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Immunohistochemical and histochemical studies of pituitary ╬▓-lipotrophs.

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Immunohistochemical and Histochemical Studies of
Pituitary 13-Lipotrophs
HENRY D. MOON, CHOH H A 0 LI AND BARBARA M. JENNINGS
Department of Pathology and Hormone Research Laboratory, University of
California, Sun Francisco, California 94122
The presence of p-lipotrophin or an immunologically similar substance was demonstrated in the intermediate lobe cells and some basophils of the
anterior lobe by both fluorescent and peroxidase-conjugated antibody methods.
These basophils were found to be corticotrophs, i.e., reactive with anti-ACTH.
ACTH, when added to anti-B-LPH, decreased the fluorescence and peroxidase reactions of intermediate lobe cells and the anterior lobe corticotrophs. The addition of MSH to anti-p-LPH produced a slight decrease in the reaction between the
intermediate lobe cells and anti-p-LPH. The intermediate lobe cells also reacted
with anti-ACTH. Both somatotrophs and lactotrophs reacted with anti-8-LPH to
give positive fluorescence or peroxidase reactions. However, the addition of STH
to anti-P-LPH abolished the reaction between anti-P-LPH and somatotrophs. Similarly, LTH, when added to anti-P-LPH, abolished the positive reaction of lactotrophs to anti-B-LPH. When STH, LTH and ACTH were added to anti-P-LPH, the
fluorescence of the somatotrophs and lactotrophs was abolished, whereas the
corticotrophs maintained their fluorescence at a reduced intensity. These results
indicate that the somatotrophs and lactotrophs do not contain p-LPH and that
corticotrophs contain P-LPH.
ABSTRACT
p-lipotrophin, a pituitary hormone with
lipolytic and melanocyte-stimulating activities, was isolated from sheep pituitaries
by Birk and Li ('64). Subsequent studies
demonstrated p-lipotrophin to be a polypeptide containing 90 amino acids (Li
et al., '66), with an amino acid sequence
identical in part to that of adrenocorticotrophic hormone (ACTH) and melanocyte-stimulating hormone (MSH) (Li,
'68). However, the specific cell(s) responsible for its production has not been identified. The present studies were conducted
to identify the lipotroph and to distinguish
it from the other cells of the pituitary
gland.
MATERIALS AND METHODS
Hormonal preparations used as antigens
for immunization were obtained from
sheep pituitaries by procedures previously
described (Cole and Li, '55; Li et al., '55;
PapItoff and Li, '58; Li et al., '66). The
antiserum was prepared in the following
manner: Young female rabbits, weighing
approximately 2.6-2.8 Kg each, were inANAT. REC., 175: 529-538.
jected with a total dose of 10 mg p-LPH,
2 mg STH, 2 mg LTH, or 2 mg ACTH suspended in complete Freund's adjuvant over
a period of about six weeks according to
the procedure of Moudgal and Li ('61).
Antisera to porcine ACTH were prepared
similarly. Blood was drawn by cardiac
puncture and the serum was obtained by
standard techniques. The antiserum to
P-LPH was tested for the presence of antibodies to STH and LTH by the procedures
reported by Ouchterlony ('49), Heidelberger and Kendall ('35) and Li et al.
('60). The results obtained by these methods indicated that anti-STII and anti-LTH
were not present in the anti-p-LPH serum.
Additional antisera consisted of anti-p-LPH
to which STH, LTH, ACTH, LPH or MSH
had been added as follows: 10 mg STH/
ml, 10 mg LTH/ml, 3 mg ACTH/ml, 6
mg p-LFH/ml, and 1 mg B-MSH/ml. 10
mg/ml STH, 10 mg/ml LTH, and 3 mg/
ml ACTH were added to one preparation of
anti-p-LPH. The concentrations of the various hormones were calculated to result in
Received Aug. 7 , '72. Accepted Nov. 15, '72.
529
530
H. D. MOON, C. H. LI AND B. M. JENNINGS
several-fold antigen excess. These anti-pLPH preparations were stirred at 22°C for
six hours, refrigerated overnight, and centrifuged. The supernatants were used for
reacting with the sections.
