.
Mishima, Y. 1966 Cellular and suhcellular differentiation of melanin phagocytosis and synthesis by lysosomes and melanosomal activity.
J. Invest. Dermatol., 46: 70-75.
Moon, H. D., C. H. Li and B. M. Jennings 1973
Immunohistochemical and histochemical studies of pituitary lipotrophs. Anat. Rec., 175: 529538.
Moriarty, G. C . , N. S. Halmi and C. M. Moriarty
1975 The effect of stress on the cytology and
immunohistochemistry of pars intermedia cells
i n the rat pituitary. Endocrinology, 96. 14261436.
Mottaz, J. H., and A. S. Zelickson 1969 Ultrastructure of hair pigment. In: Hair Growth,
Advances i n Biology of Skin. Vol. IX. W.
Montagna and R. L. Dobson, eds. Pergamon
Press, Oxford, pp. 478480.
Romeis, R . 1940 Handbuch der Mikroskopischen Anatomie des Menschen. Vol. VI. W. V.
Mollendorff, ed. Springer, Berlin, pp. 377-378.
Saland, L. C . 1968 Utrastructure of the frog
pars intermedia i n relation to hypothalamic
control of hormone release. Neuroendocrinology,
3: 72-88.
Smith, R. E., and M. G. Farquhar 1966 Lysosome function i n the regulation of the secretory process in cells of the anterior pituitary
gland. J. Cell Biol., 31: 319-347.
Stoeckel, M. E., H-D. Dellmann, A. Porte and
C. Gertner 1971 The rostral zone of the intermediate lobe of the mouse hypophysis, a
zone of particular concentration of corticotrophic cells. Z. Zellforsch., 122: 310-322.
Stoeckel, M. E., H-D. Dellmann, A. Porte, M. J.
Klein and F. Stutinsky 1973 Corticotrophic
cells i n the rostral zone of the pars intermedia
and i n the adjacent neurohypophysis of the rat
and mouse. Z. Zellforsch, 136: 97-110.
Wolff. K. 1973 Melanocyte-keratinocyte interactions in uitro. Yale J . Biol. Med., 46: 384-396.
Zucker, L. M., and T. F. Zucker 1961 Fatty, a
new mutation in the rat. J. Heredity, 52: 275278.
MELANIN IN RAT PARS INTERMEDIA
PLATE 1
L. C. Saland
EXPLANATION OF FIGURES
1
A one micrometer (1 pm) epon section of pars intermedia cells reveals
melanocytes ( M ) between lobules of gland. Some pigment-containing
cells are adjacent to parenchyma (arrows). Nuclei of pars intermedia
parenchymal cells ( N ) are clearly visible. Methylene blue-Azure I1
stain. x800.
2
A portion of a cell containing small dense granules (arrow) near its
limiting membranes dominates the field. This cell may be equivalent
to “ACTH” cells observed elsewhere (see text). Note small unmyelinated nerve fibers ( n ) containing clear and dense-cored vesicles abutting the “ A C T H cell and other pars intermedia cells ( p i ) . x 14,700.
73
PLATE 2
EXPLANATION OF FIGURES
3
Melanocytes containing melanin granules ( m ) are among pars intermedia secretory cells (pi). Note extensive Golgi zone ( G ) and stacks
of rough endoplasmic reticulum (rer) i n a cell adjacent to melanin.
X 8,400.
4 Portion of melanocytes surrounded by basal lamina and collagen ( b )
are interposed between pars intermedia secretory cells (pi). Premelaiiosomes ( p ) are observed within melanocyt~s.Note the varied densities of granules in pars intermedia secretory cells. X 17,000.
74
MELANIN I N RAT PARS INTERMEDIA
L. C . Saland
PLATE 2
75
PLATE 3
EXPLANATION OF FIGURES
5
A group of melanin-like granules is present within a pars intermedia
secretory cell. Note the lining up of granules along the plasmalemma
of an adjacent cell (arrows) and prominent rough endoplasmic reticulum (rer) and Golgi zone ( G ) in the latter cell. x 14,800.
6A Melanosomes within a melanocyte contain reaction product i n surrounding membranes and spaces after incubation for acid phosphatase.
Cytoplasmic granules of varied densities are present i n the adjacent
pars intermedia parenchymal cell (pi). x 29,500.
6B A single melanin-like granule within a pars intermedia secretory cell
exhibits acid phosphatase reaction product along its outer surface.
x 27,000.
76
MELANIN I N RAT PARS INTERMEDIA
L. C. Saland
PLATE 3
77
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