Male Reproductive Defects Caused by Puromycin-Sensitive Aminopeptidase Deficiency in Mice
Tomoharu Osada1,
Gen Watanabe,
Shunzo Kondo,
Masashi Toyoda,
Yoshiyuki Sakaki and
Takashi Takeuchi
Mitsubishi Kasei Institute of Life Sciences (T.O., S.K., M.T.,
T.T.) Tokyo,1948511, Japan
Laboratory of Functional
Genomics (T.O., Y.S.) Human Genome Center The Institute of
Medical Science University of Tokyo Tokyo 108-8639, Japan.
Laboratory of Veterinary Physiology (G.W.) Tokyo University
of Agriculture and Technology Tokyo 183-8509, Japan
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ABSTRACT
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Male reproductive performance is composed of two
principal elements, copulation and spermatogenesis. A wealth of
literature has described the intricate web of endocrine events
underlying these biological processes. In the present study we show
that puromycin-sensitive aminopeptidase (Psa)-deficient mice are
infertile, lack copulatory behavior, and have impaired spermatogenesis.
The reproductive deficits of the mutants are not restored by androgen
administration, although no aberrant localization of the sex steroid
receptors was detectable in their brains and testes. Considering the
strong expression of the Psa gene in the brain and Sertoli cells and
the degenerative morphology of Sertoli cells in Psa-deficient mice, Psa
may participate in testosterone-mediated reproductive signal pathways
in the brain and testis.
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INTRODUCTION
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Male reproduction requires the coordinated regulation of
reproductive hormones comprising the
hypothalamic-pituitary-gonadal axis. The axis is crucial for
germ cell development, reproductive organ function, and sexual
behavior. A number of molecules have been investigated to determine
their involvement in the appearance of reproductive performance in
males (for review, see Refs. 1, 2). However, their intricate
regulatory machinery is still not fully understood.
Attempts to understand the neural regulation of sexual behavior
have been based on the results of removing areas of the brain. Many
lesion studies have revealed several confined brain areas that play
important roles in the neural control of copulation (for review, see
Ref. 1). Among these brain areas, the medial preoptic area (MPOA)
appears to be an integral component of this system (3) (for review, see
Ref. 1). Chemical stimuli, such as pheromones and endogenous
testosterone, which stimulate neural activity in the MPOA via the
olfactory neural network and testosterone-responsive pathways, are
integrated in the MPOA and lead to the expression of masculine mating
behavior. A number of steroid signaling pathways and neurochemical
systems exist in the MPOA and project into other brain areas involved
in sexual behavior in males (for review, see Ref. 1). Among these
molecules, testosterone acts as a key regulator of copulation (4)
through several receptors in vivo, including the androgen
receptor (AR) and estrogen receptor-
(ER
) and ß (ERß) (for
review, see Refs. 5, 6).
Spermatogenesis requires the physical and tropic support of Sertoli
cells, although the intrinsic regulation of spermatogenic cells is
required as well (7, 8, 9). This tropic support depends on a network of
reciprocal signalings among Leydig cells, Sertoli cells, peritubular
cells, and germ cells within the testis, which is regulated upstream by
the hypophyseal hormones (for review, see Ref. 2). These cell-cell
interactions among intratesticular cells have been investigated at the
cellular level. Testosterone is a key molecule also in spermatogenesis,
is secreted from Leydig cells, stimulates Sertoli cell activity, and
regulates spermatogenesis (for review, see Ref. 2).
In the present study, we demonstrate that puromycin-sensitive
aminopeptidase (Psa)-deficient mice are infertile, lack copulatory
behavior, and have impaired spermatogenesis. Psa-deficient mice
(designated as Psagoku/goku) produced by a
gene-trap method have shown increased anxiety and impaired pain
response (10). Psa has been characterized and purified as a putative
extracellular enkephalinase in vivo (11). The intracellular
localization of Psa (12, 13) as well as our previous study using Psa
mutant mice (10), however, imply intracellular roles for Psa.
We have also found in the present study that abnormal reproductive
phenotypes are insensitive to testosterone administration, and that the
function or survival of Sertoli cells, in which Psa is expressed
strongly, might be affected. Together with the comparable levels of
ER
in the brain and AR in testes of
Psagoku/goku mice to those in normal
animals, Psa may participate in AR/ER responsive signalings.
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RESULTS
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Sterility and Lack of Copulatory Behavior in
Psagoku/goku Male Mice
Male Psagoku/goku mice did not
sire when housed with Psa+/+ females over 1
month. The testes and seminal vesicles of
Psagoku/goku mice (Fig. 1B
) were significantly reduced in weight
compared with Psa+/+ mice (seminal vesicle;
Psa+/+ mice, 175.2 ± 14.7 mg, n = 5;
Psagoku/goku mice, 96.6 ± 11.8 mg,
n = 7; P < 0.001; testes;
Psa+/+ mice, 86.0 ± 5.9 mg, n = 4;
Psagoku/goku mice, 45.3 ± 3.3 mg,
n = 6; P < 0.001). Adult male
Psagoku/goku mice, however, showed no
morphological deficiency in the external genitalia. These observations
imply that Psa is not required for sex determination.

