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Pathwaysinvolvedintesticulargermcell apoptosis
induced by H
2
O
2
in vitro
Ankur Maheshwari
1
, Man M. Misro
1
, Archana Aggarwal
1
, Rajnesh K. Sharma
2
and Deoki Nandan
1
1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India
2 Department of Zoology, Kurukshetra University, India
Seminiferous epithelium harbours a large number of
germ cells in the spermatogenic cycle, which are at dif-
ferent stages of development and maturation until they
are released into the tubular lumen as fully fledged
sperms. Earlier studies have indicated that spermato-
genic cells undergo spontaneous degeneration at
specific stages during development, and such degenera-
tion of different types of spermatogonia (A
2
–A
4
) and
meiotic cells in rats has been reported to result in the
depletion of up to 75% of the mature sperm pool
[1,2]. The plasma membrane of testicular cells is rich
in polyunsaturated fatty acids and therefore prone to
oxidation by H
2
O
2
and other reactive oxygen species
(ROS) [3]. H
2
O
2
constitutes the main ROS form in
sperms but its effective role as an endogenous messen-
ger ingermcellapoptosis is largely unknown [4]. How-
ever, H
2
O
2
is known to modulate a variety of cell
functions. It is a potent ROS, but its lower biological
activity compared to many ROS, combined with its
capacity to cross membranes and diffuse away from
the site of generation, makes it an ideal molecule in
signal transduction, and lower doses are known to
induce apoptosis [5]. As reviewed previously [6], H
2
O
2
is synthesized endogenously in certain cell systems in
response to specific cytokines or hormones ⁄ growth
factors and such endogenous H
2
O
2
can then either act
Keywords
apoptosis; germ cell; H
2
O
2
; pathways; testis
Correspondence
M. M. Misro, Department of Reproductive
Biomedicine, National Institute of Health
and Family Welfare, Baba Gang Nath Marg,
New Delhi 110067, India
Fax: +91 11 2610 1623
Tel: +91 11 2616 5959
E-mail: mm_misro@yahoo.com
(Received 17 October 2008, revised 28
November 2008, accepted 3 December
2008)
doi:10.1111/j.1742-4658.2008.06831.x
H
2
O
2
induces apoptosisin variety of cells; however, the sensitivities of
testicular germ cells to H
2
O
2
are not known. In the present study, H
2
O
2
,at
concentrations in the range 1–10 lm, was found to induce apoptosis in
testicular germ cells in vitro. Following 1 h of treatment with 10 lm H
2
O
2
,
a 10-fold rise in the percentage of apoptotic cells was observed. Induction
of germcellapoptosis was directly associated with a significant (P < 0.01)
increase in lipid peroxidation and a concomitant decrease in superoxide
dismutase and catalase activity. Examination of apoptotic signalling path-
ways revealed an increased expression of extrinsic (Fas, FasL and caspase-
8) and intrinsic (Bid, Bak, Bad, Bax and caspase-9) markers, as well as
p53, along with a simultaneous decrease in the Bcl-2 protein at the highest
concentration of H
2
O
2
exposure. Both, c-jun N-terminal kinase and p38
phosphorylated forms were found to be up-regulated. Interestingly, up-reg-
ulation of the nuclear transcription factor kappa B was also observed. The
respective transcripts for many of the above proteins followed an identical
trend. Caspase-3 activity was also estimated to be 30-fold higher. Taken
together, the above data indicate that testiculargerm cells are prone to
apoptosis at very low concentrations of H
2
O
2
, the mechanism of which
involves extrinsic and intrinsic as well other regulatory pathways.
Abbreviations
ABTS, 2,2-azino-di-(3-ethylbenzthiazoline sulfonate; DAB, diaminobenzidine; GST, glutathione S-transferase; HBSS, Hank’s balanced salt
solution; hCG, human chorionic gonadotrophin; HRP, horseradish peroxidase; ISEL, in situ end labelling; JNK, c-Jun N-terminal kinase; MAP,
mitogen-activated protein; NF-jB, nuclear factor-kappa B; PARP, poly-(ADP)-ribose polymerase; ROS, reactive oxygen species; TAC, total
antioxidant capacity
870 FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS
as a second messenger to stimulate protein kinase
cascades coupled with apoptosis or participate in regu-
latory control of the cell cycle.
