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Relationship of three lysosomal enzymes to the Golgi zone and secretory activity in the rat pituitary and thyroid glands.

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Relationship of Three Lysosomal Enzymes to the Golgi
Zone and Secretory Activity in the R a t
Pituitary and Thyroid Glands'
Department of Pathology, Mount Sinai Hospital, New York, N. Y.
A high activity of acid phosphatase (a
characteristic lysosomal enzyme) de Duve,
'59; Novikoff, '59) has been noted in
many secreting cells, in particular in endocrine organs. Since acid phosphatase activity was found in the Golgi zone and
surrounding cytoplasmic granules, and
seemed to parallel hormone secretion in
the pituitary and thyroid glands of the
rat (Sobel, '61a, b) a role of the acid
phosphatase containing cytoplasmic granules (lysosomes?) in secretion was postulated. Because the enzyme appeared to be
associated with the Golgi zone a relationship of this apparatus to the lysosome
was implied. To investigate the assumed
relationship of the Golgi apparatus and
these acid phosphatase containing granules to lysosomes, and the relationship of
other lysosomal enzymes to secretion,
E 600 (pnitrophenyl phosphate) resistant
esterase( s) and 8-glucuronidase activities were studied histochemically. Both
enzymes are at least partly associated
with lysosomes (de Duve, '59; Wachstein
et al., '61; Goldfarb and Barka, '60).
weight and killed after 28 days. The remaining animals (10) were killed at the
same time and studied similarly.
The animals were killed by exsanguination under ether anesthesia. For histochemical studies thyroids and pituitary
glands were fixed for 24 hours in cold
formol-calcium,pH 7.0. Acid phosphatase
was demonstrated in frozen sections by an
azo-dye method (Barka, '60). Thyroid
and pituitary sections were incubated at
room temperature for 15 and 45 minutes
respectively. Esterase was demonstrated
by an azo-dye method (Davis, '59) at
pH 7.0 with and without previous incubation for one hour at 37°C with lod4 and
molar solutions of E 600 in tris buffer
at pH 7.2 (Wachstein et al., '61). Thyroid and pituitary sections were incubated at room temperature for three and
six minutes respectively. The FishmanBaker ('56) method for B-glucuronidase
was used. Thyroid and pituitary preparations were incubated at 37°C for 30 and
15 minutes respectively after 17-24 hours
at 4 A 1.5"C. Pituitary basophils were
further characterized by the periodic acidSchiff (PAS) reaction, and some acid
phosphatase preparations were counterCarworth Farms CFE (Sprague-Dawley stained by this method. The criteria of
descendant), male, albino rats of approxi- Halmi ('50, '52) and Purves and Griesbach
mately 120 gm were kept five in a cage ('5 1) were utilized in differentiating thyand had free access to water and food rotrophic from gonadotrophic basophils.
(Rockland Rat Pellets). One group of
Aldehyde fuchsin staining of the frozen
these animals (ten rats) was kept at sections was unsuccessful.
4 2 1.5"C, and killed after 21 days for
histochemical studies. Another group of
1This investieation was suuuorted bv thr 11 R
Health S&vice, Nationd' Ins&&
animals (20) was kept at room tempera- Public
Graduate Training Grant 26-115, and Grants A-3848
and A-12-62 from the National Institute of Arthritis
ture (23 2 1.5"C). Half of these ani- and
Metabolic Diseases.
mals were injected subcutaneously daily
2 Trainee in Experimental Pathology, U.S.P.H.S.,
with 10 pg of DL-thyroxine (Nutritional
3 Present address: Albert Einstein College of Me&
Department of Pathology, New York 61, N. y.
Biochemical Co.) per 100 gm of body
%T T
Esterase preparations revealed a diffuse cytoplasmic stain with increased dye
deposition in the supranuclear aspect of
the cell (fig. 1). Following incubation with
E 600 (fig. 2) most of the diffuse cytoplasmic staining was abolished and the
bulk of the dye deposit was noted in a
supranuclear zone, similar to the localization of acid phosphatase activity (fig. 3).