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Figure 1. Gross Morphology of Male Reproductive Tract and
Spermatogenic Status of Psa+/+ and
Psagoku/goku Males
A and B, Male reproductive tract of 12-week-old
Psa+/+ (A) and
Psagoku/goku (B) males. Bar
represents 1 cm. C, Cross-sections of seminiferous tubules stained with
PAS and hematoxylin from 11-week-old Psa+/+
animals at stage II-III containing step 1415 spermatids. D,
Cross-sections of tubules from aged matched
Psagoku/goku male mice. Disordered
spermatogenesis and vacuolar structure can be seen. Bars
represent 100 µm. E, Flow cytometry histogram of intratesticular
cells stained with propidium iodide. Gates represent karyotype. a and
b, Histogram showing the number of cells at each fluorescence of
Psa+/+ and
Psagoku/goku males, respectively. c,
Integrated histogram of a and b. Note the altered distribution of
fraction 1N from Psagoku/goku male mice
(red), compared with Psa+/+
animals (black) indicated by the arrow.
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To assess sexual behavior, we observed the copulatory behavior of 8- to
15-week-old males of each genotype when housed with a 5- to 6-week-old
estrous female for 2 h (Table 1
). Male sexual behavior is composed
mainly of courtship and precopulatory behavior, mounting, intromission,
and ejaculation (1, 14). We assessed the expression of ejaculation by
confirming the presence of vaginal plugs on the day after the
experiments, because it is difficult to distinguish clearly between
intromission and ejaculation by our method.
During the testing periods, Psa+/+ males
exhibited overall masculine sexual behavior within 1 h. Female
mice housed with Psa+/+ males had a high
frequency of vaginal plugs (Table 1
). In
contrast, Psagoku/goku males exhibited
adequate precopulatory behavior (allo-grooming and anogenital contact,
followed by sniffing), but did not display the onset of either mounting
behavior or intromission. Furthermore, vaginal plugs in females housed
with Psagoku/goku males were not detected
the following morning (Table 1
). This indicates that
Psagoku/goku males did not ejaculate.
Although erectile dysfunction in
Psagoku/goku males cannot be ruled out,
these observations suggest that the infertility in
Psagoku/goku males is due to a lack of
copulatory behavior.
Impaired Spermatogenesis in
Psagoku/goku Males
Spermatogenesis is a major component of male reproduction.
We found a reduction in the size of the testes of
Psagoku/goku males compared with
Psa+/+ mice (Fig. 1
, A and B). Therefore,
spermatogenesis in Psagoku/goku males was
also examined. The number of sperm flushed from the caudal epididymides
of Psagoku/goku males at 812 weeks of
age was significantly decreased compared with
Psa+/+ animals (Psa+/+,
2.91 ± 0.56 x 106 sperm per epididymis, n
= 8; Psagoku/goku, 0.251 ±
0.065 x 106 sperm per epididymis, n = 10;
P < 0.001). Furthermore, most of the sperm collected
from Psagoku/goku males exhibited poor
movement and some lacked any movement. We next examined fertilization
by Psagoku/goku sperm in vitro
(15). Sperm collected from the caudal epididymides of
Psagoku/goku males lacked the ability to
fertilize intact oocytes in vitro (Table 2
). These observations suggest that
spermatogenesis in Psagoku/goku males is
impaired by Psa deficiency.
The spermatogenic status of
Psagoku/goku males was assessed
morphologically with cross-sections of semiferous tubules in
11-week-old mice of both genotypes. Fifty sectioned tubules of each
genotype were randomly selected. The cell type and stage definitions
used herein are those of Russell et al. (16). The
cross-sections of Psa+/+ animals revealed an
organized spermatogenic cycle (Fig. 1C
). In contrast, the stages of
most tubules from Psagoku/goku males were
difficult to categorize because concentric organization of the germ
cells was not detected and there were only a few late elongated
spermatids (steps 1516) (Fig. 1D
). Moreover, vacuolar structures were
frequently observed in the tubules of
Psagoku/goku males (Fig. 1D
), suggesting
germ cell degeneration. After DNA staining of the testicular cells, the
stained cells can be sorted by the cell sorter according to the
intensity of the fluorescence emission, which corresponds to DNA
content. Among the karyotype 1N cells of Psa+/+
mice, two predominant peaks of fluorescence with wide ranges were
apparent (Fig. 1E
), one representing round spermatids and the other
elongated spermatids and spermatozoa (17). In contrast, testicular
cells of Psagoku/goku males showed an
absence of the peaks representing elongated spermatids and spermatozoa
(Fig. 1E
). These results are consistent with a reduction in spermatozoa
accumulated in the epididymis and the observation that elongated
spermatids in Psagoku/goku males are most
severely affected in histological analyses (Fig. 1D
).
Testicular Development in
Psagoku/goku Males
We assessed the development of spermatogenesis in
Psagoku/goku testes. Histological studies
of each genotype from postnatal day 16 to 1 yr of age revealed that the
spermatogenic stage from spermatogonia to spermatids at step 9 (16)
appears normal (Fig. 2
, AD). Together
with the decreased number of late elongated spermatids (Fig. 1
, D and
E), these data suggest that spermiogenesis after step 9 is affected.
The observed abnormalities in the seminiferous tubules of
Psagoku/goku mice were progressively
affected depending on the age of the mouse examined, none of which
could be categorized according to the normal spermatogenic cycle by 24
weeks of age (Fig. 2
, E and F). In addition, frequency and area of the
vacuolar structures increased (Fig. 2
, F and H). By 1 yr of age, most
seminiferous tubules in Psagoku/goku males
lacked most germ cells (Fig. 2H
). These observations indicate that the
survival of germ cells is affected in addition to the defects in
spermiogenesis.

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Figure 2. Development of the Testes of
Psagoku/goku Male Mice
Cross-sections of seminiferous tubules of
Psa+/+ (left column) and
Psagoku/goku males (right
column) at postnatal day 25 (P25) (A and B) and day 35 (P35) (C
and D), 24 weeks old (24W) (E and F), and 1 yr of age (G and H).
