In the present study, H2O2, at concentrations in the range 1–10 lm, was found to induce apoptosis in testicular germ cells in vitro.. Taken together, the above data indicate that testicu
Trang 1induced by H2O2 in vitro
Ankur Maheshwari1, Man M Misro1, Archana Aggarwal1, Rajnesh K Sharma2and Deoki Nandan1
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 (A2–A4) 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 H2O2 and other reactive oxygen species
(ROS) [3] H2O2 constitutes the main ROS form in sperms but its effective role as an endogenous messen-ger in messen-germ cell apoptosis is largely unknown [4] How-ever, H2O2 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], H2O2
is synthesized endogenously in certain cell systems in response to specific cytokines or hormones⁄ growth factors and such endogenous H2O2 can then either act
Keywords
apoptosis; germ cell; H2O2; 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
H2O2 induces apoptosis in variety of cells; however, the sensitivities of testicular germ cells to H2O2are not known In the present study, H2O2, 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 H2O2,
a 10-fold rise in the percentage of apoptotic cells was observed Induction
of germ cell apoptosis 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 H2O2 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 testicular germ cells are prone to apoptosis at very low concentrations of H2O2, 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
Trang 2as 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 pathways involved in testicular germ cell
apoptosis pertaining to specific treatments As
previ-ously shown [7], testicular cell apoptosis 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 activaactiva-tion 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 H2O2
at physiological concentrations modulates Leydig cell
function and induces oxidative stress and apoptosis
[11] However, the sensitivities of isolated germ cells to
H2O2 are not known Furthermore, chronic human
chorionic gonadotrophin (hCG) treatment has been reported to be associated with the rise in testicular
H2O2 and germ cell apoptosis [12] Therefore, in the present study, the effects of H2O2 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
H2O2treatment is associated with rise in oxidative stress
Testicular germ cells demonstrated a significant (P < 0.01) rise in lipid peroxidation following H2O2 exposure at a 2 lm concentration and above (Fig 1A) However, the effect was not significant at the lowest concentration of H2O2 (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 H2O2 (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 testicular germ cells treated with H2O2 (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 H2O2 (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.
Trang 3regulated 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) of H2O2
(Fig 2)
Low concentrations of H2O2induce germ cell
apoptosis
In situend 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 H2O2 at concentrations
of 2 lm and above Approximately, 52% of the treated
cells were found apoptotic with the highest
concentra-tion of H2O2, which was ten-fold higher compared to
controls (Fig 3A,B) Germ cell apoptosis in 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, H2O2 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)
H2O2induced germ cell apoptosis follows both
extrinsic and intrinsic pathways
To determine whether H2O2-induced redox imbalance
and subsequent testicular germ cell apoptosis 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 H2O2 treated cells (Fig 5A,B) As expected, the
anti-apopto-Fig 2 Assessment of TAC in testicular germ cells with or without
H 2 O 2 (10 l M ) C, control *P < 0.05.
A
B
C
Fig 3 ISEL of testicular germ cells treated with H2O2 (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 O2 exposed testicular germ cells C, control *P < 0.01, **P < 0.001.
Trang 4tic 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 H2O2 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 H2O2 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 pathways in H2O2induced germ cell apoptosis
H2O2 treatment of testicular germ 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 H2O2, even at a concentration of 1 lm, has the ability to induce apoptosis in testicular germ cells in vitro 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
H2O2is a by-product of cell metabolism; its cellular levels and maintenance are constantly under homeo-static regulation The cells can sense sublethal doses of
H2O2 and activate peroxide-detoxifying mechanisms; alternatively, various H2O2 producing mechanisms can
be activated by different cell death stimuli As a result
of this deliberate H2O2 production, a self-destructive programmed cell death can be triggered [13] However, different organisms or cells have variations in their sensitivities to H2O2 In the study of caspase-depen-dent⁄ independent events of apoptosis in MCF-7 breast
A
B
Fig 4 (A) A rise (three- to 30-fold) in caspase-3 activity and (B)
PARP cleavage in testicular germ cells following H2O2treatment.
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.
Trang 5carcinoma cells, H2O2 in the optimal range 250–
1000 lm was used [14] However, in Jurkat T-cells,
H2O2-induced apoptosis required 100 lm or more of
H2O2 [15] It is also reported that 50 lm of H2O2 was
sufficient to induce apoptosis in rat germ cells [16]
Although a higher concentration in the medium
is toxic and affects cell survival, H2O2, even at
physio-logical concentrations (30–50 lm), was shown to
induce apoptosis in testicular Leydig cells in vitro [11]
Germ cells constitute the major inner seminiferous tubular cell mass and the role of H2O2 as a signalling molecule in apoptotic induction among germ cells is not yet known In the present study, we attempted to test germ cell sensitivity to H2O2 with reference to apoptotic induction The findings also clearly estab-lished that testicular germ cells are much more sensi-tive to H2O2and would require a five- to 30-fold lower concentration of H2O2in vitrofor induction of apopto-sis compared to 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 H2O2 dur-ing such conditions is not yet known In the rat testis, however, hormonal alterations due to chronic hCG treatment were reported to raise H2O2 levels and induce germ cell apoptosis [12] Therefore, it is reason-able to assume that H2O2 might constitute a specific physiological link to germ cell apoptosis in the testis,
A
B
C
Fig 7 Other pathways of signal transduction in H2O2induced tes-ticular germ cell 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.