Apoptosis involves a cascade of signals and there
are different pathwaysinvolvedintesticulargerm cell
apoptosis pertaining to specific treatments. As previ-
ously shown [7], testicularcellapoptosis follows both
extrinsic and intrinsic pathways. The extrinsic pathway
involves the activation of death receptors, namely
Fas ⁄ tumour necrosis factor receptor, leading to activa-
tion of initiator caspase-8, followed by activation of
executioner caspases-3, 6, 7 and, subsequently, apopto-
sis [8]. The intrinsic or the mitochondrial pathway
involves Bcl-2 family members, leading to changes in
the mitochondrial membrane permeability and release
of cytochrome c into the cytosol [9]. Cytochrome c
sequesters with the apoptotic protease activating fac-
tor-1 and the complex activates initiator caspase-9,
which in turn activates executioner caspases, leading to
apoptosis [10]. We have previously reported that H
2
O
2
at physiological concentrations modulates Leydig cell
function and induces oxidative stress and apoptosis
[11]. However, the sensitivities of isolated germ cells to
H
2
O
2
are not known. Furthermore, chronic human
chorionic gonadotrophin (hCG) treatment has been
reported to be associated with the rise in testicular
H
2
O
2
and germcellapoptosis [12]. Therefore, in the
present study, the effects of H
2
O
2
inflicting oxidative
and apoptotic damage on the isolated testicular germ
cells, as well as the pathways associated with the signal
transduction of the apoptotic stimulus, were investi-
gated.
Results
H
2
O
2
treatment is associated with rise in
oxidative stress
Testicular germ cells demonstrated a significant
(P < 0.01) rise in lipid peroxidation following H
2
O
2
exposure at a 2 lm concentration and above (Fig. 1A).
However, the effect was not significant at the lowest
concentration of H
2
O
2
(1 lm). Both, superoxide
dismutase (SOD) and catalase from treated cells
showed a significant (P < 0.01) decline in their activi-
ties above a 2 lm concentration of H
2
O
2
(Fig. 1C,D).
Parallel to the decrease in enzyme activities, mRNA
expressions of Mn SOD and catalase were also down-
A B
C D
Fig. 1. Evaluation of oxidative stress in rat testiculargerm cells treated with H
2
O
2
. (A) Dose-dependent increase in lipid peroxidation as
measured by thiobarbituric acid reactive substance formation. (B) Transient rise followed by a steep decline in GST activity at the highest
concentration of H
2
O
2
. (C) Significant decline in SOD activity coinciding with transcript levels representing Mn SOD but not Cu ⁄ Zn SOD.
(D) Catalase activity along with its transcripts. C, control. *P < 0.01, **P < 0.001.
A. Maheshwari et al. H
2
O
2
and testicularcell apoptosis
FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS 871
regulated. On the other hand, Cu ⁄ Zn SOD transcripts
were found to be unaltered in all the treatment groups.
Interestingly, the decline in the activity of glutathione
S-transferase (GST) (Fig. 1B) matched perfectly well
with the total antioxidant capacity (TAC), which was
found to be significantly (P < 0.05) lower only in cells
treated with the highest concentration (10 lm)ofH
2
O
2
(Fig. 2).
Low concentrations of H
2
O
2
induce germ cell
apoptosis
In situ end labelling (ISEL) positive cells were recorded
out of each 100 cells examined from five randomly
selected sites on each slide. A significant (P < 0.01)
and dose-dependent increase in ISEL positive cells was
observed in cells treated with H
2
O
2
at concentrations
of 2 lm and above. Approximately, 52% of the treated
cells were found apoptotic with the highest concentra-
tion of H
2
O
2
, which was ten-fold higher compared to
controls (Fig. 3A,B). Germcellapoptosisin all the
experimentally exposed groups was further supported
by the inter-nucleosomal degradation of genomic
DNA depicting a ladder-like pattern on agarose gel
electrophoresis (Fig. 3C). Also, H
2
O
2
treated cells
revealed a three- to 30-fold rise in caspase-3 activity
compared to controls and a demonstrable increase
in poly-(ADP)-ribose polymerase (PARP) cleavage
(Fig. 4A,B).
H
2
O
2
induced germcellapoptosis follows both
extrinsic and intrinsic pathways
To determine whether H
2
O
2
-induced redox imbalance
and subsequent testiculargermcellapoptosis were
associated with changes in pro- ⁄ anti-apoptotic and
other proteins, the expression of these proteins and
their mRNAs were studied by western blotting and
RT-PCR analysis, respectively. Bax protein ⁄ mRNA
expression was low in control cells compared to H
2
O
2
treated cells (Fig. 5A,B). As expected, the anti-apopto-
Fig. 2. Assessment of TAC intesticulargerm cells with or without
H
2
O
2
(10 lM). C, control. *P < 0.05.
