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Effect of castration and testosterone replacement on high glucose 6-phosphatase activity in principal cells of the mouse epididymis.

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THE ANATOMICAL RECORD 207239-295 (1983)
Effect of Castration and Testosterone Replacement on
High Glucose 6-Phosphatase Activity in Principal
Cells of the Mouse Epididymis
KAZUO KANAI, SHINSUKE KANAMURA, J U N WATANABE,
MAR1 ASADA-KUBOTA, AND MOTOKO YOSHIKAWA
Department of Anatomy, Kansai Medical University, Fumizonecho I ,
Morigguchi, Osaka 570, Japan
ABSTRACT
Glucose 6-phosphatase activity is higher in the principal cell
than in other cell types in the terminal segment and caudal half of the middle
segment of the mouse epididymis. Effect of castration and testosterone replacement on the high enzyme activity in the principal cell was studied in the
terminal segment and the caudal half of the middle segment (cytochemical
study), and in the whole epididymis (biochemical study). Ten, 20, or 30 days
after castration, the abundant amount of reaction product seen in principal
cells from intact control animals decreased to the level in basal cells, halo cells,
and smooth muscle cells. However, in animals treated with testosterone following castration, the reaction product in principal cells remained abundant.
Changes in the biochemical activity after castration or testosterone administration following castration paralleled the cytochemical results. Thus, the high
activity in the principal cell is under the control of testosterone.
The ductus epididymidis is the site of maturation (initial and middle segments) and storage (terminal segment) of spermatozoa
(Glover and Nicander, 1971; Hamilton, 1975;
Brooks, 1981). The principal cell is the major
constituent of the ductus epididymidis. Because of well developed rough endoplasmic
reticulum and Golgi apparatus, although
there are no secretory granules and no indication of exocytosis, the principal cell is believed to be a secretory cell (Hamilton, 1975;
Bloom and Fawcett, 1975; Nicander and
Malmqvist, 1977; Flickinger, 1979; Brooks,
1981). On the other hand, morphological evidence of absorption has been demonstrated
in the principal cell (Nicander, 1965; Friend
and Farquhar, 1967; Hoffer et al., 1973;
Hamilton, 1975; Brooks, 1981). Thus, the
functions of the principal cell are not completely explored and understood.
Recently, we observed that in the terminal
segment and caudal half of the middle segment of mouse epididymis, glucose 6-phosphatase (G6Pase) activity was apparently
higher in the principal cell than in other cell
types (Kanai et al., 1981). In the present
study, we examined the effect of castration
and testosterone administration following
0 1983 ALAN R. LISS, INC.
castration on G6Pase activity in the cells of
the terminal segment and caudal half of the
middle segment (cytochemical study), and in
the whole epididymis (biochemical study) in
order to know whether the high G6Pase activity in the principal cell is androgen dependent.
MATERIALS AND METHODS
Male dd-Y mice, about 3 months old, were
used. The animals had free access to food and
water prior to the experiments. Animals were
divided into three groups. Animals of one
group were bilaterally castrated. Those of
the second group were bilaterally castrated
and immediately injected intraperitoneally
(IP)with testosterone propionate in a dose of
1 mg per kg body weight once a day. Those
of the third were intact. Castration was carried out under Nembutal anesthesia. Treated
animals were killed by cervical dislocation
10,20, or 30 days after castration.
Cytochemical Method
Terminal segments and caudal halves of
the middle segments were fixed by immerReceived October 4,1982; accepted June 6, 1983.
290
K. KANA1 ET AL.
sion in 2% glutaraldehyde-0.1 M sodium cacodylate (pH 7.2) for 45 minutes at 4"C, and
immersed in 0.1 M sodium cacodylate-8%
sucrose for 1 hour at 4°C. The tissues were
sectioned at 30 pm with a freezing microtome
and incubated for 1 hour a t room temperature in a reaction medium (3.7 mM glucose
6-phosphate, 80 mM sodium cacodylate, 3.6
mM lead nitrate, 230 mM sucrose, pH 6.71,
modified from the Wachstein and Meisel medium (1956). The sections were post-fixed in
buffered osmium tetroxide, dehydrated and
embedded in Epon. Thin sections were
stained with uranyl acetate and lead citrate,
and examined in a Hitachi 500 electron
microscope.
For light microscopic observations, fresh
frozen sections of the epididymides were incubated in the reaction medium for 1hour a t
room temperature, washed in distilled water,
immersed briefly in ammonium sulfide,
washed again in distilled water, and mounted
in glycerine jelly.