Sections were stained with PAS and hematoxylin. Bar
represents 100 µm.
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Hormonal Levels and an Attempt to Restore the Phenotype of
Psagoku/goku Male Mice by Androgen
Administration
The appearance of copulatory behavior and the induction of
spermatogenesis are regulated by androgens (4). We examined the plasma
levels of testosterone and also the hypophyseal hormones (LH, FSH, and
PRL) that regulate male reproduction cooperatively. The mean plasma
levels of testosterone, LH, FSH, and PRL in
Psagoku/goku males were approximately
57%, 64%, 86%, and 84% of those in Psa+/+
males, respectively (Table 3
).
Statistical analysis (Students t test) revealed
significant differences in the levels of LH and FSH between the
genotypes while the differences in testosterone and PRL were not
significant (Table 3
).
Next, we examined whether the disruption of male reproduction in
Psagoku/goku mice could be restored by
androgen administration. Perinatal androgens are known to be crucial
for masculine sexual behavior in rodents (18). Therefore, we prepared
two types of treated Psagoku/goku male
mice; 1) those that received neonatal injections of testosterone
propionate (TP) in olive oil (for control mice, only olive oil was
injected) and implantation of a tube containing TP in the adult, and 2)
those that underwent implantation of a TP tube in the adult without
prenatal treatment. In both treatments, tubes containing Dulbeccos
PBS were used as controls. The effects of administration of TP were
assessed after 4 weeks of implantation by measuring the plasma
testosterone levels of the type 2 treated mice
(Psa+/+, 17.10 ± 1.59 ng/ml, n = 3;
Psagoku/goku males, 21.73 ± 3.59
ng/ml, n = 6, respectively). The levels of testosterone in
Psagoku/goku males showed a significant
increase compared with those of nontreated mice (Table 3
,
P < 0.01). Both treatments resulted in restoration of
the weight of the seminal vesicles of the
Psagoku/goku males (Fig. 3
, AC). The treated
Psa+/+ males displayed intact fertility.
Conversely, three type 1, and six type 2 treated
Psagoku/goku mice displayed neither
copulation nor fertility. Furthermore, the reduction in the number of
sperm accumulated in the epididymides was not restored (Fig. 3D
).
Impaired spermatogenesis in the testes of
Psagoku/goku mice was also not restored
after the administration of TP, which is the same as shown in Fig. 1
(Fig. 3
, EG).

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Figure 3. Effects of Testosterone Administration in
Psagoku/goku Animals
A, 12W seminal vesicle with coagulating gland of
Psagoku/goku (top) and
Psa+/+ (bottom) animals. B,
Seminal vesicles from the same animals as shown in panel A collected 4
weeks after implantation of the TP-containing tube. C and D, Wet weight
of seminal vesicle (C) and number of sperm accumulating in the
epididymis (D) after TP treatment. Gray,
orange, and green bars represent mean
values from mice receiving control treatment, TP implants as adults,
and TP injection neonatally followed by TP implants as adults,
respectively. The number of mice examined is shown in
parentheses. The error bars indicate SEM.
EG, Cross-sections of seminiferous tubules of
Psa+/+ (E) and
Psagoku/goku animals receiving TP implants
as adults (F), and a Psagoku/goku animal
receiving a TP injection neonatally in addition to a TP implant as an
adult (G). Note the disorganized spermatogenesis regardless of TP
treatment (F and G). Bars represent 1 mm (A and B) and
100 µm (EG).
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These data indicate that the impairments in copulation and
spermatogenesis in Psagoku/goku males are
insensitive to testosterone.
Immunolocalization of Steroid Receptors and Other Related Molecules
in the Brain and Testis
To examine whether the failure to repair the reproductive defects
in Psagoku/goku males derives from
deficiencies in AR, ER, or both, we investigated the immunoreactivity
of AR and ER in the brain and testes of Psa+/+
and Psagoku/goku males.
The ER-mediated pathway is required, in part, for the appearance of
sexual behavior in male mice (for review, see Ref. 1). We examined the
immunolocalization of ER
positive cells in the MPOA. The MPOA has
been well established as a center for masculine sexual behavior in
rodents. No apparent differences between genotypes in localization or
intensity were detectable for cells positive for ER
in the MPOA
(Fig. 4
, A and B). Next, we examined the
immunoreactivity of other molecules associated with reproductive
performance in the male: GnRH-associated peptide (GAP; characterized as
a marker of the GnRH neurons), CRH, ßendorphin, and enkephalins
in the MPOA and medial basal hypothalamus including the paraventricular
nucleus, arcuate nucleus, and the median eminence (for review, see Ref.
19). Comparable staining intensities and localizations for these
molecules were detected for both genotypes (GAP; Fig. 4
, C and D).

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Figure 4. Immunohistochemical Analyses in Brains and Testes
Immunolocalization of ER (A and B) and GAP (C and D) in MPOA and AR
(E and F) and inhibin (G and H) in the testis of
Psa+/+ (A, C, E, and G) and
Psagoku/goku (B, D, F, and H) animals.
Comparable staining intensities were detectable for ER (A and B) and
GAP (C and D) in the MPOA of both genotypes. E and F, AR
immunoreactivity was present in the nuclei of Leydig cells and Sertoli
cells (arrows) of both genotypes. The staining intensity
did not differ apparently between Psa+/+ (E)
and Psagoku/goku male mice (F). G and H,
The staining intensity against inhibin was decreased in most of the
Sertoli cells from Psagoku/goku male mice
(H, arrows) compared with
Psa+/+ mice (G, arrows).