A
B
C
Fig. 3. ISEL of testiculargerm cells treated with H
2
O
2
. (A) A
marked rise in percentage of ISEL positive cells was observed in
(B) the treated (right panel) compared to untreated cells (left panel);
·400. (C) Ladder assay showing DNA fragmentation in H
2
O
2
exposed testiculargerm cells. C, control. *P < 0.01, **P < 0.001.
H
2
O
2
and testicularcellapoptosis A. Maheshwari et al.
872 FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS
tic Bcl-2 protein ⁄ mRNA levels were found to be dras-
tically lowered in the highest treatment group
(Fig. 5A,B). However, an opposite trend was encoun-
tered when the cells were treated with 1–5 lm H
2
O
2
.
Simultaneously, there was an increase in the expression
of other Bcl-2 member pro-apoptotic proteins such as
Bid, Bad and Bak (Fig. 5A). Besides the release of
cytochrome c, caspase-9 protein and its mRNA were
found to be up-regulated, supporting the involvement
of an intrinsic ⁄ mitochondrial pathway of apoptosis
(Fig. 6A,B). The active caspase-9 was also resolved in
the cells exposed with 2–10 lm of H
2
O
2
. On the other
hand, marginal rise in FasL, Fas and caspase-8 protein
expression coincided well with the elevated levels of
their specific transcripts in the treated cells, indicating
the course of signal transduction through an extrinsic ⁄
death receptor pathway (Fig. 6C,D).
Activation of other pathwaysin H
2
O
2
induced
germ cell apoptosis
H
2
O
2
treatment of testiculargerm cells not only acti-
vates the intrinsic and extrinsic pathways, but also other
pathways of apoptotic induction. Tumour suppressor,
p53 protein and mRNA expression was up-regulated in
all the treatment groups in a dose-dependent manner
(Fig. 7A,B). Western blots for mitogen-activated pro-
tein (MAP) kinases, c-Jun N-terminal kinase (JNK) or
phosphorylated forms of JNK and p38 depicted a rise in
expressions ⁄ phosphorylation compared to untreated
controls (Fig. 7C). A dose-dependent upward expres-
sion of nuclear factor-kappa B (NF-jB) was also
observed in the treated cells (Fig. 7C).
Discussion
The results of the present study indicate that H
2
O
2
,
even at a concentration of 1 lm, has the ability to
induce apoptosisintesticulargerm cells invitro after
1 h of exposure. The mechanism takes the usual routes
of extrinsic and intrinsic as well as other designated
pathways of metazoan apoptosis, signifying the possi-
ble role of this biomolecule in the process of germ cell
development in the testis and its regulation through
apoptosis.
H
2
O
2
is a by-product of cell metabolism; its cellular
levels and maintenance are constantly under homeo-
static regulation. The cells can sense sublethal doses of
H
2
O
2
and activate peroxide-detoxifying mechanisms;
alternatively, various H
2
O
2
producing mechanisms can
be activated by different cell death stimuli. As a result
of this deliberate H
2
O
2
production, a self-destructive
programmed cell death can be triggered [13]. However,
different organisms or cells have variations in their
sensitivities to H
2
O
2
. In the study of caspase-depen-
dent ⁄ independent events of apoptosisin MCF-7 breast
A
B
Fig. 4. (A) A rise (three- to 30-fold) in caspase-3 activity and (B)
PARP cleavage intesticulargerm cells following H
2
O
2
treatment.
C, control. *P < 0.01
A
B
Fig. 5. Expression levels of Bcl-2 family members. An increase in
pro-apoptotic proteins (Bax, Bid, Bak and Bad) and a decrease in
anti-apoptotic protein Bcl-2 at the highest (10 l
M H
2
O
2
) concentra-
tion (A) was observed. (B) Expression of Bax and Bcl-2 as detected
by RT-PCR. C, Control.
A. Maheshwari et al. H
2
O
2
and testicularcell apoptosis
FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS 873
carcinoma cells, H
2
O
2
in the optimal range 250–
1000 lm was used [14]. However, in Jurkat T-cells,
H
2
O
2
-induced apoptosis required 100 lm or more of
H
2
O
2
[15]. It is also reported that 50 lm of H
2
O
2
was
sufficient to induce apoptosisin rat germ cells [16].
Although a higher concentration in the medium
is toxic and affects cell survival, H
2
O
2
, even at physio-
logical concentrations (30–50 lm), was shown to
induce apoptosisintesticular Leydig cells invitro [11].
Germ cells constitute the major inner seminiferous
tubular cell mass and the role of H
2
O
2
as a signalling
molecule in apoptotic induction among germ cells is
not yet known. In the present study, we attempted to
test germcell sensitivity to H
2
O
2
with reference to
apoptotic induction. The findings also clearly estab-
lished that testiculargerm cells are much more sensi-
tive to H
2
O
2
and would require a five- to 30-fold lower
concentration of H
2
O
2
in vitro for induction of apopto-
sis compared t o their interstitial counterparts [11].