Many small granular foci of activity, similar to those seen on acid phosphatase preparations (fig. 3A), were noted in the
surrounding cytoplasm, usually near the
bulk of the dye deposit. In hyperplastic
glands (of rats exposed to cold) (fig. 1)
and in the smaller acini composed of
more columnar cells in all glands the enzyme activity was greater, the dye deposit was coarser and more granularity
was noted. The oval supranuclear zone
containing the bulk of the precipitate was
less well demarcated and larger in these
acini. In those animals treated with
thyroxin (fig. 1A) the flattened acinar
cells revealed a much diminished enzyme
activity. The supranuclear zones were
smaller containing finer dye deposits and
the surrounding cytoplasm revealed almost no dye deposit in these instances.
Preparations for P-glucuronidase (fig.
4) revealed diffuse cytoplasmic activity
which was more intense in the apical
aspect of the acinar cells. This pattern
was made more obvious by short incubation of the sections, and was more prominent in the columnar cells of the smaller
acini and of the hyperplastic glands. In
these instances a moderate granularity of
the dye deposit in these areas was noted.
The more flattened cells of the thyroxin
treated animals (fig. 4A) revealed a
marked diminution in enzyme activity
with a loss of this zone of accentuation.
The colloid did not stain with either of
these techniques.
As noted previously (Sobel, ’61a) the
PAS staining (basophilic) cells of the anterior pituitary often reveal an oval paranuclear clear zone. This region has been
thought to represent the Golgi zone, and
contains PAS staining material (probably
Golgi lipid) (fig. 5 ) . PAS counterstained
acid phosphatase preparations (utilizing
the Barka technique (’60)) (fig. 6 ) clearly
showed the bulk of the acid phosphatase
activity superimposed upon the PAS
stained material in the clear zone of these
cells. Many small granular areas of acid
phosphatase activity were noted in the
surrounding cytoplasm. In the thyrotrophs of the animals exposed to cold this
paranuclear vesicular zone was larger,
and contained more PAS staining material and acid phosphatase activity. The
cytoplasm of these cells exhibited more
and coarser granules of acid phosphatase
activity than in the untreated controls.
This zone appeared smaller in the thyroxin
treated animals, and showed smaller
amounts of PAS staining material and acid
phosphatase activity with fewer surrounding cytoplasmic areas of activity. The
cytoplasmic areas of activity were always
more numerous in the region closest to
the Golgi zone.
Esterase preparations (fig. 7 ) revealed
essentially the same picture as that for
acid phosphatase, though somewhat less
distinct because of diffuse background
staining. Following E 600 treatment (fig.
8 ) the diffuse cytoplasmic esterase staining was decreased and the enzyme activity in the aforementioned oval zone
and in the surrounding cytoplasmic granules was more distinctly visualized.
Preparations for P-glucuronidase (fig.
9) also revealed a diffuse cytoplasmic
staining, however, by decreasing the incubation time enzyme activity in the
‘‘Golgi zone” and surrounding cytoplasmic
granules were accentuated.
The esterase and 8-glucuronidase activities in these areas in the thyrotrophs
increased in states of increased hormone
secretion (cold treated animals) (figs. 7,
8, 9) and decreased in the thyroxin treated
animals (fig. 10) as previously described
for acid phosphatase (Sobel, ’61a).
The histochemical distribution of acid
phosphatase activity in the pituitary and
thyroid glands of the rat, and changes in
concentration of enzyme activity in different physiologic states were previously
described (Sobel, ’61a, b). At that time
the exact relationship of acid phosphatase
to the Golgi apparatus and similarly
stained granules in the cytoplasm, thought
to be lysosomes, and the interrelationship
of lysosomes to the Golgi apparatus and
secretion were considered.
It has been shown that secretion granules in these and many other organs arise
as buds from the Golgi apparatus, mature
(increasing in size and in content of secretion product) and migrate toward the
periphery of the cell enclosed in a membranous structure (Essner and Novikoff,
'61; Farquhar, '61; Herman and Fitzgerald, '61; Novikoff et al., '62; and
Novikoff, '62) which reacts like a lipoprotein to various treatments as deoxycholate
designed to liberate hormone (at least in
the pituitary) (Kamrat et al., '60). Acid
phosphatase activity has been demonstrated by the light and electron microscope in both early and late secretory
granules (containing bile) of the functional Reuber H-35 hepatoma (Essner
and Novikoff, '61 ), and in the early secretory granules of the pancreas, parotid,
sublingual, Paneth and pancreatic islet
cells (Novikoff et al., '62; Novikoff, '62).
