Dexamethasone Effects on Fas Ligand Expression in Mouse Testicular Germ Cells
The aim of this study is to investigate the effect of
Dex, a widely used GC, on apoptosis and expression of FasL protein in
the mouse testicular germ cells. The effects of dexamethasone (Dex) on
expression of Fas ligand (FasL), an important proapoptotic protein, in
the mouse testicular germ cells were investigated. Six groups, each of
8 male NMRI mice were chosen for the experiment. Experimental groups received
one of the following treatments daily for 7 days: 4, 7 and 10 mg kg-1
Dex. Control groups were treated with equivalent volumes of saline. Experimental
and control animals were sacrificed 24 h after the last injection. Immunohistochemical
procedure was used to evaluation of FasL expression and the deoxyuridine
nick-end labeling (TUNEL) was applied to assessment of the apoptotic germ
cells. FasL expression of testicular germ cells were significantly increased
in 10 mg kg-1 Dex treated mice (p<0.05), particularly at
stages VII-VIII of spermatogenic cycle. Apoptotic indexes (AIs) of germ
cells were significantly increased in 7 and 10 mg kg-1 Dex
GCs are extraordinary hormones that influence the activity of almost
every cell in the body. They modulate the expression of approximately
10% of our genes and are essential for life but are also increasingly
implicated in the pathogenesis of disease and produce many unwanted effects
when given therapeutically (Julia, 2006). In therapeutic concentrations,
GCs are strongly immunosuppressive and anti-inflammatory, which has made
them one of the most prescribed drugs worldwide.
It has been reported that GCs cause changes in plasma gonadotrophin levels
and their pituitary content and indirectly contribute to the inhibition
of reproductive functions (Calogero et al., 1999). Earlier studies
have been shown that elevation of GC concentration precedes
a decline in testosterone concentration in the male (Dong et
al., 2004; Bernier et al., 1999; Hardy et al., 2005).
Presence of testosterone is essential for normal function and survival
of the germ cells in seminiferous tubules (Sinha-Hikim and Swerdloff,
1999; Sofikitis et al., 2008). When the testicular environment
can not support spermatogenesis, specific pathways leading to germ cell
apoptosis are activated.
Abnormally accelerated apoptosis of germ cells may lead to an imbalance
of cell proliferation and death, resulting in spermatogenic impairment
(Kimura et al., 2003). Two major proapoptotic pathways have been
defined in mammalian cells. One initiated at the cell surface via Apoptosis
Stimulating Fragment (Fas) system and the second occurring in the mitochondria
including Bax (Koji and Hishikawa, 2003). The Fas system is a receptor-ligand
signaling system in which Fas ligand (FasL) binds to and activates the
Fas receptor (Fas) to initiate a cascade of intracellular events that
leads to the elimination of the Fas-bearing cells via apoptosis (Koji
et al., 2001; Kavurma and Khachigian, 2003). The Fas system is
involved in maintaining homeostasis in various systems. The
Fas system in the testis has been identified as one paracrine
signaling system by which Sertoli cells, expressing FasL, can
initiate killing of Fas-expressing germ cells (Nagata, 1997).
In present study, the effects of Dex, a widely used GC, on apoptosis
and expression of FasL protein in the mause testicular germ cells were
MATERIALS AND METHODS
This study was conducted from 8/2007 to 5/2008 in Ahwaz Jundi-Shapour
University of Medical Sciences, Faculty of Medicine, Ahwaz, Iran.
Chemicals: Primary antibody FasL secondary antibody biotinylated
anti-mouse IgG (ABC Peroxidase Mouse IgG Staining Kit) and diaminobenzidine
(DAB) were from Santa Cruz, USA. In situ cell death detection kit,
for TdT-mediated dUTP nick end labeling (TUNEL) assessment, was the product
of Roche Diagnostic, Germany. Dexamethasone was purchased from Daru Pakhsh
Company, Iran. All other solvents and reagents were of the highest grade
Animals: NMRI male mice weighing 25-30 g were kept in individual
stainless steel cages under standardized conditions (constant temperature
and humidity, 12 h light-dark cycle). They were fed with commercial chow
and tap water ad lib.
