close

Вход

Забыли?

вход по аккаунту

?

Studies of the nerve growth factor in submandibular glands of female mice treated with testosterone.

код для вставкиСкачать
Studies of the Nerve Growth Factor in Submandibular
Glands of Female Mice Treated with Testosterone ’
MILTON N. GOLDSTEIN AND JOS@ A. BURDMAN
Cell Biology Laboratories, St. jude Research Hospital, and the Department
of Anatomy, University of Tennessee School of Basic Medical Sciences,
Memphis, Tennessee
ABSTRACT
The effect of testosterone propionate administration on the quantity
and localization of a nerve growth protein was studied in the submandibular glands of
adult female Swiss mice. Testosterone treatment for 14 to 24 days resulted in a n
increase in the quantity of a nerve growth-promoting protein approaching those levels
found in normal untreated adult male Swiss mice. The nerve growth-promoting protein was localized by immunofluorescent techniques in the cytoplasm surrounding
the zymogenic granules of the serous tubular portions of the submandibular glands
in both control and treated animals. The role played by the submandibular gland i n
the synthesis or storage of this factor is discussed.
Lacassagne (’40) and Lacassagne and
Chamorro (’40) reported that the tubular portions of the submandibular glands
in male mice were more highly developed than those in female mice. The
administration of testosterone produced an
hypertrophy of the tubular portions of the
female submandibular glands so that they
soon resembled those of males. There were
marked differences in the chemistry, as
well as the histology, between female and
male submandibular glands. Junqueira et
al. (‘49) and Junqueira (‘51) reported that
the quantity of amylase, phosphatases and
other enzymes could be increased in the
cells of tubular portions of female glands
by the injection of testosterone. Maintenance of the “normal” morphology of the
tubular portion of the submandibular
gland in mice and in rats is dependent on
the hypophysis and the thyroid glands,
as well as the testis (Grad and LeBlond,
’49; Lacassagne, ’40). Levi-Montalcini and
Cohen (’60) and Levi-Montalcini (’64)
proposed that the tubular portion of submandibular glands may have endocrinelike functions. The growth stimulating
proteins found in cells of the serous tubular portions of the mouse submandibular
gland probably play an important role in
maintaining the “normal” histology and
function of other glands and tissues.
The male mouse submandibular gland
has been used in our laboratory for the
extraction of a nerve growth promoting
factor (NGF) (Levi-Montalcini and Booker,
ANAT. REC.. 151: 199-208.
’60; Cohen, ’60; Burdman and Goldstein,
’64). This nerve growth promoting factor
is a protein found in high concentration
in the adult male submandibular gland
and is also present, but in much lower concentrations, in the glands of female mice
The “site” of production of the nerve
growth-promoting protein is still unknown,
nor is the role played by the submandibular gland in its production or storage completely understood. Because of the histological and histochemical changes in the
female glands following testosterone administration, a study was made of the
nerve growth factor content in the female
mouse submandibular glands before and
after treatment with testosterone. The
present study reports on the localization
of the nerve growth factor in cells of the
serous tubules of the submandibular
glands of normal and treated female Swiss
mice and correlates the histological
changes in the treated female submandibular glands with increased levels of the
nerve growth factor. These studies show
that submandibular glands of testosteronetreated females can also be used for isolation of the nerve growth factor.
MATERIALS AND METHODS
Fifty Swiss adult female mice were
divided into three groups. One group of
20 animals was injected with 2.5 mg of
1 These studies were supported by Public Health
Service research grant no. CA 07477-01 from the
National Cancer Institute and by American Lebanese
Syrian Associated Charities.
199
200
MILTON N. GOLDSTEIN AND JOSE A. BURDMAN
testosterone propionate intraperitoneally
every other day for 14 days. A second
group of 20 animals was injected with
2.5 mg every other day for 24 days, and
a third group of ten animals was injected
with 3.5 mg of testosterone propionate
intraperitoneally every other day for 14
days. One day after the last injection of
testosterone the animals were sacrificed
and the submandibular glands removed.
One gram wet weight of glands from
each group was processed for NGF content through the alcohol precipitation steps
of the purification schedule described by
Cohen ('60). One submandibular gland
from one animal of each group was fixed
in 10% formalin, and sections were
stained with hematoxylin and eosin or
stained with fluorescein-labeled bovine
anti-NGF serum.2 The other submandibular gland was frozen without fixation and
sections stained with fluorescein-labeled
bovine anti-NGF serum in the same manner as the fixed glands. For immunofluorescent studies the globulin fraction
from bovine anti-mouse growth factor
serum containing 53,000 anti-NGF units
per ml was precipitated at 50% saturation
with ammonium sulfate. The immune
globulin was conjugated with fluorescein
isothiocyanate (Riggs et al., '58) and absorbed with rat liver homogenate to increase the specificity of the staining reaction (Hiramoto et al., '58). Paraffin and
frozen sections from testosterone-treated
females, non-treated females and male
mice were stained with this globulin, as
well as with normal bovine globulin conjugated with fluorescein isothiocyanate,
which served as a control for the immune
globulin. To demonstrate the specificity of
the reaction, cells were first exposed to
unlabeled bovine anti-NGF serum, washed
with buffer and then re-exposed to labeled
antiserum.
