Involvement of Fas/FasL system in apoptotic signaling in testicular germ
cells of male Wistar rats injected i.v. with microcystins
Qian Xionga,b, Ping Xiea,*, Huiying Lia, Le Haoa,b, Guangyu Lia,b, Tong Qiua,b, Ying Liua,b
aDonghu Experimental Station of Lake Ecosystems, State Key Laboratory for Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology,
Chinese Academy of Sciences, Wuhan 430072, People’s Republic of China
bGraduate School of the Chinese Academy of Sciences
a r t i c l e i n f o
Received 18 November 2008
Received in revised form 24 January 2009
Accepted 27 January 2009
Available online 7 February 2009
a b s t r a c t
Previous studies have shown that gonads were the second target organ of microcystins
(MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of
mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway
plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the
responses of Fas/FasL system related genes and proteins in testes of rats injected intra-
venously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was
detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end
labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL
protein expression were induced evidently from 1 h post-injection, and this high
expression level maintained throughout the experiment. In addition, the activation of
caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis.
These results suggested the likely involvement of Fas/FasL system in the MCs-induced
germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly.
? 2009 Elsevier Ltd. All rights reserved.
Microcystins (MCs), a group of cyclic heptapeptide
compounds with specific hepatotoxins produced by cya-
nobacterial species, have received worldwide concern in
the past decades (Cohen,1989; Carmichael et al.,1997) and
in a recent study by Chen et al. (2009), MCs were identified
for the first time in the serum of a chronically exposed
human population (fishermen at Lake Chaohu, China)
together with indication of hepatocellular damage. So far,
more than 80 different structural analogues of MCs have
been identified (Fastner et al., 2002), with microcystin-LR
being the most toxic (Mirura et al., 1989). MCs have been
already well characterized as strong inhibitors of protein
phosphatases1 (PP1) andphosphatases 2A(PP2A)
(Yoshizawa et al.,1990; Nishiwaki-Matsushima et al.,1992).
This inhibition could lead to hyperphosphorylation of key
proteins that regulate apoptosis (Fu et al., 2005). Previous
studies confirmed that various cell types could be induced
to undergo apoptosis by MCs, mainly characterized by cell
membrane blebbing, cytoplasmic shrinkage, nuclear chro-
matin condensation, DNA fragmentation and formation of
apoptotic bodies (McDermott et al.,1998; Ding et al., 2000).
Recently, some studies indicated that gonads might be
the second target organ of MCs (Chen and Xie, 2005; Chen
et al., 2005). Wang et al. (2008) report that contents of MCs
(RRþLR) remained relatively stable (means 21–37 ng/g) in
the rat gonad within 24 h after i.v. injection of MCs’ extracts
at a dose of 80.5 mg MC-LRequivalent/kg body weight, and
that even at 24 h, a certain amount of MCs was still present
in the gonad, suggesting the difficulty of MCs’ elimination
from gonad. Ding et al. (2006) revealed various toxic effects
of MC-exposure on the reproductive system of male mice
* Corresponding author. Tel./fax: þ86 27 68780622.
E-mail address: email@example.com (P. Xie).
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Toxicon 54 (2009) 1–7
structure, increased space between the seminiferous
tubules, decreased numbers of interstitial cells, Sertoli cells
and mature sperm. Li et al. (2008) also found MC-LR can
exert a generally chronic toxicity to male rat reproductive
system and specific damage to the testes, including
increased sperm abnormality, decreased sperm motility
and concentration. As is known, the Fas-signaling system is
implicated in the elimination of germ cells after testicular
injury. When exposed to specific testicular toxicants, germ
cell and Sertoli cell injuries were observed, followed by
up-regulation of Fas and FasL in injured cells. This up-
regulation of Fas–FasL resulted in an elevation of germ cell
apoptosis (Lee et al., 1997, 1999; Boekelheide et al., 2000).