Bars represent 100 µm (AD) and 20 µm (EH).
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AR immunoreactivity was present in the nuclei of spermatogonia, Sertoli
cells, Leydig cells, and elongated spermatids in the seminiferous
epithelia in the testes of Psa+/+ and
Psagoku/goku mice. This observation is
consistent with previous studies (20, 21). The staining intensity of
most Sertoli cells was comparable between genotypes (Fig. 4
, E and F).
Next, we examined the expression of inhibin
, a subunit of inhibin
that is expressed in Sertoli cells and has important roles in
spermatogenesis. The immunoreactivity of inhibin
in most Sertoli
cells from Psagoku/goku mice revealed a
reduction in intensity compared with Psa+/+
(Fig. 4
, G and H), while the immunoreactivity of WT-1, which is also
expressed in Sertoli cells and is a differentiation marker, did not
show apparent differences, suggesting that Sertoli cells in
Psagoku/goku mice differentiate
developmentally (data not shown). The observation was coincident with
the results of decreased levels of inhibin
in Western blot analysis
(Fig. 5
). We measured the intensity of
the bands and compared them between genotypes. We found that those for
Psagoku/goku mice were significantly
reduced (Psa+/+ mice, 222.2 ± 3.0, n
= 3; Psagoku/goku mice, 151.1 ±
12.6, n = 3; P < 0.01), while the intensity of
the bands against anti WT-1 did not exhibit significant differences
(Psa+/+ mice, 222.8 ± 20.8, n = 3;
Psagoku/goku mice, 246.9 ± 2.8,
n = 3; P = 0.31). We also detected decreased
levels of inhibin ß (Fig. 5
) (intensity,
Psa+/+ mice, 228.0 ± 9.3, n = 3;
Psagoku/goku mice, 121.0 ± 33.7,
n = 3; P < 0.05). These results suggest that the
expression of AR is normal, while functions including the expression of
inhibin are impaired in Sertoli cells in
Psagoku/goku mice.

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Figure 5. Immunoblot Analysis of Inhibin in the Testes
Proteins extracted from the testes of two
Psa+/+ and
Psagoku/goku male mice were analyzed by
immunoblot analysis. Upper, middle, and lower
panels show the results using anti- WT-1, inhibin , and
inhibin ß antibodies, respectively. Note the reduced expression
levels of inhibin and ß in
Psagoku/goku males.
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Electron Microscopic Analysis and Expression of the Psa Gene in
Testes
Transmission electron microscopic analysis (Fig. 6
, AD) was performed to characterize
the aberrant status of the testes of
Psagoku/goku animals. In 11-week-old
Psagoku/goku animals, the morphology of
the Leydig cells was not severely affected (data not shown). In
contrast, some Sertoli cells were found to have degenerated
morphologically compared with Psa+/+ animals
(Fig. 6
, A and B). Sertoli cells from Psa+/+
mice adhere tightly to germ cells and provide physical and tropic
support for the developing spermatogenic cells (22). The abnormal
Sertoli cell morphology in Psagoku/goku
animals is likely to lead to impaired physical interactions with
encompassing spermatogonia, spermatocytes, and spermatids (Fig. 6
, A
and B).

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Figure 6. Transmission Electron Microscopic Analysis and the
Expression of Psa in Testes
AD, Transmission electron micrograph of testes derived from
11-week-old Psa+/+ (A) and
Psagoku/goku male mice (BD). Note the
morphologically degraded Sertoli cell (B, encompassing
arrowheads) and germ cell abnormalities (C, multinuclear
cells and D, vacuolation of the cytoplasm of spermatids are shown by
asterisks, respectively) in
Psagoku/goku mice. E, Low-magnification
view of a cross-section of seminiferous tubules from an adult
Psa+/goku animal stained with X-gal to see
Psa expression. F, High-magnification view showing an example of Psa
expression in Sertoli cells and Leydig cells (blue). The
section was stained doubly with X-gal (blue) and
antiinhibin as a marker for Sertoli cells (brown).
Bars represent 100 µm (E) and 10 µm (F).
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This observation is consistent with the appearance of multinuclear
cells that may result from opened intracellular bridges connecting
spermatids (Fig. 6C
). In spermatogenic cells, most of the observed
elongated spermatids were not advanced beyond step 10, and, if
advanced, the heads of the elongated spermatozoa were mostly affected
morphologically and no juxtaposed mitochondria were detected. Moreover,
extensive vacuolation was seen within the cytoplasm of the elongating
spermatids (Fig. 6D
). These observations may result from impaired
Sertoli cell function and reflect the decreased number of sperm in the
epididymides of Psagoku/goku mice, and
their inability to fertilize eggs in vitro.
Next, expression of the Psa gene in the testes was examined. Because
the Escherichia coli ß-galactosidase gene
(lacZ) was inserted into the Psa gene, we can monitor the
expression of the Psa gene using ß-galactosidase activity (10). X-gal
staining was performed to examine ß-galactosidase activity, and the
activity was detected predominantly in Sertoli cells and Leydig cells
(Fig. 6
, E and F). Very weak activity was observed in spermatogonia and
spermatocytes, but not in spermatids.
Together with Psa gene expression in Sertoli cells, Psa may play
critical roles in either Sertoli cell survival or functions that
support germ cells.