Peroxidase-labeled sheep anti-rabbit Y
globulin was prepared by the method of
Nakane and Pierce ('67).
Portions of the pituitary glands of six
male sheep were fixed in buffered neutral
formalin for paraffin embedding; unfixed
portions of the glands were frozen in liquid
nitrogen and subsequently used for freezedried or cryostat sections. Preliminary
studies indicated that the paraffin sections
were superior for both immunofluorescence and histochemical studies; thus the
definitive studies were conducted on paraffin sections.
Serial sections of the paraffin-embedded
thick, and
pituitary tissue were cut, 6
each section was mounted on a slide. Sections for immunofluorescence studies were
selected at intervals of two or more slides
apart so that intervening contiguous sections could be used for histochemical
studies or additional immunofluorescent
slides.
For immunofluoresc:ence studies, the
deparaffinized sections were treated with
rabbit antisera followed by fluoresceinconjugated goat anti-rabbit y globulin. Control sections were treated with ( 1 ) specific
antisera, unconjugated anti-rabbit y globulin, followed by fluorescein-conjugated
anti-rabbit y globulin; (2) pooled untreated
rabbit sera and fluorescein-conjugated
anti-rabbit y globulin; or only with ( 3 )
fluorescein-conjugated anti-rabbit y globulin. Some of the sections reacted with antip-LPH and fluorescein-conjugated antirabbit y globulin were examined, photomicrographed and relayered with anti-STH,
anti-LTH or anti-ACTH followed by fluorescein-conjugated anti-rabbit y globulin. The
sections were studied with a Zeiss fluorescence microscope with an Osram HBO
high-pressure mercury lamp and also under
dark field with ordinary light.
Additional sections were treated with
anti-p-LPH followed by peroxidase-conjugated sheep anti-rabbit y globulin. The
sections were then reacted for peroxidase
by the method of Graham and Karnovsky
('66) using 3,3'-diaminobenzidine (tetrahydrochloride) as the substrate.
The immunofluorescent slides were restained by a periodic acid-Schiff (PAS),
orange G method (Pearse and van Noorden, '63), or by carmoisine, orange G and
wool green (Erookes, '68 ). Also, additional
sections contiguous to those reacted with
antisera were stained by one of the above
methods. Photomicrographs were taken of
identical areas of immunofluorescent sections and histochemically stained sections.
Also, photomicrographs were taken of the
sections reacted with specific antisera and
peroxidase-labeled antibody.
RESULTS
SpecSc cell types of the pituitary gland
were identified by their fluorescence in
sections treated with anti-STH, anti-LTH
or anti-ACTH followed by fluorescein-conjugated sheep anti-rabbit y globulin and by
the presence of peroxidase reaction product
following treatment by the peroxidaselabeled antibody method. The cells were
further characterized by their reactions in
immunofluorescent sections restained histochemically.
Somatotrophs
In immunofluorescence studies with
anti-STH, the somatotrophs were found to
be large, brightly fluorescent cells with
abundant cytoplasm occurring in loose
clusters, slender cords and frequently as
isolated cells (fig. 1 ) . When examined
with dark-field illumination, the cells had
bright luminescent cytoplasm. Similar results were obtained in the sections treated
by peroxidase-labeled antibody. In restained immunofluorescent sections, the
somatotrophs were orange G-positive and
PAS-, carmoisine- and wool green-negative
(fig. 2 ) .
Lactotrophs
The lactotrophs, in immunofluorescence
studies with anti-LTH, were found to be
slightly smaller than the somatotrophs.
They appeared very frequently in compact
folliculoid masses and columns rather than
as individual cells; they were more numerous than somatotrophs (fig. 3 ) . In darkfield illumination with ordinary light, they
were luminescent but to a lesser degree
53 1
PITUITARY ,B-LIF’OTROPHS
than somatotrophs. In the sections reacted
with anti-LTH and peroxidase-conjugated
anti-rabbit y globulin, the lactotrophs appeared in patterns similar to those observed in the immunofluorescent slides.