Fas (APO-1, CD95) is a transmembrane receptor protein
that belongs to the tumor necrosis factor/nerve growth
factor receptor family (Watanabe-Fukunaga et al., 1992;
Nagata and Golstein, 1995). FasL is a tumor necrosis factor-
related type II transmembrane protein (Suda et al., 1993).
Binding of FasL to Fas can induce trimerization of Fas
receptors, which recruit Fas-associated death domain
(FADD) through shared death domains (DD). FADD also
contains a death effector domain (DED) at N-terminal
region. Fas/FADD complex then binds to procaspase-8
through interactions between DED of the FADD and these
caspase molecules. Aggregation of procaspase-8 causes
self-activation, and generatesactive caspase-8, the initiator.
At this point, there are two possible pathways through
which caspase-8 can enter into: one is to directly activate
procaspase-3 by proteolytic cleavage, and the other is to
cleave Bid, a proapoptotic member of the Bcl-2 family. The
cleaved Bid induces the release of cytochrome c from
mitochondria, which interacts with apoptotic protease
activating factor-1 (Apaf-1) and dATP to form the apopto-
som. Apoptosom is a large oligomeric protein complex
which can activate procaspase-9. Caspase-9 then activates
dependent apoptotic pathway (Budihardjo et al., 1999;
Cohen, 1997). Both pathways converge on caspase-3 and
other executioner caspases and then nucleases that drive
the terminal events of programmed cell death. In the whole
process of apoptosis regulation, the expression of Fas, FasL,
FADD, caspase-8, Apaf-1, caspase-9 and caspase-3 is of vital
Based on these factors, it is of consequence to find out
whether Fas/FasL system is involved in the mechanism of
MCs-induced injury or apoptosis in testes. The primary
aims of this study were to evaluate the time-dependent
responses of the Fas/FasL system related genes and proteins
in testes of rats exposed toMCs, and further toelucidate the
underlying toxicological mechanisms.
2. Materials and methods
The cyanobacterial material used in this experiment was
collected fromsurface blooms (phytoplankton cells) of Lake
Dianchi, Yunnan in China during May and June, 2006.
According to microscopic examinations, the predominant
species was Microcystis aeruginosa. Freeze-dried crude
algae were extracted three times with 75% (V/V) methanol.
The extract was centrifuged and the supernatant was
applied to a C18reversed phase cartridge, which had been
preconditioned by washing with methanol and then
distilled water. The cyanobacterial material was analyzed
for MCs content via a reverse-phase high-performance
liquid chromatography (HPLC, LC-10A, Shimadzu Corpor-
ation, Nakagyo-ku, Kyoto, Japan) equipped with an ODS
column (Cosmosil 5C18-AR, 4.6?150 mm, Nacalai, Japan)
and an SPA-10A UV–vis spectrophotometer set at 238 nm.
comparing the peak areas of the test samples with those of
the standards available (MC-LR and MC-RR, Wako Pure
Chemical Industries, Japan). Crude MCs’ extracts were
finally suspended in salt solution (0.9% NaCl).
Male Wistar rats weighing 180–200 g were supplied by
Hubei Laboratory Animal research Center (Hubei, China).
The rats were housed under controlled conditions of 12 h
light/dark cycle, 50?5% humidity and 23?1?C. The
animals were allowed free access to food and water. All
animal procedures were approved by the Institutional
Animal Care and Use Committee (IACUC) and were in
accordance with the National Institutes of Health Guide for
the Care and Use of Laboratory.
2.3. LD50of cyanobacterial crude extracts
Male Wistar rats (n¼40) were divided into five groups,
and rats in each group were administered by an i.v. injec-
tion of MCs at different dose of MC-LRequivalent/kg body
weight. Calculating the death number and mortality of rats
in each group during 24 h, we obtained the LD50level for
24 h by using the formula LD50¼log?1[Xm?I(Sp?0.5)]
(Xm: the log of max dose; p: mortality; Sp: the sum of
mortality in each group; I: the difference between the logof
adjacent two group dose). The LD50studies were approved
by the IACUC.