The submandibular glands of adult
Swiss female mice of the same age as the
experimental animals were processed in
the same manner and used as controls.
NGF was also isolated from untreated
adult Swiss male mouse submandibular
glands following the same procedure and
tested for NGF activity. The sedimented
fractions containing the NGF from treated
and non-treated mice were re-suspended
in normal saline and serial dilutions were
made in Hanks' solution from the clear,
pink supernatant. Nerve growth stimulating activity was assayed in cultures of
eight day chick embryo spinal ganglia.
Ganglia were explanted in Carrel flasks
on perforated cellophane in plasma clot
cultures containing one part chicken
plasma, one part growth factor to be assayed and one drop diluted bovine thrombin in Hanks' solution and incubated at
37°C for 20 hours. In all of the experiments, the growth promoting activity of
the submandibular gland fractions was
classified between 1+ and 4+, depending
on the density of the outgrowing axons
(Burdman and Goldstein, '64).
RESULTS
A detailed description of the histology
of the male and female mouse submandibular glands has already been described
by Lacassagne ('4O),
Lacassagne and
Chamorro ('4O), Junqueira et al. ('49)
and Junqueira ('51). The following is a
description of the localization of the nerve
growth promoting protein in the submandibular glands of normal female and male
Swiss mice and of testosterone-treated female Swiss mice. The nerve growth promoting protein was found only in the cells
of the serous tubular portions of the submandibular glands. When sections were
made from freshly frozen glands which
were then fixed with 95% alcohol at 37°C
or when similar sections were fixed in cold
acetone, only cells of the tubular portions
of the glands fluoresced when they were
stained with the anti-nerve growth factor
fluorescein-labeled bovine serum. After the
submandibular glands were fixed in formalin or in Bouin's and then embedded in
paraffin, a much better preservation of the
general architecture of the gland was obtained and similar localization of the NGF
was observed in the cells of the serous tubules. Excitation with ultraviolet light of
tissue fixed in formalin and embedded in
paraffin, however, resulted in autofluorescence of the basement membranes of cells
of the tubular portions of the glands. This
autofluorescence was due to fixation and
paraffin embedding and did not represent
2This serum was kindly supplied by Dr. R. K.
Richards of Abbott Laboratories.
20 1
STUDIES O F NERVE GROWTH FACTOR
localization of the NGF since it was also
observed in control sections not stained
with anti-nerve growth factor serum.
At low magnification note (figs. 1 and 2)
all of the cells of the tubules appeared to
fluoresce brilliant green. Note the intense
autofluorescence of the basement membranes in these photographs and in the
subsequent photographs (figs. 2-6). No
fluorescence was observed in control sections of kidney, liver and pancreas which
were stained in a similar manner with the
anti-nerve growth factor bovine labeled
antiserum.
When sections of normal and testosterone-treated mice were studied at high
magnification, the fluorescence which appeared to be generalized in the tubular portions of the gland at low magnification
were now found to be localized as halos
of fluorescent material surrounding the
zymogenic secretory granules of these
cells. The marked difference in the size
of the normal female and of the testosterone-treated female can be seen in figures 5 and 6. Note also the increase in
the number of the zymogenic granules and
of the “fluorescent material surrounding
the granules in the testosterone-treated
female. The fluorescence of the basement
membranes (figs. 5 and 6 ) is due to nonspecific autofluorescence caused by the
fixation and embedding process.
To test the specificity of the staining
reactions of the anti-nerve growth factor
serum, sections of freshly frozen material and of fixed and embedded material
were first exposed to unlabeled anti-nerve
growth factor bovine serum. Sections were
thoroughly washed with buffer and then
restained with fluorescein labeled antinerve growth factor serum. Sections of the
freshly frozen submandibular glands
which were subsequently fixed and stained
did not fluoresce. Only the basement
membranes fluoresced in sections of paraffin-embedded material. This demonstrated
that the staining procedure was specific
for the nerve growth factor and demonstrated its localization in cells of the tubular portions of the gland.