When the immunofluorescent slides were
restained with PAS and orange G , the
lactotrophs were orange G-positive, but in
slides stained with carmoisine, orange G
and wool green, the lactotrophs were carmoisine-positive (fig. 4).
LTH. In the immunofluorescent slides restained histochemically, the intermediate
lobe cells were faintly PAS- and wool
green-positive.
The posterior lobe cells did not react
with any of the antisera.
Lipotrop hs
The cells of the intermediate lobe had
brightly fluorescent cytoplasm in the sections treated with anti-p-LPH and fluorescein-conjugated anti-rabbit y globulin
Corticotrop hs
(fig. 7). These cells were also brightly
The corticotrophs of the anterior lobe, fluorescent with preparations of anti-p-LPH
in the immunofluorescence and peroxidase- to which STH or LTH had been added. In
labeled antibody studies with anti-ACTH, sections treated with anti-p-LPH to which
were found in small groups, cords, or as p-LPH had been added, there was little or
isolated stellate cells (fig. 5). Their num- no fluorescence of the intermediate lobe
ber varied considerably from one anterior cells (table 1). In sections treated with
pituitary to another. When examined by anti-p-LPH to which ACTH had been
dark field and ordinary light, the cortico- added, there was a definite reduction in the
trophs were not luminescent. In immuno- intensity of the fluorescence of the interfluorescent sections stained with PAS and mediate lobe cells. There was a slight deorange G , the corticotrophs were PAS-posi- crease in fluorescence when MSH was
tive (fig. 6). There were other cells that added to anti-p-LPH. When sections were
gave a less intense PAS reaction. Presum- treated with anti-p-LPH to which STH,
ably these latter cells were gonadotrophs LTH and ACTH had been added, the interand thyrotrophs, since they did not react mediate lobe cells showed a reduced
with anti-ACTH.
fluorescence.
The cells of the intermediate lobe gave
In the sections treated with anti-p-LPH
varying reactions with anti-ACTH. With and peroxidase-labeled sheep anti-rabbit y
anti-ovine ACTH, the fluorescent and per- globulin, the peroxidase reaction product
oxidase reactions were moderately posi- was observed in the intermediate lobe cells
tive, whereas with anti-porcine ACTH, (fig. 8); similar results were obtained with
strong reactions were observed. No reac- preparations of anti-p-LPH to which STH
tion occurred with either anti-STH or anti- or LTH had been added. In sections treated
TABLE 1
Anti-pLPH and pituitary cells
Antisera
Anti-R-LPH
Anti-b-LPH
+ LPH
Anti-a-LPH
+ ‘STH
Anti-6-LPH
+ LTH
Anti-@LPH
+ ACTH
Anti-p-LPH
Interned. lobe
Somatotroph
Lactotroph
Corticotroph
++++
+++
++
++++
0
0
0
0
0
++
+++
+++
+ MSH
Anti-i-iPH
+ ACTH + p-MSH
+++
+++
++
+++
+
+++
++
++
+
+
+
+
+
++
0
0
Anti-6-LPH
LTH
+‘STH
+ ACTH
0
++
+++
+
++
532
€1. D. MOON, C. 13. LI AND
with anti-p-LPH containing ACTH or
p-MSH, there was a reduction in the
amount of peroxidase reaction product. In
sections treated with anti-p-LPH to which
p-LPH had been added, there was minimal
peroxidase reaction product in the intermediate lobe. In the immunofluorescent
sections restained with PAS and orange G,
the intermediate lobe cells were faintly
PAS-positive. With carmoisine, orange G
and wool green, the cells were stained by
wool green.
In the anterior lobe there were three
types of cells that reacted with anti-p-LPH.