2.4. MCs exposure
Healthy male Wistar rats weighing 200?20 g were
divided in equal numbers into two groups randomly. One
group received intravenous injection (i.v.) of 1 mL MCs
extracts at LD50of 80.5 mg MC-LRequivalent/kg body weight.
An equivalent volume of 0.9% saline solutionwas applied to
control ones. Six sampling points were set during a period
of 24 h in the experiment (1, 2, 4, 6, 12 and 24 h). Five rats
from each group were killed at each time points and the
testes were quickly removed, minced and stored frozen at
liquid nitrogen for later analysis.
2.5. Terminal deoxynucleotide transferase-mediated deoxy-
UTP nick end labeling (TUNEL)
For TUNEL staining (Gavrieli et al., 1992), the standard
protocol for frozen sections was followed (ApopTag, Oncor,
Gaithersburg, MD). Frozen cross-sections (8 mm) from
testes were prepared, fixed in 10% neutral buffered
Formalin for 10 min at room temperature, rinsed in PBS,
Q. Xiong et al. / Toxicon 54 (2009) 1–72
postfixed in acetone for 5 min at ?20?C, and then incu-
bated in 2% H2O2for 15 min to quench endogenous perox-
idases. Five slides were prepared per testis and the number
of TUNEL-positive cells was counted in each seminiferous
tubule (ST). To quantitate the incidence of apoptosis at each
time point, the TUNEL-positive cells within a ST cross-
section were counted. All TUNEL-positive cells within the
percentage of apoptotic germ cells in both the control and
MCs-treated rat testes was determined by counting a total
of 1000 germ cells (including both apoptotic and non-
apoptotic cells) from STs with cross-section.
2.6. Total RNA isolation
Total RNA was isolated from 50 to 100 mg sections of
testis using Trizol reagent (Invitrogen, America) and
quantified by determination at OD260. RNA was extracted
according to the manufacturer’s protocol, resuspended in
50 ml RNase-free water, and stored at ?80?C. Quantifica-
tion was done using Eppendorf Biophotometer (Germany).
The purified total RNA (2 mg) was then reverse transcribed.
Reverse transcription was performed with oligo (dT) 18
primer using first strand cDNA synthesis kit (TOYOBO,
Japan). The resultant cDNA was then diluted 20 fold and
kept at ?20?C.
2.7. Quantitative real-time PCR (Q-PCR)
All the primers used in Q-PCR were listed in Table 1. The
primers were designed based on the gene sequences of
Rattus norvegicus present on the NCBI homepage (http://
www.ncbi.nlm.nih.gov). The specification of each pair of
primers was confirmed by randomly sequencing six clones,
and further confirmed by the melting curve analysis using
Q-PCR. The amplification efficiency of each pair of primers
was tested by constructing corresponding plasmid. Only
primers with similar amplification efficiency were used in
this experiment. GAPDH was used as the internal control
gene for Q-PCR assay. Q-PCR was conducted with the SYBR
Green qPCR kit (Finnzymes, Finland) on a Chromo4 Real-
Time Detection System (MJ Research, Cambridge, MA). The
reactions were performed in a 20 ml volume mix containing
10 ml SYBR Green I mixture,1 ml primers,1 ml cDNA and 1 ml
sterile, distilled–deionized water. Cycling conditions were
as follows: 3 min at 95?C, 44 cycles of 15 s at 95?C, 20 s at
62?C or 64?C, and 15 s at 72?C. Melting curve analysis of
amplification products was performed at the end of each
PCR reaction to confirm that a single PCR product was
detected. Each sample was run in three tubes, and PCR
reactions without the addition of the template used as
blanks. After completion of the PCR amplification, data
were analyzed with the Option Monitor software 2.03
version (MJ Research, Cambridge, MA).
2.8. Western blot analysis
For Western blotting sample preparation, small tissue
sections were homogenized in ice-cold protein extraction
buffer (Wuhan Boster Biological Technology Company,
China). After centrifugation at 12,000 g (4?C) for 10 min to
remove debris, the supernatant was carefully recovered.