The evidence was especially convincing
for the latter three cell types and the
Reuber hepatoma where secretion product
easily could be discerned in the acid phosphatase containing granules. Bullivant
('60) also showed acid phosphatase reaction product associated with secretory
granules (on the periphery of mature
zymogen granules of freeze substituted
and methacrylate embedded mouse pancreas). To the best of my knowledge
changes in lysosomes (acid phosphatase
containing organelles, dense bodies) have
not been described in secretion in instances where they have not accumulated
secretion product. Thus, it is probable
that the acid phosphatase containing bodies that I described were secretion granules. These granules in the thyrotrophs
were usually most densly clustered about
the Golgi zone, raising the possibility in
this instance that the more mature granules (at a greater distance from the
Gold apparatus) did not retain acid phosphatase activity. This was substantiated
by the observation of an inverse relationship between acid phosphatase activity
and cytoplasmic PAS staining (hormone
content) of the thyrotrophs. In thyroxin
treated animals these cells exhibited an
increased cytoplasmic PAS staining and
decreased acid phosphatase activity (most
secretory granules mature), conversely
the thyrotrophs of cold treated animals
revealed a decreased PAS staining and
increased acid phosphatase activity (most
early secretory granules) (Purves and
Griesbach, '51; Sobel, '61a). By analogy
with other organs it seems probable that
the acid phosphatase containing organelle
concentrates secretion product, and that
it often loses enzyme activity in this
process. It seems likely that acid phosphatase (lysosomal hydrolases? ) is involved in secretory material accumulation.
Although possible it appears improbable
that what is being observed is a parallel
lysosomal budding from the Golgi apparatus with secretion granules or that
there is transfer of acid phosphatase activity or secretory material between separate lysosomes and secretory granules.
Are these organelles (secretory granules) really lysosomes? In further delineating this relationship other lysosomal
enzymes (E 600 resistant esterase(s),
thought to be a cathepsin (Hess and
Pearse, '58) and i3-glucuronidase) were
studied under similar conditions.
Esterase preparations were previousIy
studied in the thyroid (Weber, '54; Pepler
and Pearse, '57) and pituitary (Pearse,
'56) in various states of secretory activity.
The distribution of enzymatic actvity conesponded to that which we observed. Weber
('54) described an increased enzyme activity in the thyroid of rats treated with
thyrotrophic hormone and a marked diminution in activity in rats treated with
thyroxin. Pepler and Pearse ('57) were
unable to confirm these observations.
These workers also noted total inhibition of thyroid esterase activity by
E 600. We did not obtain total inhibition
at this concentration. Our findings were
similar to those of Weber. Pearse ('56)
observed esterase activity in the Golgi
zone of the cells of the anterior pituitary
and a parallelism of cytoplasmic esterase
staining with secretory activity. In a n
informative article describing an improved
but still not entirely adequate technique
for 8-glucuronidase staining Fishman and
Baker (’56) inadequately described the
distribution of this enzyme in the rat pituitary and thyroid glands. To the best of
our knowledge this has not been described
Esterase and 8-glucuronidase are concentrated in the Golgi zone (best seen in
pituitary preparations), and in granules
in the surrounding cytoplasm. Their activities vary with functional activity of the
endocrine glands in the same manner as
acid phosphatase, further favoring the relationship of the Golgi apparatus to
lysosomes, both these organelles to secretion and the probable lysosomal nature of
the secretion granule.
The distribution of E 600 resistant esterase(s) activity was identical to that of
acid phosphatase in the rat pituitary and
thyroid glands. Staining for P-glucuronidase was diffuse, but was more accentuated in the same location.
These enzyme activities appeared to be
associated with the Golgi zone and surrounding cytoplasmic granules.
The histochemically determined enzyme activity in these areas paralleled
hormone secretion.
It is suggested that secretion granules
in these organs are probably lysosomes.
The author wishes to thank Dr. T. Barka
for his interest and encouragement, and
Dr. M. Wachstein for the E 600 used in
this work, and his helpful advice. The
advice and assistance of Drs. H. Popper
and R. Jahiel are gratefully acknowledged.
Barka, T. 1960 A simple azc-dye method for
histochemical demonstrations of acid phosphatase. Nature, London, 187: 248-249.