In experimentally induced conditions of ischaemia
or cryptorchidism in rats, the major pathway of cell
removal from the testis was mainly through apoptosis
[17,18]. However, the role of intratesticular H
2
O
2
dur-
ing such conditions is not yet known. In the rat testis,
however, hormonal alterations due to chronic hCG
treatment were reported to raise H
2
O
2
levels and
induce germcellapoptosis [12]. Therefore, it is reason-
able to assume that H
2
O
2
might constitute a specific
physiological link to germcellapoptosisin the testis,
A
B
C
Fig. 7. Other pathways of signal transduction in H
2
O
2
induced tes-
ticular germcell apoptosis. (A) Dose-dependent increase in expres-
sion of p53 protein coinciding with (B) a marked rise in transcript
levels in all treated groups and (C) a change in expression levels of
other proteins (JNK, p-JNK, p-p38, NF-jB). C, Control.
A
B
C
D
Fig. 6. Pathways of signal transduction in H
2
O
2
induced testicular
germ cell apoptosis. Western blots showing expression of (A)
(intrinsic) caspase-9 and cytochrome c and (C) (extrinsic) caspase-8,
FasL and Fas, which are supported by RT-PCR analysis of (B)
caspase-9 and (D) caspase-8, FasL and Fas. C, control.
H
2
O
2
and testicularcellapoptosis A. Maheshwari et al.
874 FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS
for which the modalities need to be further worked
out.
The redox status inside the cell is crucial to the correct
functioning of many enzymes, and can be used to alter
enzyme activity; thus, alteration of redox status could act
as a signalling mechanism. It may either trigger or block
the apoptotic death program depending on the severity
of the oxidative stress [4]. However, H
2
O
2
was found to
trigger apoptosis possibly by modulating the oxidative
stress and functioning of different antioxidant enzymes,
as observed in the present study (Fig. 1). Germ cells trea-
ted with H
2
O
2
(10 lm) for 1 h demonstrated a significant
(P < 0.001) increase in lipid peroxidation, and a
decrease in antioxidant enzyme activity and TAC (Figs 1
and 2). With the rise in lipid peroxidation, a fall in
enzyme (SOD, catalase) activities was observed, and was
statistically significant (P < 0.01) even at 2 lm of H
2
O
2
(Fig. 1). Mn SOD demonstrated a downward trend and
the unaltered transcript levels of Cu ⁄ Zn SOD, as pres-
ently observed, need to be investigated further (Fig. 1C).
Because Mn SOD mostly represents mitochondrial frac-
tion, its down-regulation at the transcriptional level
probably contributes to the overall insufficient avail-
ability of the enzyme. The increase in transcript levels at
5 lm may be considered as an aberration of the decreas-
ing trend of Mn SOD expression with respect to the
increase in H
2
O
2
concentration. However, the expression
was significantly low (P < 0.01) compared to untreated
cells. A slight deviation in GST activity (Fig. 1B) was
noted, which declined significantly (P < 0.01) only in
cells exposed to the highest concentration of H
2
O
2
.By
contrast, a moderate increase in GST activity was
observed at lower concentrations of H
2
O
2
(1–2 lm). Such
an increase in GST activity may be considered as one of
the measures to counteract the oxidative stress induced
by H
2
O
2
at lower, but not at the highest, concentrations.
In the present study, ‘laddering’ of the DNA in the
exposed cells was seen in all the treatment groups in a
dose-dependent manner (Fig. 3C). Furthermore, ISEL
positive cells in different treatment groups increased
from 8% to 52% in a dose-dependent manner
(Fig. 3A,B). An increase in caspase-3 activity (from
three- to 30-fold compared to controls) and PARP
cleavage (Fig. 4A,B) in the treated cells is associated
with DNA fragmentation and supported by the fact
that PARP is essential in rat germinal cells for repair
of DNA damage inducedby either gamma-irradiation
or H
2
O
2
[16].
Spermatocytes and spermatids constitute the bulk of
the germ cells isolated and H
2
O
2
was seen to mediate
the up-regulation of Fas protein and its transcripts in
these cells (Fig. 6C). Interestingly, H
2
O
2
was found to
up-regulate FasL mRNA expression, irrespective of the
concentration used, whereas only a marginal increase in
protein was observed (Fig. 6C,D) and the significance of
this increase in FasL expression is yet to be explored.