Experimental protocol: The animals were randomly divided into
6 groups of 8 each. The experimental groups (E1-E3) received intrapritoneal
injections of 4 (E1), 7 (E2) and 10 (E3) mg kg-1 Dex (dissolved
in 0.9% saline) on 7 consecutive days. Control groups (C1-C3) were injected
with 0.9% saline in equal volumes as for the experimental groups. Since
TUNEL and immunohistochemical analysis showed no significant differences
between three controls, all data were combined into one control group
(C). One day after the last injection animals were sacrificed by decapitation
under ether anesthesia. The testes were excised, fixed in formalin 10%
for 48 h and embedded in paraffin wax. Five micrometer thick sections
were prepared by using Leitz microtome for subsequent immunohistochemistry
and deoxy-UTP-digoxigenin nick end labeling (TUNEL) studies.
Immunohistochemistry: Five-micron thick tissue sections were deparaffinized
in xylol and hydrated in decreasing series of ethanol. Endogenous peroxidase
activity was blocked by incubation in methanol containing 0.3% H2O2
for 15 min at room temperature. The sections were then treated with citrate
buffer (pH = 6) for 15 min at 98°C as antigen retrieval. Then the
sections incubated overnight at 4°C with primary antibodies, including
the monoclonal antibody against FasL at 1/100 diluted in phosphate buffered
saline (PBS; pH = 7.4) containing 10% normal goat serum (NGS). After washing
twice with PBS the sections were incubated with secondary antibody biotinylated
anti-mouse IgG at 1/100 for 50 min. Then the specimens were incubated
with peroxidase-conjugated avidin-biotin for 30 min at room temperature.
After washing, the sections were incubated with diaminobenzidine (DAB
substrate) as chromogen and counterstained with haematoxyline. Three immunohistochemical
sections from each animal were blindly assessed and staining intensity
was estimated using a semiquantitative score, H-score, as earlierly described
(Pallares et al., 2005; Ariel et al., 2001). The H-score
(Histo-score) was calculated for each section by application of the following
algorithm: H-score = ΣPi(i+1), where, i is the intensity of staining
(0: no staining, 1: weak, 2: moderate and 3: strong) and Pi is the percentage
of stained cells for each intensity (0 to 100%). For each mouse, at least
10 tubules/stage were used. The stages were identified according to the
criteria proposed for paraffin sections. This method provides 12 stages
of the spermatogenic cycle in mice. H-score assessment was repeated at
least 3 times for each section by 3 workers.
TUNEL assay: The deoxyuridine nick-end labeling (TUNEL) assay
for apoptotic cell detection was performed with the in situ cell
death detection kit. Briefly, deparaffinized tissue sections were predigested
with 20 μg mL-1 proteinase K for 20 min and incubated
in PBS containing 3% H2O2 for 10 min to block the
endogeous peroxidase activity. After incubating in 0.1% triton X-100 in
0.1% sodium citrate for two min on ice (4°C), the sections were incubated
with the TUNEL reaction mixture, fluorescin-dUTP for 60 min at 37°C.
The slides were then rinsed three times with PBS and incubated with secondary
antifluorescine-POD-conjugate for 30 min. After washing three times in
PBS, diaminobenzidine-H2O2 (DAB) chromogenic reaction
was added to the sections. As a control for method specificity, the step
using the TUNEL reaction mixture omitted in negative control section and
nucleotide mixture in reaction buffer was used instead. A cell was considered
TUNEL-positive when the nuclear staining was intense, dark brown and homogenous.
Apoptotic index (AI) was calculated by dividing the number of TUNEL-positive
germ cells in a randomly focused seminiferous tubule by the total number
of germ cells in that tubule and the result was multiplied by 100 (Yu
et al., 2001; Glicella et al., 2005). The AIs of 10 randomly
selected tubules for each spermatogenic stage were evaluated and the mean
AI of each case was calculated.