Protein concentrations were determined using the Brad-
ford assay (Bradford, 1976). All samples were stored at
?80?C prior to electrophoresis.
Aliquots from supernatant containing 20 mg of proteins
were mixed with equal volume of 2? sample buffer. The
sample was boiled for 5 min and subjected to 12% SDS-
PAGE. After electrophoresis, the resolved proteins were
transferred to nitrocellulose membrane using an electro
blotting apparatus (Bio-Rad, America). Membranes were
blocked at room temperature for 2 h in blocking buffer
containing 5% nonfat dry milk to prevent non-specific
binding of reagents, and then incubated with anti-Fas (sc-
716; Santa Cruz Biotechnology; 1:200), anti-FasL (sc-834;
Santa Cruz Biotechnology; 1:200), anti-caspase-8 (Wuhan
Boster Biological Technology Company, China; 1:100), anti-
caspase-3 (H-277; Santa Cruz Biotechnology; 1:200) or
GAPDH (Wuhan Boster Biological Technology Company,
China) at 4?C overnight. The membranes were washed in
TBST (50 nmol/L Tris-Cl, pH 7.6, 150 mmol/L NaCl, 0.1%
Tween 20) for 30 min and incubated with IgG (H þL)
conjugated secondary antibody (Wuhan Boster Biological
Technology Company, China; 1:1000) for 1 h at room
temperature. The protein signal was developed using NBT/
BCIP system. The results of Western blots were quantified
with Gene Snap software (Syngene, America).
2.9. Statistical analysis
Significance of differences between the treated and
control groups was analyzed by Student’s t test. Statistical
significance was concluded at P<0.05 and 0.01.
Q-PCR primers used in this experiment.
Target genePrimer sequence (50–30) Size (bp)
Q. Xiong et al. / Toxicon 54 (2009) 1–73
3.1. TUNEL staining
As shown in Fig. 1(A), only a few apoptotic germ cells
were observed in the basal compartment of STs in the
control rats. In contrast, a remarkable increase in the
number of apoptotic germ cells (spermatogonia, sper-
matocytes and round spermatids) was observed in the
STs of MCs-treated rats (Fig. 1(B)). The percentage of
apoptotic germ cells in MCs-treated rats was significantly
higher than that in the control group (Fig. 2). The level
of apoptosis peaked at 6 h after treatment and the
elevation of the incidence of apoptosis was evident from
1 h onward.
3.2. mRNA level of the related genes of Fas/FasL system
Fig. 3 showed the transcriptional changes of Fas, FasL
and their downstream effectors-FADD, Caspase-8, Apaf-1,
Caspase-9 and Caspase-3 in the rat testes within 24 h. The
transcription of Fas was significantly induced since 2 h
post-injection, and reached a peak value at 6 h and then
returned to the original level at 24 h. FasL mRNA was
significantly increased at 1 h post-injection and peaked at
4 h, and returned to the control level at 24 h. The expres-
sion of FADD was suppressed evidently in the first two
hours and then significantlyenhanced at 4 h post-injection.
The transcription of caspase-8 was significantly elevated at
all time points. Apaf-1 mRNA was induced at 6 h and
showed a tendency to recover after 12 h. The transcription
of caspase-9 was induced from 2 h to 6 h and returned to
the original level since 12 h. The mRNA expression of the
terminal executioner-caspase-3 was induced from 4 h to
6 h but was depressed at 24 h. The mRNA expressions of
Fas, FasL, FADD, caspase-8, Apaf-1, caspase-9 and caspase-3
were all significantly increased at 6 h, among which Fas,
Apaf-1 and caspase-3 reached to the maximum at this time
3.3. Western blot analysis
The protein expressions of Fas, FasL, Caspase-8 and
Caspase-3 weredetected byWestern blot.BothFas and FasL
proteins were induced obviously at 1 h after MCs treat-
ment, and the high expression level remained throughout
the experiment. Quantification of the two proteins revealed
that the expressions of both Fas and FasL were significantly
increased from 1 h post-injection, and reached to the
maximum at 6 h and 4 h, respectively. Associated with this
increase was the activation of caspase-8, the initiator cas-
pase in this apoptotic pathway. The terminal executioner,
caspase-3 was also examined by Western bolt analysis. The
results showed that caspase-8 and caspase-3 cleavage was
detectable from 1 h onward using Western blots (Fig. 4). In
Fig. 1. TUNEL labeling of sections from treated and control rats. (A) Control
section showing very few cells positive for TUNEL (brown deposits).