Following the binding of FasL with Fas, activation of
caspase-8 occurs, ensuring the direct activation of cas-
pase-3 [8,19]. The present findings depicted a marked
rise in caspase-8 transcripts, with a marginal increase in
its protein expression. Caspase activation is often con-
sidered principally at the protein level with respect to
controlling proteolytic cascades, but some studies have
reported increases in caspase mRNA subsequent to
apoptotic stimuli [20,21]. This suggests that H
2
O
2
expo-
sure to testiculargerm cells probably induces the extrin-
sic pathway of apoptotic induction.
The Bcl-2 family of proteins is a widely recognized
group of apoptotic regulators. This family consists of
both pro-(Bax, Bad, Bak, Bid) and anti-apoptotic
(Bcl-2, Bcl-xL, Bcl-2l2) proteins that modulate the
execution phase of the cell death pathway, and the
expression of these proteins changes intesticular germ
cell apoptosis [22–24]. Evidence for the role of these
proteins in spermatogenesis is provided by studies con-
ducted in different ‘knockout mice’ models in which
the expression of selected members of Bcl-2 family is
disrupted [25]. Transgenic mice over-expressing bcl-2
or bax-deficient male mice are infertile, which is attrib-
uted to the disruption of spermatogenesis leading to
accumulation of gem cells [23]. The pro-apoptotic
Bid ⁄ Bax ⁄ Bad moves from the cytosol to the outer-
mitochondrial membrane following an apoptosis
inducing signal and the interaction between these Bcl-2
family proteins induces a conformational change,
which results in cytochrome c release from the mito-
chondria. Cytochrome c association with Apaf-1 and
procaspase-9 promotes the activation of caspase-9,
followed by downstream events leading to apoptosis
[7–10]. We have shown that all the pro-apoptotic
proteins, Bax, Bid, Bak and Bad, are up-regulated
(Fig. 5) along with caspase-9 mRNA and protein, indi-
cating the involvement of the intrinsic pathway in the
H
2
O
2
-induced testiculargermcellapoptosis (Fig. 6A,B).
The ability of Bcl-2 (an anti-apoptotic protein) as an
antioxidant to prevent apoptosis has been reported
previously [26,27]. Bcl-2 appeared to prevent lipid
peroxidation associated with apoptosis without reduc-
ing intracellular ROS levels, suggesting that Bcl-2 may
indirectly regulate antioxidant defences to prevent cell
death. H
2
O
2
induces oxidative stress in cells and
appears to be responsible for the decrease of Bcl-2 pro-
tein and mRNA levels in the highest treatment group
(10 lm). Static expression in the lower treatment groups
(1–5 lm) possibly indicates its involvement as an intra-
cellular antioxidant to counteract the oxidative stress.
A. Maheshwari et al. H
2
O
2
and testicularcell apoptosis
FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS 875
The down-regulation of Bcl-2 in the group exposed to
10 lm H
2
O
2
probably reflects the collapse of the anti-
apoptotic measure beyond a critical concentration.
As already discussed, H
2
O
2
exposure leads to redox
alterations with elevated oxidative stress, and this geno-
toxic stress may affect p53 tumour suppressor
gene ⁄ protein. As noted previously [28], p53 represents
the sensor of DNA damage whose activation provides
the cell with an opportunity to repair any cellular dam-
age prior to commitment to undergo apoptosis, and
p53-induced apoptosis results from cellular redox alter-
ations. The absence of p53 expression correlates with
an attenuation of germcellapoptosis after mono-
(2-ethylhexyl)-phthalate exposure [29]. A dose-depen-
dent increase in p53 protein and its transcript was
observed ingerm cells after H
2
O
2
exposure in the pres-
ent study (Figs 7A,B). Previous data have suggested a
potential role for the nuclear transcription factor,
NF-jB, in regulating rodent spermatogenesis, but its
physiological significance in the testis still remains elu-
sive [30]. However, during testicular stress in humans,
Sertoli cell NF-jB proteins exert pro-apoptotic effects
on germ cells, which raises the possibility that the phar-
macological inhibition of NF-jB could be a therapeutic
target in transient stress situations involving excessive
germ cell death [31]. NF-jB activation in the present
study was consistent with the increase in concentration
of H
2
O
2
exposure, which may act as a proapoptotic
factor (Fig. 7C). Our findings are also in agreement
with similar studies in Jukat and HeLa cells, where it
was demonstrated that increased p53 expression was
dependent on the functional activation of NF-jB
because the apoptosis-inducing ability of NF-jB pre-
sumably relies on an induced elevated expression of
death effector genes such as p53 [15]. This is supported
by the fact that radiation induced NF-jB activation is
proapoptotic because c-irradiation preferentially targets
rapidly dividing cells towards apoptosis [32].