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DISCUSSION
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The Basis of Suppression of Copulatory Behavior in
Psagoku/goku Mice
Masculine sexual behavior is regulated by androgens (Ref. 4 ;
for review, see Ref. 1). Replacement of testosterone to castrated male
animals can restore copulatory behavior (for review, see Ref. 1).
However, in the present study the administration of androgen in
neonates and adults failed to restore copulatory behavior in
Psagoku/goku males. Psa gene is expressed
strongly in almost all neurons in the brain (10). These facts
strongly suggest that the lack of copulatory behavior in
Psagoku/goku males results from defects in
the brain, and that the defects are independent of those in the
testes.
Immunohistochemical analyses revealed no significant alteration
in the MPOA region of Psagoku/goku mice
with respect to the distribution and staining intensity of ER and other
molecules. Based on these observations, we hypothesize that Psa may be
a component of the AR/ER-responsive pathways. The reproductive
phenotypes of Psagoku/goku mice resemble
those described for ER
gene-deficient mice. ER
deficiency leads
to greatly reduced copulatory behavior (23, 24). Furthermore, previous
studies suggest that AR activation is necessary to express sexual
behavior as well as ER
-mediated signaling (25). Based on the
observations in this manuscript, Psa may assemble and modulate both the
AR and ER-mediated signaling that give rise to masculine copulatory
behavior. Previous studies have reported that testosterone stimulates
growth in female rats and that testosterone deficiency leads to
behavioral depression in mice due to castration (26, 27). These
physiological effects of testosterone (including estrogens)-responsive
pathways possibly support our hypothesis because
Psagoku/goku mice exhibit dwarfism and
behavioral impairment-associated anxiety (10).
The Basis of Spermatogenetic Disruption in
Psagoku/goku Mice
In this study, the Psa gene was found to be
expressed strongly in Leydig and Sertoli cells in the testes. Psa
gene expression was not detected in elongated spermatids and
spermatozoa that had advanced beyond step 10, the stage that is
severely affected in Psagoku/goku mice.
Moreover, decreased levels of inhibin were observed in Sertoli cells of
Psagoku/goku mice. Together with
morphological degeneration in Sertoli cells of
Psagoku/goku mice, Psa deficiency is
likely to affect not only inhibin expression but also other Sertoli
cell-secreting molecules. These observations further suggest that Psa
in Sertoli cells is required for Sertoli cell function and
survival.
The same explanation given for the impaired copulatory behavior
described above can explain the deficits in spermatogenesis of
Psagoku/goku males. Sertoli cells express
AR and ERß in mice (28). Moreover, morphological changes in
testicular cells by pharmacological destruction of the Leydig cells can
be prevented by testosterone administration (29). These studies
indicate that AR/ER signaling plays important roles in functions of
Sertoli cells for spermatogenesis. According to this hypothesis,
deficits in spermatogenesis in
Psagoku/goku males are likely to arise
from hampered AR/ER signaling within Sertoli cells.
Putative Role of Psa in Vivo
A possible role of Psa in steroid signal pathways can be
hypothesized. The efficient signal transduction of the steroid hormones
requires a number of accessory proteins (30). Upon 17ß-estradiol
binding, ER dissociates from the complex and works as a transcriptional
factor (31). The mechanisms of signal transduction by steroid receptors
are complex and not fully understood. The degenerative pathways for the
heterocomplex and receptor-hormone complex are still unclear. A
previous study suggested that the ubiquitin-proteasome pathway may
contribute to part of the chaperone machinery (32). Psa protein
contains two motifs showing significant similarity to a sequence in the
26S proteasome subunits (13). This suggests that Psa participates in
either the proteolysis of the chaperone complex, posttranscriptional
ER-ligand complexes, or both. In fact, proteasome appears to be
responsible for the degradation of key regulatory proteins, such as
transcriptional factors (33). Thus, ER may fail to interact with
hormones because of the excess amount of chaperone complex in
Psagoku/goku animals. Otherwise, a lack of
degradation of the receptor-steroid complex may result in an excess
flow of the transcriptional stimulation of AR/ER, which leads to a
disruption in the normal regulation of reproductive performance in
Psagoku/goku males.
Further analyses of Psagoku/goku mice will
resolve the nature of the testosterone-insensitive deficits and Sertoli
cell degeneration resulting from Psa deficiency.
Psagoku/goku mice provide a tool with
which to investigate the role of Psa in the molecular machinery
underlying male reproductive performance.
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MATERIALS AND METHODS
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Mice
Psagoku/goku mice were generated by
intercrossing goku heterozygous mice. F2021 heterozygotes
obtained by backcrossing with BALB/cA strain mice were used as parents.
All animals were maintained one to three mice per cage with a 12-h
light/12-h dark cycle. Food and water were given ad libitum.
All experiments involving animals were performed in accordance with
standard ethical guidelines for the care and use of laboratory animals
(NIH Standards for Treatment of Laboratory Animals, 1985) and approved
by the Ethical Committee of our institute.
Copulatory Behavior and Fertilization Analyses
Five to 6-week-old female BALB/cA mice (Clea Japan, Tokyo,
Japan) were treated by ip injection with PMSG (5 IU; Serotropin,
Teikoku-Zouki Co. Ltd., Tokyo, Japan) 2 days before the
observation of copulatory behavior. On the day of an experiment, estrus
was induced in the females by the injection of human CG (hCG, 5 IU;
Gonatropin, Teikoku-Zouki Co. Ltd.) eight hours before the
experiment. At 2200 h, a female was placed in a separate cage with
an 8- to 15-week-old virgin male mouse. The cages were videotaped for
2 h under dim light. The observed behaviors were assessed as
described by McGill (14) (for review, see Ref. 1). The females were
checked for vaginal plug formation the following morning. Duplicate
observations were carried out weekly.