One type was identified as somatotrophs
on the basis of their size, pattern of distribution, dark-field luminescence and histochemical reactions. The somatotrophs
were brightly fluorescent with anti-p-LPH
and fluorescein-conjugated anti-rabbit Y
globulin (fig. 9). When sections were
treated with anti-p-LPH to which STH had
been added, the somatotrophs showed no
fluorescence. p-LPH, when added to antip-LPH, likewise abolished the positive fluorescence of the somatotrophs. When sections were treated with anti-p-LPH to
which LTH had been added, the somatotrophs were again fluorescent. The addition of ACTH or p-MSH to anti-p-LPH resulted in some reduction in the intensity
of fluorescence. Relayering of immunofluorescent anti-p-LPH sections with anti-STH
and fluorescein-conjugated anti-rabbit y
globulin did not increase the number of
fluorescent cells. The results obtained with
the peroxidase-conjugated antibody method were essentially identical to those obtained with the immunofluorescent sections. When the immunofluorescent slides
were restained histochemically, these cells
were orange Gpositive by both PAS-orange
G and carmoisine, orange G , wool green
techniques (fig. 10).
The second type of anterior lobe cell
reacting with anti-p-LPH was the lactotroph. This identification was based on
pattern of arrangement, moderate luminescence by dark-field examination and
histochemical reactions. The lactotrophs
exhibited a lesser degree of fluorescence
than the somatotrophs in the sections reacted with anti-p-LPH and fluoresceinconjugated anti-rabbit y globulin. The
lactotrophs did not fluoresce in sections
B. M. JENNINGS
treated with anti-p-LPH to which either
LTH or LPH had been added. The addition
of STH to anti-p-LPH produced no change
in the fluorescence of lactotrophs. The
addition of ACTH or p-MSH to anti-p-LPH
resulted in a significant reduction in the
fluorescence of lactotrophs. Relayering of
immunofluorescent anti-p-LPH sections
with anti-LTH did not increase the number of fluorescent cells. In the sections
treated by the peroxidase-conjugated antibody method, the results were the same as
those obtained by the immunofluorescent
method. When the immunofluorescent sections were restained with PAS and orange
G, the cells were orange G-positive. In the
sections restained with carmoisine, orange
G and PAS, these cells were carmoisinerather than orange G-positive.
The third type of cell reacting with antip-LPH was identified as the corticotroph.
This was based on their occurrence in
small groups, cords, and as isolated cells,
lack of luminescence by dark-field examination and postive PAS reaction, i.e., the
same features which characterized the
cells reacting with anti-ACTH. p-LPH,
when added to anti-p-LPH, abolished the
fluorescence of the corticotrophs. The addition of ACTH to anti-p-LPH resulted in
definite reduction in fluorescence of the
corticotrophs; however, there was continued residual fluorescence of the corticotrophs. STH, LTH and ACTH, when added
to anti-p-LPH, abolished the fluorescence
of somatotrophs and lactotrophs; the corticotrophs fluoresced with a reduced intensity. The addition of STH or LTH to anti-pLPH produced a slight change in the
fluorescence of these cells. The addition of
p-MSH to anti-p-LPH resulted in slight reduction of fluorescence. When both ACTH
and p-MSH were added to anti-p-LPH, the
fluorescence of the corticotrophs was
greatly reduced. The peroxidase-conjugated antibody studies yielded the same
results. In the restained immunofluorescent sections, the cells were PAS- and wool
green-positive.
DISCUSSION
The immunofluorescent reactions of the
cells of the intermediate lobe and some
basophils of the anterior pituitary indicate
that both cell types contain pLPH or an
533
PITUITARY j3-LIPOTROPHS
immunologically related substance. Further evidence for this view is provided by
the studies with anti-p-LPH and peroxidase-conjugated anti-rabbit y globulin in
which peroxidase reaction product was observed in the intermediate lobe cells and
some basophils of the anterior pituitary.
The immunofluorescent reactions of these
two cell types with anti-ACTH also indicate
that both are “corticotrophs,” or at least
contain a substance reacting with antiACTH.