Bullivant, S. 1960 The staining of thin sections of mouse pancreas prepared by the
Fernhndez-MorAn helium. 11. Freeze-substitution method. J. Biophys. and Biochem. Cytol.,
8: 639-647.
Davis, B. J. 1959 Histochemical demonstration
of erythrocyte esterases. Proc. SOC. Exper.
Biol. and Med., 101: 90-93.
de Duve, C. V. 1959 Lysosomes, A New Group
of Cytoplasmic Particles. In: Subcellular Particles, T. Hayashi, ed. The Ronald Press Co.,
New York, pp. 128-159.
Essner, E., and A. B. Novikoff 1961 “Membrane Flow”: Endoplasmic Reticulum, Golgi
Apparatus and Secretory Granules. Abstracts,
First Annual Meeting of the American Society
for Cell Biology, p. 55.
Farquhar, M. G. 1961 Origin and fate of
secretion granules in cells of the anterior pituitary gland. Trans. N. Y. Acad. Sci., 23: 346351.
Fishman, W.H.,and J. R. Baker 1956 Cellular
localization of P-glucuronidase in rat tissues.
J. Histochem., 4: 570-587.
Goldfarb, S., and T. Barka 1960 Cytologic
localization of P-glucuronidase. Ibid., 8: 226227.
Halmi, N. S. 1950 Two types of basophils in
anterior pituitary of rat and their respective
cytophysiological significance. Endocrinology,
47: 289-299.
1952 Two types of basophils in the
rat pituitary: “Thyrotrophs” and “Gonadotrophs” vs. Beta and Delta Cells. Ibid., 50:
Herman, L., and P. J. Fitzgerald 1961 The
fine structure of the Golgi body following thyroid stimulation and pancreatic regeneration.
Trans. N. Y. Acad. Sci., 23: 332-345.
Hess, R.,and A. G. E. Pearse 1958 The histochemistry of indoxylesterase of rat kidney
with special reference to its cathepsin-like activity. Brit. J. Exp. Path., 39: 292-299.
Xamrat, V. B.,D. F. Hoelzl Wallach, J. F. Crigler,
Jr. and A. J. Ladman 960 The intracellular
localization of hormonal activity in transplantable thyrotrophin-secreting pituitary tumors in mice. J. Biophys. and Biochem. Cytol.,
7: 219-226.
Novikoff, A. B. 1959 Approaches to the I n
Vivo Function of Subcellular Particles. In:
Subcellular Particles, T. Hayashi, ed. The
Ronald Press Co., New York, pp. 1-22.
1962 Cytochemical staining methods
for enzyme activities: Their application to
the rat parotid gland. Jewish Mem. Hosp.
Bull., 7: 70-93.
Novikoff, A. B., E. Essner, L. Biempica and
P. Iaciofano 1962 Lysosomes and secretory
granules : Electron microscopic examination
of enzyme reaction product. Abstract, Thirteenth Annual Meeting of the Histochemical
Society, J. Histochem., 10: in press.
Pearse, A. G. E. 1956 The esterases of the
hypophysis and their functional significance.
J. Path. Bact., 72: 471487.
Pepler, W. J., and A. G. E. Pearse 1957 A
histochemical study of the esterases of rat
thyroid and their behaviour under experimental conditions. Brit. J. Exp. Path., 38:
Purves, H. D., and W. E. Griesbach 1951 The
site of thyrotrophin and gonadotrophin production in the rat pituitary studied by McManusHotchkiss staining for glycoprotein. Endocrinology, 49: 244-264.
Sobel, H. J. 1961a The localization of acid
phosphatase activity in the rat pituitary and
thyroid glands, and its relation to secretory
activity. Ibid., 68: 801-808.
1961b The relation of acid phosphatase activity of pituitary gonadotrophs and
acidophik to secretory activity in the rat.
Ibid., 69: 1108-1110.
Wachstein, M., E. Meisel and C. Falcon 1961
Histochemistry of thiolacetic acid esterase:
A comparison with nonspecific esterase with
special regard to the effect of fixatives and in.
hibitors on intracellular localization. J. Histochem., 9: 325-339.
Weber, G . 1954 L'attivita esterasica nella
tiroide di ratto nella norma e i n diverse situazioni funzionali sperimentali (trattamento con
tireotropina ipofisana e tiroxina). Arch. de
Vecchi, 22: 929-937.