The sperm collected from the caudal epididymides of each genotype at
812 weeks of age were prepared for in vitro fertilization
as previously described (15). Eggs were collected from BALB/cA females
in which superovulation was induced (same procedure as the induction of
estrus in females). Sixteen hours after the injection of hCG,
insemination in vitro at a concentration of 250 sperm/µl
was carried out, and the inseminated eggs were incubated in 5%
CO2 at 37 C. Six hours after insemination, eggs
containing polar bodies and two pronuclei were used for further
analysis. On the following day, eggs that had developed to the two-cell
stage were designated as fertilized eggs.
Histological and Immunohistochemical Analysis
The testes were removed and fixed overnight in Bouins solution
(Sigma, St. Louis, MO) at 4 C, dehydrated in ethanol,
cleared with xylene, and then embedded in paraplast. The specimens were
cut into 7-µm sections and stained with hematoxylin and eosin
(Sigma), or with hematoxylin and periodic acid
Schiff (PAS). For X-gal staining or immunohistochemistry, the
testes and brains derived from 1020-week-old mice were fixed for
5 h at 4 C in 4% paraformaldehyde in PBS (pH 7.6) and embedded in
Tissue-Tech O.C.T. compound (Sakura Finetechnical Co., Ltd.,
Tokyo, Japan). The testes and brains were cut into 7-µm and 40-µm
sections, respectively. X-gal staining was performed to monitor Psa
expression using ß-galactosidase activity (10). The sections was
stained overnight at 37 C as previously described (34). Paraffin
sections were used for immunohistochemistry using antiinhibin
antibody without X-gal staining. Immunohistochemistry was performed
with antiporcine inhibin
(diluted at 1:8,000, Ref. 35), anti-AR
(diluted at 1:5,000, PG21; a gift from G. Greene, University of
Chicago), anti-GAP (diluted at 1:10,000, YANAIHARA Institute),
anti-ER
(diluted at 1:5,000, ER21; a gift from G. Greene, University
of Chicago), anti-CRH (diluted at 1:5,000, Peninsula Laboratories, Inc., Belmont, CA), anti-ß-endorphin (diluted at
1:5,000, Sigma), or antienkephalins (diluted at 1:5000,
Chemicon International, Inc., Temecula, CA). The sections were probed
two to three overnights with first antibody for brain sections and one
overnight for testicular sections at 4 C. A
Vectastain ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA) was used for the subsequent
immunodetection.
Western Blot Analysis
Testes removed from 8-month-old mice were suspended in 2x
sample buffer (100 mM Tris-HCl, pH 6.4, 4% SDS, 0.2%
bromophenol blue, and 20% glycerol), sonicated, and boiled for 5 min.
The protein concentrations of the cell lysates were measured with a
protein assay kit (Bio-Rad Laboratories, Inc., Hercules,
CA). Lysates containing protein (50 µg) were resolved in 7.5%
SDS-polyacrylamide gels and electroblotted to a nitrocellulose
membrane. The filters were probed with first antibodies at 4 C
overnight and incubated with HRP-conjugated antibody to rabbit-IgG at
room temperature for 1 h. The immune complexes were detected by
ECL detection (Amersham Pharmacia Biotech, Arlington
Heights, IL). As first antibodies, antibodies against porcine inhibin
and ß (diluted at 1:1,000, Ref. 35) and rat WT-1 (C-19, diluted
at 1:1,000, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) were used for the analysis. The results of blotting were scanned
with a scanner, and the intensities of the bands were analyzed by the
software NIH image.
Flow Cytometric Analysis
Cytological analysis of testicular cells by FACScan was
performed as described previously (17). Four 8- to 12-week-old mice of
each genotype were prepared for assay. For FACScan analysis, testicular
cells were added to a concentration of 1 x
106 cells/ml in separation medium and stained
with propidium iodide. The cells were analyzed with a FACScan apparatus
(Becton Dickinson and Co., Rutherford, NJ).
Electron Microscopy
Analysis by transmission electron microscopy was performed as
described previously (36). Testes from 11- and 17-week-old mice of each
genotype were removed and fixed in 2% glutaraldehyde in PBS (pH 7.6).
For quick and solid fixation, a 27G needle was inserted through the
tunica albuginea of the testes and a small volume of glutaraldehyde was
injected before immersion fixation.
RIA
Male mice caged in groups of one to three sexually identical
mice were used for the hormonal assay. The mice were decapitated
without anesthetic and blood samples rapidly collected (within 30 sec).
RIAs of FSH, LH, and PRL in mouse plasma were performed in duplicate
using reagents distributed by the NIDDK National Hormone and Pituitary
Program. Results are expressed in terms of rat FSH-RP-2, rat LH-RP-2,
and PRL (AFP-6476C). Plasma levels of testosterone were determined with
a double-antibody RIA system using 125I-labeled
radioligand as described by Taya et al. (37). Antiserum
against testosterone (GDN250; Ref. 38) kindly provided by G. D.
Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado
State University, Fort Collins, CO) was used. In assays for
testosterone, PRL, LH, and FSH detection, the interassay coefficients
of variation (CVs) were 15.5%, 18.7%, 13.5%, and 9.9%,
respectively, and the intraassay CV values were 6.0%, 12.6%, 5%, and
9.2%, respectively.