Statistical analysis: The data were analyzed using one-way ANOVA
followed by LSD test and presented as the mean±SD. p<0.05 was
Expression of FasL: C group showed weak immunoreactivity
in cell membrane of spermatogonia at different stages of spermatogenic
cycle (Fig. 1A). In E1 group spermatogonia showed strong
immunostaining at stages VII-VIII (Fig. 1B). The pattern
of FasL expression in other stages was similar to the C group. No detectable
immunostaining was observed in primary spermatocytes or spermatids. In
E2 group immunoreactivity was observed only at stages VII-VIII and IV-VI.
Spermatogonia showed strong or moderate immunostaining and other germ
cells were moderate or weak at stages IV-VI. In stages VII-VIII spermatogonia
and primary spermatocytes were strongly stained and spermatids showed
strong or moderate immunostaining (Fig. 1C). In E3 group
all stages of spermatogenic cycle showed positive immunostaining. In stages
I-III, strong staining in spermatogonia and weak immunostaining in primary
spermatocyte and spermatids were observed. In stages IV-VI, spermatogonia
and primary spermatocytes showed strong or moderate immunostaining and
moderate or weak immunostaining was observed in spermatids. Stages VII-VIII
showed the strongest immunostaining. Strong immunoreactivity was observed
in spermatogonia and primary spermatocytes. Spermatids showed strong or
moderate immunoreactivity. In stages IX-XII, moderate staining in spermatogonia
and weak or moderate immunostaining in spermatids were observed (Fig.
1D). No expression of FasL was observed in Sertoli cells or Leydig
cells in control and three experimental groups. The results of H-score
assessments of FasL expression are reported in Fig. 2.
FasL-immunodetection in control
and experimental testes. A: Control testis; with moderate immunostaining
in spermatogonia (x1000), B: E1 testis; showing strong imunoreactivity
in spermatogonia (x1000), C: E2 testis; with moderate immunoreactivity
only at stages VII-III (x400) and D: E3 testis; showing strong immunoreactivity
at stages VII-VIII (x400). SG: Spermatogonia, PS: Primary spermatocytes,
H-score assessment of FasL expression
in different stages of spermatogenic cycle in control and experimental
groups. Values are expressed as Mean±SD for 8 mice, *p<0.05,
**p<0.01, ***p<0.001, vs. C (control)
Assessment of germ cell apoptosis: In C group, spermatogonia showed
a low frequency of apoptosis in different stages (Fig. 3A).
In E1 group, TUNEL-reactivity was observed in some of the spermatogonia
and primary spermatocytes (Fig. 3B). There was no significant
change in the AI of germ cells between E1 and C group (p>0.05). In
E2 group, all types of germ cells showed TUNEL-positive reaction (Fig.
3C). The AIs of germ cells were significantly increased in all stages
of spermatogenic cycle apart from stages I-III. In E3 group, all types
of germ cells showed TUNEL-positive staining (Fig. 3D).
The AI of germ cells were significantly increased in all stages of spermatogenic
cycle, particularly in stages of VII-VIII. The results of the AIs of germ
cells are reported in Fig. 4.
TUNEL staining in control and experimental
testes, A: Control testis; a few numbers of spermatogonia and primary
spermatocytes showed TUNEL staining (x1000), B: E1 testis; a few
numbers of spermatogonia, primary spermatocytes and spermatids showed
TUNEL-positive reaction (x1000), C: E2 testis; TUNEL-positive reaction
in spermatogonia, primary spermatocytes, spermatids and sertoli
cells (x400) and D: E3 testis; the majority of germ cells and Sertoli
cells showed TUNEL-positive reaction (x400). SG: Spermatogonia,
PS: Primary spermatocytes, ST: Spermatid, S: Sertoli cell
||Effects of Dex on apoptotic index (AI) of germ cells
in different stages of spermatogenic cycle in mice. Values are expressed
as Mean±SD for 8 mice, *p<0.05, **p<0.01, ***p<0.001
vs. C (control)
Sertoli cells showed TUNEL-positive staining in both E2 and E3 groups.