Biochemical Methods
Whole epididymides were homogenized a t
4°C in 50 volumes of 0.25 M sucrose in a
Potter-Elvehjem type homogenizer for 1.5
minutes a t 2000 rpm. The homogenates were
centrifuged at 2000 rpm for 15 minutes at
4"C, and assayed for G6Pase activity according to the method described by Leskes et al.
(1971). Levamizole (10 mM) was added to the
reaction mixture for inhibiting alkaline
phosphatase activity (Van Belle, 1972). Incubation time was 30 minutes. The inorganic
phosphorus released was determined by the
method of Fiske and SubbaRow (1925). Protein was estimated according to the method
of Lowry et al. (1951). Enzyme activity was
expressed as nanograms of phosphorus liberated per minute per milligram of protein of
ductus epididymidis. All of the data were
subjected to statistical analysis (Student's
t-test).
RESULTS
treated with testosterone following castration.
In normal animals, the reaction product for
G6Pase activity was observed in the endoplasmic reticulum and nuclear envelope of
all cell types composing the terminal segment and caudal half of the middle segment.
The amount of reaction product was apparently more abundant in principal cells than
in basal cells, halo cells, and smooth muscle
cells (Fig. 1).Ten, 20, or 30 days after castration, the abundant reaction product seen in
principal cells from intact control animals
decreased to the level of basal cells, halo cells,
and smooth muscle cells and, therefore, the
amount of reaction product appeared similar
in the four cell types (Figs. 2, 3). In animals
treated with testosterone following castration, however, the reaction product in principal cells remained abundant until 30 days
after castration (Fig. 4). The deposition of
final product was also observed in some lysosomes of principal cells and basal cells.
Golgi apparatus, mitochondria, plasma
membrane, and other organelles in the four
cell types showed no reaction product.
In order to ascertain whether the reaction
product is due to G6Pase activity, control
experiments were carried out as described
previously at light and electron microscopic
levels (Kanamura, 1975). Immersion of the
glutaraldehyde-fixed sections in 0.1 M acetate buffer (pH 5.0) a t 37°C for 15 minutes
before incubation in the reaction medium,
and the use of P-glycerophosphate in place of
glucose 6-phosphate in the reaction medium,
resulted in complete absence of the reaction
product except in lysosomes of principal cells
and basal cells. Pre-incubation of the sections
in 0.25 M sucrose containing 1mM Cu2+, 10
mM Zn2', 10 mM F- or 10 mM CN-, and
then incubation in the reaction medium containing equal moles of these ions, caused a
loss of the histochemical staining. These results indicate that the reaction product in the
endoplasmic reticulum and nuclear envelope
is due to G6Pase activity, but the deposition
of final product in lysosomes of principal cells
and basal cells is probably related to acid
phosphatase activity.
Cytochemical Results
Castration caused regressive changes in
the fine structure of principal cells, i.e., reduction in the height and number of apical miBiochemical Results
crovilli, and in the amount of rough endoProtein
(pg)
per
wet weight (mg) of
plasmic reticulum. These regressive changes
epididy
mis
appeared more pronounced with time after
castration. However, no morphologic changes
Ten days after castration, the value deoccurred in principal cells from animals creased (from 90.3 f 1.45 to 55.2 f 7.53, the
GGPASEIN EPIDIDYMIS
Figs. 1-4. Cytochemical demonstration of G6Pase activity in the cells of the mouse epididymis. Sections (30
fim) cut from glutaraldehyde-fixed tissues were incubated in a modified Wachstein and Meisel medium for 1
hour. Figures 2-4 appear on following pages.
Fig. 1. Principal cells (PI, a basal cell (B), and smooth
mean of 5 animals S.E.M., P < 0.01) and
then did not change significantly. Testosterone administration after castration maintained the protein level of the intact control
animals.
G6Pase activity
The value decreased to 35% (P < 0.001) of
that of the intact animals 10 days after castration, and thereafter remained unaltered
291
muscle cells (S) of caudal half of the middle segment
from a n intact control animal. The reaction product for
GGPase activity is seen in the endoplasmic reticulum
and nuclear envelope of these cells. Note that deposition
of reaction product is apparently more abundant in principal cells than in basal cell and smooth muscle cells.
x5,500.
(Fig. 5). In animals treated with testosterone
following castration, the value was unchanged except €or a decrease (P < 0.05)
between 10 and 20 days. When the activity
was expressed as nanograms of phosphorus
per minute per wet weight of epididymis, the
difference in the pattern of changes in the
activity between castrated animals and testosterone-treated animals after castration
became more distinct. This is due to the
292
K. KANA1 ET AL.
GGPASE IN EPIDIDYMIS
293
Fig. 4. Principal cells (P), a basal cell (B), and smooth
muscle cells (S) of caudal half of the middle segment
from an animal treated with testosterone (1 mgkg, once
a day) during 30 days after castration. The reaction
product for the activity is apparently more abundant in
principal cells than in basal cell and smooth muscle cells
(cf. Fig. 1).~ 8 , 0 0 0 .
marked decrease in protein concentration in
the epididymis after castration.
tains the abundant amount of reaction product in principal cells, if animals are castrated.