MAP kinases are also activated primarily by cyto-
kines or exposure to extracellular stress including
H
2
O
2
. JNK and p38 MAP kinases are important medi-
ators of stress-inducing signals and oxidative stress
may be responsible for activation of MAP kinases,
leading to apoptosis [33–35]. The results obtained in
the present study also demonstrate that H
2
O
2
induces
the activation of JNK and p38 in the treated testicular
germ cells. This is in agreement with the previously
reported evidence indicating that p38 MAP kinase
plays an important role in the regulation of mitochon-
dria-dependent apoptosis, which is crucial with respect
to male germcell death in rats after hormonal
deprivation by potent gonadotrophin-releasing hor-
mone antagonist treatment [36,37].
In conclusion, the present study is the first to dem-
onstrate that H
2
O
2
induces germcellapoptosis and
triggers all the possible pathways of metazoan apop-
tosis, a representative model of which is depicted in
Fig. 8. The death signal triggered by H
2
O
2
at very low
concentrations signifies its importance as a regulatory
biomolecule intesticulargermcell apoptosis, although
Fig. 8. A proposed model depicting the pathways of H
2
O
2
-induced germcell apoptosis. The metazoan apoptosis model as proposed by
Hengartner [10] has now been updated to include the findings on the pathways leading to testiculargermcell apoptosis, as observed in
the present study.
H
2
O
2
and testicularcellapoptosis A. Maheshwari et al.
876 FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS
its role and interactions in normal testicular physiology
need to be explored further.
Experimental procedures
Animals
Adult male albino rats (Holtzman strain) weighing 180–
200 g were used. The animals were maintained under con-
trol temperature (25 ± 2 °C) and constant photoperiod
(12 : 12 h light ⁄ dark cycle) with food and water available
ad libitum. Animal experiments were carried out in strict
compliance with the Institutional Guidelines for Animal
Care.
Isolation of testicular cells
A two-step enzymatic method was used with a few modifi-
cations [38]. Briefly, Hank’s balanced salt solution (HBSS)
containing 0.44 mm KH
2
PO
4
, 137 mm NaCl, 5.36 mm KCl,
4.2 mm NaHCO
3
, 0.44 mm KH
2
PO
4
and 5 mm glucose was
prepared and sterilized by passing through a 0.22 lm filter
(Millipore, Billerica, MA, USA). The excised testis was
rinsed in HBSS and the tunica albuginea along with other
visible connective tissues were removed. The seminiferous
tubule mass was transferred to fresh HBSS containing
0.25 mgÆmL
)1
collagenase and kept for 15 min at 34 °C
with constant shaking. The dispersed seminiferous tubules
were allowed to sediment and washed three times in the
fresh HBSS, which largely removed contamination from
interstitial and blood cells. The isolated tubules were sub-
jected to trypsin (1.25 mgÆmL
)1
) and DNaseI (50 lgÆmL
)1
)
treatment for 10 min. The resultant crude cell suspension
was filtered through organza, washed after centrifugation at
500 g for 5 min at room temperature and resuspended in
medium-199 containing 100 lgÆmL
)1
streptomycin sulfate
and 100 IUÆmL
)1
penicillin. The cell viability of the final
crude population that contained mostly maturing germ cells
was in excess of 95%.
H
2
O
2
treatment
The isolated testiculargerm cells (5 · 10
6
cellsÆmL
)1
per
tube) were incubated for 1 h at 34 °C with or without
H
2
O
2
in medium-199. Incubations with H
2
O
2
were made at
final concentrations of 0, 1, 2, 5 and 10 lm. The treated
cells were washed in NaCl ⁄ P
i
and stored at )20 °C until
further use. Approximately 75% cells were viable in the
group exposed to 10 lm H
2
O
2
.
DNA ladder assay
To determine the inter-nucleosomal DNA fragments gener-
ated during cell death, total cellular DNA was isolated by
treating the cell pellet with 500 lL of lysis buffer (50 mm
Tris–Cl, pH 8.0, 20 mm EDTA, 10 mm NaCl and 1% SDS)
for 30 min at 4 °C. The suspension was centrifuged at
12 000 g for 10 min and the supernatant was extracted with
phenol ⁄ chloroform ⁄ isoamyl alcohol (25 : 24 : 1) and centri-
fuged at 12 000 g for 5 min. The upper phase was collected,
treated with a 1 : 10 volume of 3 m sodium acetate
(pH 5.2) and an equal volume of absolute ethanol for 2 h
at )20 °C to precipitate DNA, and centrifuged at 15000 g
at 4 °C for 30 min. The pellet was washed with 70% etha-
nol, air dried and resuspended in TE buffer containing
RNase (20 lgÆmL
)1
) for 30 min at 37 °C. Equal amount of
samples were loaded to 1.5% agarose gel containing
0.5 lgÆmL
)1
of ethidium bromide and visualized through
gel documentation system (UVP Inc., Upland, CA, USA).