Administration of Androgen
Neonatal mice were injected with androgen as described
previously (18). Male and female mice heterozygous for the
goku mutation were housed for breeding and the pups on
postnatal days 03 were injected sc with 100 µg of TP in 0.02 ml of
olive oil. As control experiments, only olive oil was injected. For the
administration of androgen in adults, an sc implant of 2-cm Silastic
tube (outer diameter, 3.18 mm; inner diameter, 1.57 mm, Silastic
medical grade tubing, Dow Corning Corp.; Midland, MI)
containing either TP or PBS as a control was undertaken in 6- to
8-week-old mice.
Duplicate observations (weekly observations) of copulatory behavior
were conducted 2 weeks after TP implantation as described in
Copulatory Behavior and Fertilization Analyses above, and
then the morphology of the testes and plasma testosterone levels were
analyzed 4 weeks after TP implantation as described in
Histological and Immunohistological Analysis and
RIA, respectively.
Statistical Analysis
The experimental data were analyzed by Students t
test. Values of P < 0.05 were considered as
statistically significant. All values in the text are expressed as
mean ± SEM.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Dr. G. Greene (Chicago
University) for generously providing the ER (ER21) and AR (PG21)
antibodies; Dr. N. Harada (Fujita Health University) for the aromatase
antibody; the National Hormone and Pituitary Program (Torrance, CA) for
providing the RIA materials for rat FSH and LH, and mouse PRL; Dr.
G. D. Niswender (Colorado State University) for providing the
antiserum to testosterone (GDN250); Dr. H. Fujimoto (Mitsubishi Kasei
Institute of Life Sciences); Dr. H. Kawano (Tokyo Metropolitan
Institute of Neuroscience) for their valuable advice during the course
of this work; Dr. S. Ogawa (Rockefeller University) and Dr. W. Wurst
(Max-Plank Institute) for their helpful advice concerning the
immunohistochemistry technique; and Dr. S. Hayashi (Yokohama City
University) for his critical review of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Takashi Takeuchi, Ph.D., Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. E-mail: take{at}libra.ls.m-kagaku.co.jp
1 Present address, The Institute for Biogenesis Research, Department of
Anatomy and Reproductive Biology, John A. Burns School of Medicine,
University of Hawaii, 1960 East-West Road, Honolulu, Hawaii 96822. 
Received for publication May 31, 2000.
Revision received January 30, 2001.
Accepted for publication February 6, 2001.
 |
REFERENCES
|
---|
-
Meisel RL, Sachs BD 1994 The physiology of male sexual
behavior. In: Knobil E, Neil JD (eds) The Physiology of Reproduction,
ed 2. Raven Press, New York, pp 3105
-
Sharpe RM 1994 Regulation of spermatogenesis. In: Knobil E,
Neil JD (eds) The Physiology of Reproduction, ed 2. Raven Press, New
York, pp 13631434
-
Bean NY, Nunez AA, Conner R 1981 Effects of medial preoptic
lesions on male mouse ultrasonic vocalizations and copulatory behavior.
Brain Res Bull 6:109112[Medline]
-
Matochik JA, Sipos ML, Nyby JG, Barfield RJ 1994 Intracranial
androgenic activation of male-typical behaviors in house mice:
motivation versus performance. Behav Brain Res 60:141149[CrossRef][Medline]
-
Couse JF, Korach KS 1998 Exploring the role of sex steroids
through studies of receptor deficient mice. J Mol Med 76:497511[CrossRef][Medline]
-
Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ,
Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking
estrogen receptor
and ß. Science 286:23282331[Abstract/Free Full Text]
-
Skinner MK 1991 Cell-cell interactions in the testis. Endocr
Rev 12:4577[Medline]
-
Edelmann W, Cohen PE, Kane M, Lau K, Morrow B, Bennett S,
Umar A, Kunkel T, Cattoretti G, Chaganti R, Pollard JW, Kolodner RD,
Kucherlapati R 1996 Meiotic pachytene arrest in MLH1-deficient mice.
Cell 85:11251134[Medline]
-
Liu D, Matzuk MM, Sung WK, Guo Q, Wang P, Wolgemuth DJ 1998 Cyclin A1 is required for meiosis in the male mouse. Nat Genet 20:377380[CrossRef][Medline]
-
Osada T, Ikegami S, Takiguchi-Hayashi K, Yamazaki Y,
Kato-Fukui Y, Higashinakagawa T, Sakaki Y, Takeuchi T 1999 Increased
anxiety and impaired pain response in puromycin-sensitive
aminopeptidase gene-deficient mice obtained by a mouse gene-trap
method. J Neurosci 19:60686078[Abstract/Free Full Text]
-
Hersh LB, McKelvy JF 1981 An aminopeptidase from bovine brain
which catalyzes the hydrolysis of enkephalin. J Neurochem 36:171178[Medline]
-
Dyer SH, Slaughter CA, Orth K, Moomaw CR, Hersh LB 1990 Comparison of the soluble and membrane-bound forms of the
puromycin-sensitive enkephalin-degrading aminopeptidases from rat.