The addition of p-LPH to anti-p-LPH
abolished both the fluorescent and peroxidase reactions of the intermediate lobe
cells and corticotrophs of the anterior pituitary. When ACTH was added to anti-pLPH, there was a significant reduction in
the fluorescence of the intermediate lobe
cells; reduced fluorescence was observed in
the corticotrophs of the anterior lobe. Similar results were obtained with the peroxidase-conjugated antibody studies. These
findings suggest that these cells contain
ACTH and that at least part of the fluorescent and peroxidase reactions are due to a
cross-reaction between anti-p-LPH and
ACTH. On the other hand, the intermediate lobe cells did maintain a definite, although decreased, reaction with anti-p-LPH
preparations containing ACTH, indicating
that they contained p-LPH as well as
ACTH. This view is consistent with the results obtained when p-MSH was added to
anti-p-LPH, i.e., there was only a slight
reduction in the fluorescent and peroxidase
reactions of intermediate lobe cells. The
continued though reduced fluorescence of
corticotrophs, when treated with anti-pLPH to which STH, LTH and ACTH had
been added, suggests that the corticotrophs
contain p-LPH.
Hess et al. (’68) and Baker et al. (’70)
reported that the intermediate lobe reacted
with antisera to ACTH. Phifer and Spicer
(’70) presented strong evidence that the
immunohistologic demonstration of ACTH
in the intermediate lobe was not due to a
cross-reaction with MSH. These investigators used an antiserum to 17-39 ACTH,
which has no amino acid sequences in
common with any known MSH. On the
other hand, Baker (’72), using anti-17-39
ACTH, obtained evidence that the intermediate lobe cells did not contain ACTH.
The present studies provide evidence that
the intermediate lobe cells and probably
the anterior lobe corticotrophs contain
p-LPH. Since p-LPH, ACTH and MSH
have an identical acid sequence in parts of
their structure, it seems reasonable to suppose that the intermediate lobe cells and
the anterior lobe corticotrophs would be
programmed to synthesize a group of
chemically related substances.
The immunofluorescent and peroxidaseconjugated antibody reactions of the
somatotrophs and lactotrophs with anti-pLPH were unexpected and cannot be explained. The dissimilarity in the amino
acid sequence of p-LPH and the two anterior lobe hormones, STH and LTH,
makes it seem unlikely that either hormone
would cross-react with anti-p-LPH. Nonetheless, the somatotrophs and lactotrophs
did fluoresce with anti-p-LPH. The fact
that STH, when added to anti-p-LPH, selectively abolished the reaction between the
somatotrophs and anti-p-LPH indicates
that the positive fluorescent and conjugated antibody reactions are due to the
presence of STH and not p-LPH in these
cells. Similarly, the selective inhibition of
the reaction between lactotrophs and antip-LPH by LTH likewise indicates that positive reactions are due to the presence of
LTH and not p-LPH in the lactotrophs.
ACKNOWLEDGMENTS
This work was supported in part by
United States Public Health Service grant
GM-2907and the A. B. Miller Fund of the
University of California, San Francisco.
LITERATURE CITED
Baker, B. L. 1972 On the origin of corticotropin in the rat hypophysis. Anat. Rec., 172:
264-265,
Baker, B. L., S. Pek, A. R. Midgley, Jr. and B. D.
Gersten 1970 Identification of the corticotropin cell in rat hypophyses with peroxidaselabeled antibody. Anat. Rec., 166: 557-567.
Birk, Y., and C. H. Li 1964 Isolation and
properties of a new, biologically active peptide
from sheep pituitary glands. J. Biol. Chem.,
239: 1048-1052.
Brookes, L. D. 1968 A stain for differentiating
two types of acidophil cells in the rat pituitary.
Stain Technol., 43: 41-42.
Cole, R. D., and C. H. Li 1955 Studies on pituitary lactogenic hormone. XIV. A simplified procedure of isolation. J. Biol. Chem., 213:
197-201.
534
H. D. MOON, C. H. LI AND B. M. JENNINGS
Graham, R. C., Jr., and M. J. Karnovsky 1966
The early stages of absorption of injected
horseradish peroxidase in the proximal tubules
of mouse kidney: Ultrastructural cytochemistry
by a new technique. J. Histochem. Cytochem.,
14: 291-302.