AIs of Sertoli cells were significantly increased in E2 (36.4±6.5%
vs. 0%; p<0.01) and E3 (44.8±9.3% vs. 0%; p<0.01) groups.
Leydig cells showed TUNEL-negative reaction in all groups.
The results of this study demonstrated that Dex dose-dependently
increased FasL expression in testicular germ cells. Stages VII-VIII showed
the most susceptible to apoptotic effects of Dex. Sinha et al.
(1997) have previously reported that deprivation of gonadotropins and
testosterone by GnRH antagonist treatment is followed by a stage-specific
increase in germ cell apoptosis. There is abundant evidence suggesting
that stages VII-VIII of the rat spermatogenic cycle exhibit the strongest
levels of immunohistochemically detectable androgen receptors expression
and are considered to be androgen dependent stages (Bremner et al.,
1994; Nisrine et al., 2005).
It is possible that high susceptibility to apoptosis evidenced by FasL
expression does not necessarily commit all FasL -immunopositive cells
to the apoptotic cell death. For this reason we applied the TUNEL method.
This method is based on the detection of oligonucleosomal DNA fragments
which are characteristic of cells in the later stages of the apoptotic
process (Walker et al., 1994). The measurement of apoptosis showed
an increase in germ cell apoptosis after Dex treatment.
The reason responsible for the increased apoptosis induced by Dex in
germ cells is not known. Although the endocrine control in testicular
function is clear, the complexity of the intratesticular events highlights
the importance of regulatory mechanisms and interactions. Intratesticular
androgens, secreted by Leydig cells, play an important paracrine role
in preventing germ cell degeneration (Tapanainen et al., 1993).
Dex and other synthetic GCs exert a direct inhibitory effect on testosterone
production by Leydig cells in vitro (Bernier et al., 1999).
In addition to the inhibition of androgen biosynthesis, excessive exposure
to GC in rodents initiate apoptosis in Leydig cells (Gao et al.,
In contrast previous studies, which showed Dex induces apoptosis in rat
Leydig cells (Gao et al., 2002, 2003), this study demonstrated
that Leydig cells were TUNEL negative in Dex treated mice. Thus, the increase
of FasL expression in testicular germ cells is probably related to the
inhibition effect of Dex on androgen biosynthesis in Leydig cells. In
present study androgen-independent stages of spermatogenic cycle also
showed TUNEL-reactivity and expression of the proapototic protein. Thus,
the increasing of apoptosis can not be exclusively due to hormonal influence.
Sertoli cells showed TUNEL-positive reaction and AI of Sertoli cells
were significantly increased in E2 and E3 groups. Sertoli cells, the supportive
cells in the seminiferous epithelium, orchestrate spermatogenesis by providing
structural and nutritional support to germ cells (Boekelheide et al.,
2000). In the rat, physiological apoptosis occurs continuously to limit
the size of the germ cell population to numbers that can be adequately
supported (Franca et al., 1993). Presence apoptosis in Sertoli
cells, which demonstrated in this study, causes these cells loss their
supporting action on germ cells and this may stimulate apoptotic signaling
in germ cells. In this study, there was no detectable FasL imunoreactivity
in Sertoli cells, while these cells showed TUNEL-positive staining. Additionally,
in E2 group all stages of spermatogenic cycle showed TUNEL-positive reaction,
while FasL expression was observed only in stages IV-VIII. These data
suggest that other apoptotic factors are involved in Dex induced apoptosis
in testicular tissue. Further experiments are needed to clarify the mechanisms
of the effect of Dex on different apoptotic signaling pathways in testicular
This research was a part of Ph.D project and supported by a grant
(u-86044) from the research council of the Ahwaz Jundi-Shapour University
of Medical Sciences.
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