(B) Increased number of apoptotic germ cells (arrows) is observed in the STs
of MCs-treated rats 4 h after exposure. (C) A marked increase in the number
of apoptotic germ cells (arrows) is observed in the STs of MCs-treated rats
6 h after exposure.
C 1h 2h 4h6h12h24h
Apoptotic cells (%)
Time after dosing (h)
Fig. 2. Time course of changes in percentages of apoptotic cells in testes of
control and MCs-treated rats. (*indicates significant change at P< 0.05,
**indicates significant change at P<0.01).
Q. Xiong et al. / Toxicon 54 (2009) 1–74
addition, procaspase-3 and procaspase-8 expression was
significantly elevated. The procasepase-8 and procaspase-3
expression peaked at 4 h and 6 h, respectively.
The principal finding of the present study was that
enhanced apoptosis of germ cells in the testes of MCs-
treated rats was associated with up-regulation of the Fas-
signaling system. The TUNEL assay demonstrated the
presence of a few apoptotic cells and apoptotic STs in the
control testes, which was in agreement with previous
reports (Lee et al., 1997, 1999; Richburg et al., 1999; Rich-
burg, 2000). In contrast, a significant increase in the
percentage of apoptotic germ cells was observed in the
testes of MCs-treated rats. From these results, it could be
deduced that MCs exposure induced germ cell apoptosis in
testes of male Wistar rats. Monitoring of time course
changes could facilitate analysis of toxicological mecha-
nisms including cause and effect relationship (Kijima et al.,
2004). In the present study, the mRNA expressions of Fas,
FasL and their downstream effectors-FADD, caspase-8,
Apaf-1, caspase-9 and caspase-3 were all significantly
increased within 24 h post-injection as determined by
Q-PCR. The simultaneous expression of these apoptosis-
related genes might suggest a multiple wave like propa-
gation of apoptosis signal from FasL, Fas, FADD down to
caspase-3 via a pathway including caspase-8, Apaf-1, cas-
pase-9. Moreover, we found that the expression profiles of
Fas and FasL in protein level were correlated closely with
that of mRNA level. Significant elevation of FasL and Fas
protein with similar folds to that of mRNA was observed in
testis from 1 h onward. Accompanied with this increase
was the activation of caspase-8, the initiator caspase and
caspase-3, the terminal executioner. As is known, activation
of caspase-8 is required for Fas-induced apoptosis (Juo
et al.,1998), while activation of caspase-3 is a central event
upon which numerous signaling pathways converge and
through which multiple downstream substances such as
caspase-activated DNase are cleaved (Jacobson et al., 1997;
Kim et al., 2000, 2001). The time-dependent up-regulation
of mRNA and activation of proteins suggest that the
Ratio compared with controls
Time after dosing (h)
Time after dosing (h)
Fig. 3. Time course of Fas/FasL system related mRNA expression by Q-PCR analysis in rat testis after treatment with 80.5 mg MC-LRequivalent/kg body weight.
(*indicates significant change at P <0.05, **indicates significant change at P<0.01).
Q. Xiong et al. / Toxicon 54 (2009) 1–75
apoptogenic effect of MCs might be mediated via the
up-regulation of the Fas-signaling system and the activa-
tion of caspases.