ISEL assay
ISEL was carried out in accordance with the manu-
facturer’s instructions (R&D Systems, Minneapolis, MN,
USA). Briefly, pre-washed cells were smeared on poly-l-
lysine coated slides and fixed in 4% formaldehyde. Cells
were treated with cytonin for 10 min followed by quenching
with H
2
O
2
. Biotinylated nucleotides were incorporated into
the 3¢-OH ends of the DNA fragments by TdT, and
detected by using streptavidin-horseradish peroxidase
(HRP). The colour was developed by diaminobenzidine
(DAB) solution and later counter stained with methyl
green. ISEL positive cells were examined using a Nikon
microscope (Nikon, Tokyo, Japan) and the percentage
apoptosis was calculated.
Lipid peroxidation and antioxidant enzymes
activity
Treated or untreated testiculargerm cells were sonicated
for 30 s and divided into two equal parts. One part was
assayed for lipid peroxidation through the formation of
thiobarbituric acid reactive substances in the reaction
mixture, as described previously [39]. The second part was
centrifuged at 10 000 g for 5 min and the supernatant was
assayed for antioxidant enzyme activity. SOD was mea-
sured as described previously [40]. Catalase was estimated
by the degradation of hydrogen peroxide (6 mm), as
described previously [41]. GST activity towards 1-chloro-
2,4-dinitrobenzene was measured in accordance with the
method of Habig et al. [42]. Protein was estimated by the
Bradford assay.
TAC
Testicular germ cells treated with or without H
2
O
2
were
assessed for TAC in accordance with the manufacturer’s
instructions (Cayman Chemical Company, Ann Arbor, MI,
A. Maheshwari et al. H
2
O
2
and testicularcell apoptosis
FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS 877
USA). The assay relies on the ability of combined antioxi-
dants (vitamin, protein, lipids, glutathione, uric acid, etc.)
present in the cell lysate to inhibit the oxidation of 2,2-azi-
no-di-(3-ethylbenzthiazoline sulfonate) (ABTS) to the oxi-
dized form of ABTS by metmyoglobin. The amount of
oxidized ABTS produced was measured at 750 nm. Trolox
standards were used and total antioxidant capacity (mm)
was estimated using the standard value.
Caspase-3 activity
Activity of caspase-3 was assayed in accordance with the
manufacturer’s instructions supplied with the caspase-3 col-
orimetric assay kit (Alexis, San Diego, CA, USA). Briefly,
germ cells were resuspended in cold lysis buffer and incu-
bated for 10 min. Cell lysates were centrifuged for 2 min at
10 000 g at 4 °C. Supernatant (75 lg of protein per 50 lL)
was added to 50 lL of reaction buffer containing 200 lm
of chromogen (Ac-DEVD-pNA), kept at 37 °C for 2 h and
terminated. The increase in A
405
due to the release of
p-nitroanilide was measured using micro-titre plate reader
(BioTek Inc., Winooski, VT, USA).
Western blot analysis
Primary antibodies (rabbit polyclonal) for anti-PARP,
anti-caspase-9, anti-Bid, anti-Bak, anti-Bad, anti-JNK,
anti-NF-jB, anti-cytochrome c, anti-Fas, anti-FasL and
anti-p53-HRP and primary antibodies (mouse monoclonal)
for anti-caspase-8, anti-pJNK, anti-Bcl-2 and anti-b-actin
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) were
utilized. Anti-phospho-p38, a rabbit polyclonal primary
antibody was obtained from R&D Systems. Goat anti-rab-
bit ⁄ mouse-HRP conjugate secondary serum was obtained
from Santa Cruz Biotechnology. Whole cell lysates were
prepared in 200 lL of lysis buffer containing 20 mm Hepes
(pH 7.4), 2 mm EDTA, 50 mm b-glycerophosphate, 1%
Triton X-100, 150 mm NaCl, 10% glycerol and protease
inhibitor cocktail (Roche, Basel, Switzerland). Lysates were
clarified by centrifugation at 15 000 g at 4 °C for 20 min
and the protein concentration of the supernatant was deter-
mined by the Bradford assay. For SDS ⁄ PAGE, protein
lysates were mixed with Laemmli sample buffer (Bio-Rad,
Herculus, CA, USA) and boiled for 10 min. Total protein
was separated on a 12 ⁄ 15% gel and transferred to nitrocel-
lulose membrane (Millipore). After blocking in NaCl ⁄ Tris-
T (20 mm Tris–HCl, 137 mm NaCl, 0.1% Tween 20,
pH 7.6) with 5% skimmed milk, membranes were incubated
with the primary antibodies (1 : 1000) diluted in NaCl ⁄ -
Tris-T for 2 h at room temperature. Next, membranes were
washed three times with NaCl ⁄ Tris-T, and incubated for an
additional 2 h with HRP linked secondary antibody
(1 : 2000) diluted in NaCl ⁄ Tris-T. Again, membranes were
washed three times with NaCl ⁄ Tris and labelled protein
bands were visualized with the DAB system (Bangalore
Genei, Bangalore, India). b-actin was used to monitor equal
loading of protein. Densitometric analysis (see Table S1)
was performed with the help of Image analysis software
(lab works image analysis software, version 4.0; UVP
Inc.).