Trang 6for 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, H2O2was 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 H2O2(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 H2O2
(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 H2O2concentration 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 H2O2 By
contrast, a moderate increase in GST activity was
observed at lower concentrations of H2O2(1–2 lm) Such
an increase in GST activity may be considered as one of
the measures to counteract the oxidative stress induced
by H2O2at 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 induced by either gamma-irradiation
or H2O2[16]
Spermatocytes and spermatids constitute the bulk of
the germ cells isolated and H2O2 was seen to mediate
the up-regulation of Fas protein and its transcripts in
these cells (Fig 6C) Interestingly, H2O2 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 H2O2 expo-sure to testicular germ 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 in testicular 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
H2O2-induced testicular germ cell apoptosis (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, suggestreduc-ing that Bcl-2 may indirectly regulate antioxidant defences to prevent cell death H2O2 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
Trang 7The down-regulation of Bcl-2 in the group exposed to
10 lm H2O2 probably reflects the collapse of the
anti-apoptotic measure beyond a critical concentration
As already discussed, H2O2 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 germ cell apoptosis after
mono-(2-ethylhexyl)-phthalate exposure [29] A
dose-depen-dent increase in p53 protein and its transcript was
observed in germ cells after H2O2exposure 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 H2O2 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
H2O2 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 H2O2 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 germ cell 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 H2O2 induces germ cell apoptosis 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 H2O2at very low concentrations signifies its importance as a regulatory biomolecule in testicular germ cell apoptosis, although
Fig 8 A proposed model depicting the pathways of H 2 O 2 -induced germ cell apoptosis The metazoan apoptosis model as proposed by Hengartner [10] has now been updated to include the findings on the pathways leading to testicular germ cell apoptosis, as observed in the present study.
Trang 8its 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 ± 2C) 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 KH2PO4, 137 mm NaCl, 5.36 mm KCl,
4.2 mm NaHCO3, 0.44 mm KH2PO4and 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 34C
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%
H2O2treatment
The isolated testicular germ cells (5· 106
cellsÆmL)1 per tube) were incubated for 1 h at 34C with or without
H2O2in medium-199 Incubations with H2O2were made at
final concentrations of 0, 1, 2, 5 and 10 lm The treated
cells were washed in NaCl⁄ Pi and stored at )20 C until
further use Approximately 75% cells were viable in the
group exposed to 10 lm H2O2
DNA ladder assay
To determine the inter-nucleosomal DNA fragments
gener-ated during cell death, total cellular DNA was isolgener-ated 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 4C 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 4C 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 37C 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 H2O2 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 testicular germ 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 H2O2 were assessed for TAC in accordance with the manufacturer’s instructions (Cayman Chemical Company, Ann Arbor, MI,
Trang 9USA) 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 4C 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 37C for 2 h and
terminated The increase in A405 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 4C 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-Tris–HCl, 137 mm NaCl, 0.1% Tris-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¢- to 3¢)
Reference ⁄ accession no.
Mg 2+
concentration (m M )
Annealing temperature ( o C)
Product size (bp)
Reverse: CTGAAGATAGTAAGCGTGCTCCC
Cu ⁄ Zn SOD Forward: GAGCATGGGTTCCATGTCCAT
Reverse: ACTTTCTTCATTTCCACCTTTGCC
Catalase Forward: CCGACGAGATGGCACACTTTGACA
Reverse: CGCGAGCACGGTAGGGACAGTTC
Caspase-8 Forward: CTGGGAAGGATCGACGATTA
Reverse: CATGTCCTGCATTTTGATGG
Caspase-9 Forward: AGCCAGATGCTGTCCCATAC
Reverse: CAGGAGACAAAACCTGGGAA
Reverse: GCTGTTGTGCTCGATCTCATCG
Reverse: CATATCTGGCCAGTAGTGCAGTAATTC
Reverse: AATCCACACAGAGTACTTGCGCT
Trang 10RNA isolation and RT-PCR analysis
Total RNA was extracted using TRI-Reagent (Ambion,
Austin, TX, USA) cDNA was synthesized from 2 lg of
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 95C
for 15 min; (b) 30 cycles of 95C for 30 s, 60–65 C for
1 min and 72C for 1 min; and (3) a final extension for
10 min at 72C The sequence, source, annealing
temper-ature, Mg2+ 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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