Heidelberger, M., and F. E. Kendall 1935 The
precipitin reaction between Type I11 pneumococcus polysaccharide and homologous antibody. 111. A quantitative study and a theory
of the reaction mechanism. J. Exp. Med., 61:
563-591.
Hess, R., D. Barratt and J. Gelzer 1968 Immunofluorescent localization of p-corticotropin
in the rat pituitary. Experientia, 24: 584-585.
Li, C. H. 1968 Current concepts on the chemical biology of pituitary hormones. Perspect.
Biol. Med., 11: 498321.
Li, C. H.,L. Barnafi, M. Chr6tien and D. Chung
1966 Isolation and structure of @-LPH from
sheep pituitary glands. Proc. 6th Pan-Am.
Congr. Endocrinol., Excerpta Medica Int. Congr.
Series, 11 2: 349-364.
Li, C. H., I. I. Geschwind, J. S . Dixon, A. L.
Levy and J. I. Harris 1955 Corticotropins
(ACTH). I. Isolation of a-corticotrouin from
sheep pituitary glands. J. Biol. Chem., 213:
171-185.
C . H., N. R. Moudgal and H. Papoff 1960
Immunochemical investigations of human pituitary growth hormone. J. Biol. Chem., 235:
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growth hormone. Arch. Biochem. Biophys., 93:
122-127.
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J. Cell Biol., 33: 307418.
Ouchterlony, 8. 1949 Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand.,
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Papkoff, H., and C. H. Li 1958 The isolation
and characterization of growth hormone from
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Pearse, A. G. E., and S . van Noorden 1963 The
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Li,
PLATE 1
EXPLANATION OF FIGURES
1
Large fluorescent somatotrophs in anterior lobe. Rabbit anti-STH,
fluorescein-conjugated goat anti-rabbit y globulin.
2 Same section as 1, after staining with carmoisine, orange G, wool
green. Orange G-positive (dark) cells are identical with fluorescent
cells in preceding figure.
3
Compact clusters of fluorescent lactotrophs. Rabbit anti-LTH, fluorescein-conjugated goat anti-rabbit y globulin.
4 Same section as 3, after staining with carmoisine, orange G, wool
green. Carmoisine-positive (dark) cells are identical with the fluorescent cells in 3.
5
Fluorescent corticotrophs in anterior lobe. Rabbit anti-ACTH, fluorescein-conjugated goat anti-rabbit y globulin.
6
Same section as 5, after staining with PAS and orange G. PASpositive (dark) cells are identical with fluorescent cells in 5 . Figures
1-6 x 400.
PITUITARY PLIPOTROPHS
H. D. Moon, C. H. Li and B. M. Jennings
PLATE 1
535
PLATE 2
EXPLANATION O F FIGURES
7 Fluorescent intermediate lobe cells occupy most of the field. A few
large fluorescent anterior lobe cells at lower right; no fluorescence of
posterior lobe at upper left. Rabbit anti-p-LPH, fluorescein-conjugated
goat anti-rabbit y globulin. x 240.
8
Positive peroxidase reaction in the intermediate lobe and in some
anterior lobe cells at lower right. Rabbit anti-p-LPH, peroxidase-conjugated sheep anti-rabbit y globulin. x 240.
9 Large .fluorescent anterior lobe cells are somatotrophs. The smaller,
less fluorescent cells (arrows) are lactotrophs. Rabbit anti-p-LPH,
fluorescein-conjugated goat anti-rabbit y globulin. x 400.
10 Same section as 9, after staining with carmoisine, orange G, wool
green. Orange G-positive cells (somatotrophs) are identical with
large fluorescent cells in 9 and have abundant dark cytoplasm. Carmoisine-positive cells (lactotrophs) have less abundant, paler cyte
plasm than somatotrophs. X 400.
536
PITUITARY PLIPOTROPHS
H. D. Moon, C . H. Li and B. M. Jennings
PLATE 2
537
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