A comparison of the volumes of the cells
of the serous tubular portions of normal
female mice and testosterone-treated female mice revealed that the cells in the
testosterone-treated female had increased
approximately seven fold in volume. Accompanying this increase in volume was
also an increase in the content of the
nerve growth factor in the glands (note
below).
The content of nerve growth promoting
protein did not vary significantly among
the three experimental groups of female
mice receiving testosterone. NGF fractions isolated from the submandibular
glands of untreated female mice produced
a 4+ reaction in cultures of eight day
chick spinal ganglia at a dilution of
1 : 10,000. A 4f reaction was obtained
with similar fractions from male mouse
submandibular glands at dilutions greater
than 1:50,000 (note table 1). At a dilution of 1: 50,000 extracts of female glands
produced only a 1+ reaction of the chick
spinal ganglia. After the female mice had
been injected with testosterone propionate,
the content of the nerve growth promoting
protein in the glands of female mice increased to those levels found in normal
TABLE 1
Growth response of chicken embryo spinal ganglia to partially purified mouse submandibular
gland nerve gTowth extracts
Dilutions
Control
Female
Testosterone
Treated
female
1/1,000
1/5,000
1/25,000 1/250,000 1/350,000 1/500,000
Outgrowth
inhibition
4+
Outgrowth
inhibition
Outgrowth
inhibition
4
Outgrowth
inhibition
Outgrowth
inhibition
4+
3+
+
-
-
-
3+
2+
It
3+-4+
3+
2+
Control
Male
1
NGF activity increased 10-fold.
202
MILTON N. GOLDSTEIN AND JOSk A. BURDMAN
adult male mice. The fractions which were
assayed after testosterone treatment in the
female mice now produced a 4 f reaction
in chick spinal ganglia cultures at dilutions of 1:150,000 and a l+ reaction
could be obtained after a dilution of
1:500,000. A 4+ reaction was obtained
from extracts of the male glands after
dilution of 1:200,000 and the activity of
extracts from male glands disappeared
after dilution of 1:650,000. Short-term
treatment with testosterone resulted in an
increase to those levels normally found in
untreated adult male mice. In a similar
study Levi-Montalcini found that the quantity of nerve growth promoting protein in
the submandibular glands of testosteronetreated female mice even surpassed that
found in male mice.3
DISCUSSION
Although i t is apparent from these studies that testosterone administration results
in an increase in the content of the NGF
in the tubular portions of the submandibular glands of female mice, the mechanism
by which the hormone stimulates production of NGF is still unknown. The alterations in the histology and histochemistry
suggest that other growth stimulating factors discovered by Levi-Montalcini and
Cohen (’60) and Levi-Montalcini (’64)
may also be increased in the glands of the
female after testosterone administration.
The question raised by Levi-Montalcini
and co-workers, “Is the growth factor synthesized in the submandibular glands or
is it synthesized at a distant site and then
stored in these glands?” has not been answered. In an attempt to answer this
question, antiserum is being injected into
male mice so that all circulating NGF will
be bound to the antiserum. In this manner
it may be possible to prevent its localization in the submandibular glands. If the
growth factor is synthesized at a distant
site and is prevented from localizing in the
submandibular glands for a sufficiently
long period of time, one would expect the
growth factor to disappear or the quantity
of NGF protein to be markedly reduced.
The increase in the quanity of NGF
after testosterone treatment correlates with
an increase in the size of the pyramidalshaped cells of the submandibular gland.
There was also an increase in the number
of zymogenic granules and the NGF was
localized around these granules. The results indicate that testosterone can be used
to increase the amount of NGF in adult
female mice and that the female mouse
treated in this manner can be used as a
source of nerve growth promoting protein.
The submandibular glands of mice are
able to synthesize or store other proteins
which are important for survival, growth
and continued function of certain differentiating cells. Similar growth promoting
proteins may eventually be isolated from
other glands and tissues.
ACKNOWLEDGMENTS
The authors would like to thank their
colleagues, Dr. Raymond Hiramoto and
Dr. Leon Journey, for their criticism and
suggestions during preparation of the
manuscript.
LITERATURE CITED
Burdman, Josh A., and Milton N. Goldstein 1964
Long-term tissue culture of neuroblastomas.
111. In vitro studies of a nerve growth-stimulating factor in sera of children with neuroblastoma. J. Nat. Cancer Inst., 33: 123-133.
Cohen, Stanley 1960 Purification of a nervegrowth promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum.
Proc. Nat. Acad. Sci., 46: 302-311.
Grad, B., and C. P. LeBlond 1949 The necessity of testis and thyroid hormones for the
maintenance of the serous tubules of the submaxillary gland in the male rat. Endocrinol.,
4 5 : 250-266.