All photomicrographs, except figures 5 and 6, are of uncounterstained
enzyme preparations. Figures 1 through 4 and 3A are of hyperplastic thyroids of rats exposed to cold for 21 days. Upper inserts (1A and 4 A ) are of
thyroids of rats treated with thyroxin f o r 28 days. Lower inserts ( 1 B and
4B) are of thyroids of control animals. All photomicrographs of thyroid
(figs. 1 through 4)are a t a magnification of 120 x, except figure 3A, 540 X,
those of pituitary (figs. 5 through 12) are at 1,200 X.
Esterase preparation showing diffuse cytoplasmic staining with suggestion of increased dye deposition in lumenal aspect of cell (arrow).
The acini are small, composed of cuboidal to columnar cells and generally contain little colloid. Few larger colloid-rich acini are present.
A. (Upper insert) Esterase preparation showing larger acini, composed of flattened cells and containing large amounts of colloid.
Esterase staining is weak.
B. (Lower insert) Esterase preparation showing larger colloid-filled
acini composed of more fiattened cells. (Compare fig. 1). The enzymatic activity is intermediate between that i n figures 1 and A.
Figure 1 and inserts were incubated for the same period of time.
M E 600 at 37°C for one
Similar to figure 1, but incubated in
hour prior to staining. Sharp localization of dye deposit is seen in
lumenal aspect of acinar cells (arrows).
Acid phosphatase preparation showing localization of activity similar
to that in figure 2, (closed arrow). There are a few large acini
composed of flattened cells showing little enzyme activity in lower
left and a t open arrows.
A. (Insert) Higher magnification of a n acinus from this preparation
showing cytoplasmic granules (closed arrows). The bulk of the
acid phosphatase activity is noted in a supranuclear zone (open
Preparation for p-glucuronidase similar to figure 1. Increased activity
in lumenal aspect of cells (arrows).
A. (Upper insert) Preparation for pglucuronidase similar to figure
B. (Lower insert) Similar to figure 1B.
Figure 4 and inserts were incubated for the same period of time.
Harold John Sobel
Anterior pituitary of rat stained with PAS, showing two PAS staining
(basophilic) cells (arrows) near sinusoid ( S ) . Negative images of
nuclei (open arrows). Oval paranuclear zone or Golgi zone (closed
arrows) containing PAS staining material.
PAS counterstained acid phosphatase preparation showing three PAS
staining cells (arrows). The bulk of the acid phosphatase activity is
in the oval paranuclear zone (closed arrows) apparently superimposed
on the PAS staining materials. Many small granular dye deposits are
noted in the surrounding cytoplasm. The negative images of the nuclei are at open arrows.
Esterase preparation thyrotroph of rat exposed to cold 21 days. Note
large coarsely granular central deposition of dye (Golgi zone) and
smaller dye deposits in surrounding cytoplasm. There is a faint diffuse cytoplasmic staining as well.
M E GOO a t 37°C
Preparation similar to figure 7, but incubated
for one hour prior to staining. Coarsely granular central deposition of
dye (arrow) is more distinct, as are surrounding cytoplasmic granules.
Diffuse cytoplasmic staining is almost absent.
Preparation for P-glucuronidase similar to figure 7. Note bulk of activity in oval paranuclear area (closed arrows). A few fine granular
areas of activity are noted i n the surrounding cytoplasm of the upper
cell. Negative images of nuclei at open arrows.
10 Esterase preparation of pituitary of rat treated with thyroxin. Note
small oval paranuclear dye deposits (arrows). There are few granular
areas of activity in the surrounding cytoplasm.
11 Esterase preparation of pituitary of control rat treated similarly to
that in figure 8. Note paranuclear dye deposit (arrows) and granular
areas of activity in surrounding cytoplasm intermediate between that
in figures 8 and 10.
12 Preparation for P-glucuronidase of pituitary of control rat. Note that
oval paranuclear area of activity (closed arrow) is smaller than that
in figure 9. Negative image of nucleus at open arrow. Fine cytoplasmic granules of activity are noted i n surrounding cytoplasm.
Harold John Sobel
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pituitary, enzymes, secretory, gland, activity, golgi, lysosomal, three, rat, zone, thyroid, relationships
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