Further, the biochemical activity of whole
epididymis parallels generally these cytochemical results. Therefore, the high G6Pase
activity in principal cells of the mouse epididymis is androgen dependent.
A marked regressive change occurred in
the endoplasmic reticulum of principal cells
after castration. Therefore, the reduction of
G6Pase activity in principal cells after castration may be related to the regressive
change in the endoplasmic reticulum. Brooks
(1976a,b, 1978, 1979, 1981) showed that castration results in a decline in the activity of
a number of enzymes associated with glycolysis, tricarboxylic acid cycle, lipid oxidation,
and others in the epididymis. Alkaline phosphatase (Allen and Slater, 1957; Ono, 1973;
Sreenivasulusetty et al., 1977), aliesterase
DISCUSSION
As revealed in the present cytochemical
results, castration results in an apparent reduction of the amount of reaction product for
G6Pase in principal cells to the level in basal
cells, halo cells, and smooth muscle cells.
However, testosterone administration main-
Figs. 2, 3 . Principal cells (P) and basal cells (B) of
caudal half of the middle segment from animals 10 days
(Fig. 2) and 30 days (Fig. 3 ) after castration. Note a
reduction in the amount of reaction product for the activity in principal cells compared to that in principal cells
in Figure 1. The amount of reaction product appears
similar in principal cells and basal cells. ~ 5 , 5 0 0 .
294
K. KANA1 ET AL.
I
0
10
20
30
Days a f t e r castration
Fig. 5. Effect of castration and testosterone administration following castration on G6Pase activity. Testosterone was injected in a dose of 1 mgkg once a day. Each
point represents the mean of 5 animals and the stan-
dard error is shown as a vertical line. * P < 0.05, ** P
i0.01, *** P < 0,001: significantly different from value
of animals castrated and injected with testosterone.
(Allen and Slater, 1957), acid phosphatase
(Allen and Slater, 1958; Sreenivasulusetty et
al., 1977) and hyaluronidase (Sreenivasulusetty et al., 1977) activities in the epididymis
or principal cell also decrease after castration. Brooks (1981)considered that the reduction in enzyme activity reflects a decrease in
the number of enzyme molecules rather than
in the catalytic activity of each enzyme molecule, because the reduction in activity of the
affected enzymes is relatively slow (about 12
days) after castration. The reduction in enzyme molecules is effected when their rate of
degradation is more than their rate of synthesis in the endoplasmic reticulum. In the
regressed endoplasmic reticulum after castration, the decline in the enzyme activities
occurs probably by such a mechanism. This
situation is probably applicable to G6Pase.
G6Pase activity has been detected in a variety of cell types of various organs. The activity is high in hepatocytes (Tice and
Barrnett, 1962; Kanamura, 1971a; Leskes et
al., 19711,proximal convoluted tubule cells of
the kidney (Kanamura, 1971b1, and jejunal
epithelial cells (Hugon et al., 1970, 1971).
The activity of hepatocytes and proximal
convoluted tubule cells is concerned with
supplying glucose to the blood from the liver
and kidney (Krebs, 1963; Nordlie, 1969), and
that of jejunal epithelial cells might relate to
the absorption of nutrient. However, the
function of this enzyme is unknown in other
various cell types containing low activity. In
the terminal segment and caudal half of the
middle segment, the activity is apparently
higher in the principal cells than in basal
cells, halo cells, and smooth muscle cells.
Therefore, it is supposed that the enzyme in
principal cells performs a specific function
like the enzyme in hepatocytes and proximal
convoluted tubule cells.
As revealed in the present results, the high
G6Pase activity in principal cells appears to
be under the control of testosterone. Therefore, it is suspected that the enzyme plays
some role in the maturation and storage of
the spermatozoa in the ductus epididymidis.
The spermatozoa use fructose in the semen
as an energy source for their movement
(Mann, 1964).However, the spermatozoa may
need glucose during their maturation and
storage in the ductus epididymidis. The appearance of glucose has been reported in the
G6PASE IN EPIDIDYMIS
epididymal fluid (Cooper, 1982). The role of
G6Pase in the principal cell may be to release glucose into the epididymal fluid surrounding the spermatozoa.
ACKNOWLEDGMENTS
This work was partially supported by a
grant (no. 5677028) from the Japanese Ministry of Education.
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principaux, effect, testosterone, high, castration, mouse, activity, phosphatase, epididymal, replacement, cells, glucose
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