Hiramoto, Raymond, Milton Goldstein and David
Pressman 1958 Reactions of antisera prepared against HeLa cells and normal fetal liver
cells with adult human tissues. Cancer Res.,
18: 668-669.
Junqueira, L. C. U. 1951 Cytological, cytochemical and biochemical observations on secreting and resting salivary glands. Exptl.
Cell Res., 2: 327-338.
Junqueira, L. C. U.,A. Faier, M. Rabinovitch and
L. Frankenthal 1949 Biochemical and histochemical observations on the sexuel dimorphism of mice submaxillary glands. J. Cell.
and Comp. Physiol., 34: 129-158.
Lacassagne, A. 1940 Dimorphisme sexuel de la
glande sous-maxillaire chez la souris. Compt.
Rend. SOC.Biol., 133: 180-181.
Lacassagne, A,, and A. Chamorro 1940 Reaction a la testosterone de l a glande sous-maxillaire atrophiee consecutivement a l’hyophysectomie chez la souris. Compt. Rend. SOC.Bid.,
134: 223-224.
3
Personal communication.
STUDIES OF NERVE GROWTH FACTOR
Levi-Montalcini, Rita 1964 Growth control of
nerve Cells by a protein factor and its antiserum. Science, 143: 105-110.
Levi-Montalcini, Rita, and Barbara Booker 1960
E~~~~~~~~growth of the sympathetic ganglia
evoked by a protein isolated from mouse salivary glands. Proc. Nat. Acad. Sci., 46: 373-384.
Levi-Montalcini, Rita, and Stanley Cohen 1960
Effects of the extract of the mouse submaxil-
203
lary salivary glands on the sympathetic system
of mammals. Ann. N. y. Acad. Sci,. 85: 324341.
Riggs, J. L., R. J. Swinwald, J. Burckhalter, C. M.
Deans and T* G. Metcalf 1958 Isothiocyanate compounds as flmrescent labelhlg agents
for immune serum. Am. J. Pathol., 34: 10811097.
PLATE 1
EXPLANATION O F FIGURES
The following photographs were prepared from formalin-fixed, paraffinembedded 5 p sections of male and female Swiss mouse submaxillary
glands.
This section has been stained with fluorescein-labeled anti-mouse
submandibular gland nerve growth factor bovine serum. Note that
the cells of the serous tubular portions of the submandibular gland
fluoresce but the cells of the mucous alveolar portions do not. Arrows
point to fluorescein labeled cells of tubular portions of the normal
female mouse submandibular gland. X 300.
This is a section prepared from a normal Swiss male mouse. There
is a marked difference i n size of the serous tubular portions of the
submandibular glands compared to those in the untreated female
mice. In paraffin fixed sections the basement membranes of both
the male and female fluoresce a brilliant yellow-green. This negative
autofluorescence is contrasted by the brilliant green fluorescence of
cells in the tubular portions of the gland. X 300.
A stained section of a normal female submandibular gland photographed at higher magnification. Note the position of the nuclei i n
the pyramidal-shaped cells of the serous tubular portions of the gland.
Compare the size of the serous tubular cells with those from a testosterone-treated female i n figure 4. Photographed at low magnification.
x 500.
Section from a female mouse submandibular gland. The mouse had
been treated with 3.5 mg of testosterone propionate daily for 14 days.
Note the marked increase in the size of the cells of the tubular portions of the gland. In many sections the mucous portions of the gland
cannot be seen because of the increase in number and size of serous
tubular components. x 250.
204
STUDIES OF NERVE GROWTH FACTOR
Milton N. Goldstein and Jose A. Burdman
PLATE 1
205
PLATE 2
EXPLANATION OF FIGURES
5 At high magnification the difference i n size of the serous tubular portions of the untreated female and the treated female gland is very
apparent. Compare with figure 6. At this magnification the halos of
fluorescent nerve growth promoting protein can be seen in the cytoplasm of the pyramidal-shaped cells. Note that this material surrounds the secretory granules i n the cytoplasm and is not localized
in any one portion of the cell. The mucous alveolar portions of the
glands are not stained. Autofluorescence of the basement membranes
is very prominent. x 900.
6
206
Note the increased number of secretory granules and the distribution
of the fluorescein-labeled nerve growth protein around these granules.
Section of female mouse submandibular gland - treated with 2.5 mg
of testosterone for 24 days. x 900.
STUDIES OF NERVE GROWTH FACTOR
Milton N. Goldstein and Jose A. Burdman
PLATE 2
207
Документ
Категория
Без категории
Просмотров
2
Размер файла
657 Кб
Теги
factors, submandibular, growth, testosterone, nerve, mice, gland, female, studies, treated
1/--страниц
Пожаловаться на содержимое документа