It is generally believed that Sertoli cells are the main
cells expressing FasL in STs (Xu et al.,1999; Hu et al., 2003;
Koji and Hishikawa, 2003; D’Abrizio et al., 2004) and Fas is
localized to specific germ cell subtypes (Xu et al.,1999). The
Fas-signaling system is initiated differentially depending
on the primary cellular site of toxicity. Exposure to toxi-
cants, such as 2, 5-hexanedione or mono-2-ethylhexyl
phthalate, which target the Sertoli cells and disrupt their
function, will lead to up-regulation of FasL followed by Fas
(Lee et al., 1997, 1999). Radiation-induced injury or testic-
ular hyperthermia, which both directly affect germ cells
failed to up-regulate FasL while up-regulating Fas mRNA
expression (Lee et al., 1999). Our studies revealed an
evident induction of both Fas and FasL mRNA in testis after
MCs treatment, when compared to control groups. The
highest expression of FasL mRNA was detected at 4 h after
MCs treatment, while the expression of Fas mRNA reached
to the maximum at 6 h when the level of apoptosis also
peaked. This result was in accordance to that of Lee et al.
(1999), who reported an early onset of FasL mRNA up-
regulation after Sertoli cell injury. Actually, large vacuoles,
distended endoplasmic reticulum and increased lipid
droplets were found in the Sertoli cell through electron
microscopy at 6 h post-exposure in this experiment
(unpublished data). Therefore, we supposed that MCs
damaged Sertoli cells in the testis of rats. This corroborated
previous reports that MCs could damage Sertoli cells in the
testes (Ding et al., 2006).
In normal state, Sertoli cells maintain the homeostasis
by providing hormonal, nutritional and physical support to
most healthygermcells, and killing a few Fas-positive germ
cells with FasL. Toxicants that injure or disrupt the func-
tions of Sertoli cells can effectively reduce their supportive
capacity and, as a result, germ cell cannot be supported
adequately. The Fas-signaling pathway between Sertoli
cells and germ cells plays a key role in mediating germ cell
apoptosis in the testis after Sertoli cell injury (Richburg
et al., 1999; Richburg, 2000; Giammona et al., 2002; Rich-
burg and Nanez, 2003). In the present study, both mRNA
and protein expression of Fas and FasL in the testis of male
Wistar rats were significantly induced. According to the
working model for the Fas-signaling system in the testis
proposed by Lee et al. (1999), it is most probable that after
injury, the disfunctional Sertoli cells increased the expres-
sion of FasL which translocates and diffuses towards the
apical side of STs to facilitate the elimination of the inad-
equately supported germ cells that express Fas. In this way,
the germ cells’ population can be reduced to a level which
the Sertoli cells can support. Thereby, a new equilibrium
state can be achieved to match the reduced supportive
capacity of the disfunctional Sertoli cells with fewer germ
cells (Richburg et al., 1999; Richburg, 2000).
In summary, the present study indicated for the first
time that MC-exposure could induce germ cell apoptosis in
rat testes. And the enhanced apoptosis of germ cells was
124612 241246 1224
Time after dosing (h)
c 1h 2h 6h 12h 24h
Fig. 4. Western blot analysis of proteins from rat testes with antibodies to Fas, FasL, caspase-8, caspase-3 and GAPDH. (A) Western blot analysis was performed
with antibody against Fas, FasL, caspase-8, caspase-3 and GAPDH. Each figure corresponds to a representative experiment out of three experiments. (B) Each
column and bar represent the mean ?SD of three individual samples. Mean protein expression in each treated groups is shown as a fold increased compared to
mean expression in control groups which has been ascribed an arbitrary value of 1. (*indicates significant change at P<0.05, **indicates significant change at
Q. Xiong et al. / Toxicon 54 (2009) 1–76
associated with the increased expression of Fas/FasL system
related genes at both mRNA and protein level, indicating
the likely participation of the Fas/FasL system in MCs-
induced germ cell apoptosis. Our study also suggested that
MCs had Sertoli cell toxicity.
The authors would like to thank Dr Alan Harvey and an
anonymous reviewer for their very useful comments and
suggestions on the manuscript. This work was supported
by a fund from the National Basic Research Program of
China (973 Program) (Grant No. 2008CB418101).
Conflicts of interest
The authors declare that there are no conflicts of
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