J Neurochem 54:547554[Medline]
-
Constam DB, Tobler AR, Rensing-Ehl A, Kemler I, Hersh LB,
Fontana A 1995 Puromycin-sensitive aminopeptidase. Sequence, analysis,
expression and functional characterization. J Biol Chem 270:2693126939[Abstract/Free Full Text]
-
McGill TE 1961 Sexual behavior in three inbred strains of
mice. Behavior 19:341350
-
Toyoda Y, Yokoyama M, Hoshi T 1971 Studies on the
fertilization of mouse eggs in vitro. I. In vitro
fertilization of eggs by fresh epididymal sperm. Jpn J Anim Reprod 16:147151
-
Russell LD, Ettlin RA, Hikim APS, Clegg ED 1990 In: Histological and Histopathological Evaluation of the
Testis. Cache River Press, Clearwater, FL
-
Malkov M, Fisher Y, Don J 1998 Developmental schedule of the
postnatal rat testis determined by flow cytometry. Biol Reprod 59:8492[Abstract/Free Full Text]
-
Livne I, Silverman AJ, Gibson MJ 1992 Reversal of reproductive
deficiency in the hpg male mouse by neonatal
androgenization. Biol Reprod 47:561567[Abstract]
-
Silverman AJ, Livne I, Witkin JW 1994 The
gonadotropin-releasing hormone (GnRH) neuronal system;
Immunocytochemistry and in situ hybridization. In: Knobil E,
Neil JD (eds) The Physiology of Reproduction, ed 2. Raven Press, New
York, pp 13631434
-
Vornberger W, Prins G, Musto NA, Suarez-Quian CA 1994 Androgen
receptor distribution in rat testis: new implications for androgen
regulation of spermatogenesis. Endocrinology 134:23072316[Abstract]
-
Zhou X, Kudo A, Kawakami H, Hirano H 1996 Immunohistochemical
localization of androgen receptor in mouse testicular germ cells during
fetal and postnatal development. Anat Rec 245:509518[CrossRef][Medline]
-
Lu Q, Gore M, Zhang Q, Camenisch T, Boast S, Casagranda F, Lai
C, Skinner MK, Klein R, Matsushima GK, Earp HS, Goff SP, Lemke G 1999 Tyro-3 family receptors are essential regulators of mammalian
spermatogenesis. Nature 398:723728[CrossRef][Medline]
-
Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn
DB, Korach KS 1996 Targeted disruption of the estrogen receptor gene in
male mice causes alteration of spermatogenesis and infertility.
Endocrinology 137:47964805[Abstract]
-
Ogawa S, Lubahn DB, Korach KS, Pfaff DW 1997 Behavioral
effects of estrogen receptor gene disruption in male mice. Proc Natl
Acad Sci USA 94:14761481[Abstract/Free Full Text]
-
Szczypka MS, Zhou QY, Palmiter RD 1998 Dopamine-stimulated
sexual behavior is testosterone dependent in mice. Behav Neurosci 112:12291235[CrossRef][Medline]
-
Dubuc PU 1976 Body weight regulation in female rats following
neonatal testosterone. Acta Endocrinol (Copenh) 81:215224[Medline]
-
Bernardi M, Genedani S, Tagliavini S, Bertolini A 1989 Effect
of castration and testosterone in experimental models of depression in
mice. Behav Neurosci 103:11481150[CrossRef][Medline]
-
Robertson KM, ODonnell L, Jones ME, Meachem SJ, Boon WC,
Fisher CR, Graves KH, McLachlan RI, Simpson ER 1999 Impairment of
spermatogenesis in mice lacking a functional aromatase (cyp 19) gene.
Proc Natl Acad Sci USA 96:79867991[Abstract/Free Full Text]
-
Sharpe RM, Maddocks S, Kerr JB 1990 Cell-cell interactions in
the control of spermatogenesis as studied using Leydig cell destruction
and testosterone replacement. Am J Anat 188:320[Medline]
-
Knoblauch R, Garabedian MJ 1999 Role for hsp90associated
cochaperone p23 in estrogen receptor signal transduction. Mol Cell Biol 19:37483759[Abstract/Free Full Text]
-
Elliston JF, Fawell SE, Klein-Hitpass L, Tsai SY, Tsai M-J,
Parker MG, OMalley BW 1990 Mechanism of estrogen receptor-dependent
transcription in a cell-free system. Mol Cell Biol 10:66076612[Medline]
-
Segnitz B, Gehring U 1997 The function of steroid hormone
receptors is inhibited by the hsp90-specific compound geldanamycin.
J Biol Chem 272:1869418701[Abstract/Free Full Text]
-
Hochstrasser M 1995 Ubiquitin, proteasome, and the regulation
of intracellular protein degradation. Curr Opin Cell Biol 7:215223[CrossRef][Medline]
-
Motoyama J, Kitajima K, Kojima M, Kondo S, Takeuchi T 1997 Organogenesis of the liver, thymus and spleen is affected in jumonji
mutant mice. Mech Dev 66:2737[CrossRef][Medline]
-
Otsuka M, Kishi H, Arai K, Watanabe G, Taya K, Greenwald GS 1997 Temporal changes in inhibin, steroid hormones, and steroidogenic
enzymes during induced follicular atresia in the hypophysectomized
cyclic hamster. Biol Rep 56:423429[Abstract]
-
Takeuchi T, Yamazaki Y, Katoh-Fukui Y, Tsuchiya R, Kondo
S, Motoyama J, Higashinakagawa T 1995 Gene trap capture of a novel
mouse gene, jumonji, required for neural tube formation. Genes Dev 9:12111222[Abstract]
-
Taya K, Watanabe G, Sasamoto S 1985 Radioimmunoassay for
progesterone, testosterone, and estrogen-17ß using
125I-iodohistamine radioligands. Jpn J Anim
Reprod 31:186197
-
Gay VL, Kerlan JT 1978 Serum LH and FSH following passive
immunization against circulating testosterone in the intact male rat
and in orchidectomized rats bearing subcutaneous Silastic implants of
testosterone. Arch Androl 1:257266[Medline]