Table 1. Primer-specific conditions used for PCR amplification of candidate genes.
Name
Primer
sequence (5¢-to3¢)
Reference ⁄
accession
no.
Mg
2+
concentration
(m
M)
Annealing
temperature
(
o
C)
Product
size (bp)
Mn SOD Forward: CTTCAGCCTGCACTGAAGTTCAAT
Reverse: CTGAAGATAGTAAGCGTGCTCCC
[43] 2.5 65 326
Cu ⁄ Zn SOD Forward: GAGCATGGGTTCCATGTCCAT
Reverse: ACTTTCTTCATTTCCACCTTTGCC
[43] 2.5 62 277
Catalase Forward: CCGACGAGATGGCACACTTTGACA
Reverse: CGCGAGCACGGTAGGGACAGTTC
[26] 2.5 62 972
Bax-a R&D Systems (RDP-43) U49729 2.5 62 482
Bcl-2 R&D Systems (RDP-44) U34964 2.5 60 293
p53 R&D Systems (RDP-144) X13058 2.5 62 397
Caspase-8 Forward: CTGGGAAGGATCGACGATTA
Reverse: CATGTCCTGCATTTTGATGG
[20] 2.5 62 123
Caspase-9 Forward: AGCCAGATGCTGTCCCATAC
Reverse: CAGGAGACAAAACCTGGGAA
[20] 2.5 65 132
Fas Forward: GCAATGCTTCTCTCTGTGACCACT
Reverse: GCTGTTGTGCTCGATCTCATCG
[44] 3.5 65 351
FasL Forward: GGAATGGGAAGACACATATGGAACTGC
Reverse: CATATCTGGCCAGTAGTGCAGTAATTC
[45] 1.5 65 238
b-Actin Forward: CTGTGCCCATCTATGAGGGTTAC
Reverse: AATCCACACAGAGTACTTGCGCT
[46] 2.5 60 539
H
2
O
2
and testicularcellapoptosis A. Maheshwari et al.
878 FEBS Journal 276 (2009) 870–881 ª 2009 The Authors Journal compilation ª 2009 FEBS
RNA isolation and RT-PCR analysis
Total RNA was extracted using TRI-Reagent (Ambion,
Austin, TX, USA). cDNA was synthesized from 2 lgof
total RNA using the omniscript RT kit (Qiagen, Hilden,
Germany). Two microliters of the RT reaction was then
used for PCR using the HotStar HiFidelity DNA poly-
merase (Qiagen). The PCR reactions were carried out
under the temperature profile: (a) denaturation at 95 °C
for 15 min; (b) 30 cycles of 95 °C for 30 s, 60–65 °C for
1 min and 72 °C for 1 min; and (3) a final extension for
10 min at 72 °C. The sequence, source, annealing temper-
ature, Mg
2+
concentration and product size of primers
are shown in Table 1. After amplification, the products
were separated on 1.5% agarose and documented with
the help of a gel documentation system (UVP Inc.).
b-actin was used as an internal control. Densitometric
analysis was performed as previously described (see
Table S2).
Statistical analysis
All the experiments were repeated three times. Error bars
represent the SD. Statistical analysis was performed using
an unpaired Student’s t-test. P < 0.05 was considered
statistically significant.
Acknowledgements
A Junior Research Fellowship to Ankur Maheshwari
from the Council of Scientific and Industrial Research
(CSIR), New Delhi, India, is greatly acknowledged.
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induced by H
2
O
2
in vitro
Ankur Maheshwari
1
, Man M. Misro
1
,. there
are different pathways involved in testicular germ cell
apoptosis pertaining to specific treatments. As previ-
ously shown [7], testicular cell apoptosis follows