Toxicology Letters 225 (2014) 433–444

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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Differential proteome and gene expression reveal response to carbon

ion irradiation in pubertal mice testes
Hongyan Li a,b,c,d , Yuxuan He e , Hong Zhang a,b,c,∗ , Guoying Miao a,d,f
a
Department of Heavy Ion Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China
b
Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, China
c
Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, China
d
University of Chinese Academy of Sciences, Beijing 100049, China
e
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
f
Department of Radiotherapy, Gansu Provincial Hospital, Lanzhou 730000, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• Proteomics of pubertal mice testes

after carbon ion radiation were
examined.
• Differential proteins in 2-DE gels
were investigated 14 days after irra-
diation.
• Eight proteins were identified among
the differentially expressed protein
spots.
• The relationship between mRNA and
the abundance of proteins were con-
firmed.
• These proteins may lead to new
insights into the information of CIR
toxicity.

a r t i c l e i n f o a b s t r a c t

Article history: Heavy ion radiation, a high linear energy transfer (LET) radiation, has been shown to have adverse effects
Received 25 August 2013 on reproduction in male mice. The aim of this study was to profile and investigate the differentially
Received in revised form expressed proteins in pubertal male mice testes following carbon ion radiation (CIR). Male mice under-
30 December 2013
went whole-body irradiation with CIR (1 and 4 Gy), and MALDI-TOF/TOF analysis was used to investigate
Accepted 1 January 2014
the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused
Available online 15 January 2014
by irradiation after 14 days. 8 differentially expressed proteins were identified and these proteins were
mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant
Keywords:
Proteome
capacity and mitochondrial respiration, which play important roles in the inhibition of testicular func-
Pubertal mice tion in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and
Testis the abundance of proteins. Our results indicated that these proteins may lead to new insights into the
Carbon ion radiation molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves
diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins.
© 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: HIR, heavy ions radiation; 2-DE, two-dimensional gel electrophoresis; MALDI-TOF-TOF, matrix-assisted laser desorption/ionization tandem time-of-flight
mass spectrometry; HIRFL, heavy ion research facility in Lanzhou; CCB, colloidal coomassie blue G 250; TBS, tris buffer saline; DAB, diaminobezidine; HRP, horseradish
peroxidase; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GRP78, 78 kDa glucose-regulated protein; VCP, valosin-containing protein, isoform CRA a; ACO,
aconitate hydratase–mitochondrial precursor; PKM1/M2, pyruvate kinase isozymes M1/M2 isoform 1; GSTA3, glutathione-S-transferase A3; GSTP1, glutathione S-transferase
P 1; SOD1, Cu/Zn superoxide dismutase.
∗ Corresponding author at: Chinese Academy of Sciences, Department of Heavy Ion Radiation Medicine, Institute of Modern Physics, Lanzhou 730000, China.
Tel.: +86 931 4969344; fax: +86 931 8272100.
E-mail address: zhangh@impcas.ac.cn (H. Zhang).

0378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxlet.2014.01.001
434 H. Li et al. / Toxicology Letters 225 (2014) 433–444
1. Introduction
The biological effects of high linear energy transfer (LET) heavy-
ion radiation are more pronounced than low-LET radiation, such
as X-rays or ␥-rays (Duan et al., 2008). Heavy ions have greater
potential for application in various disciplines, especially in heavy
ion radiotherapy which involves trends in tumor localization radio-
therapy and its related biological research, and has become a hot
topic in research related to radiobiology and radiation medicine
(Tsujii and Kamada, 2012; Ohno, 2013). However, the negative
effects of heavy ion therapy cannot be ignored, as studies have
shown that heavy ions have a stronger cell killing effect than X- and
␥-rays (Sekine et al., 2011; Varès et al., 2011). In addition, ionizing
radiation can also cause damage to the body’s tissues and organs,
and can generate reactive oxygen free radicals, resulting in bond
breakage in biological molecules, the role of bioorganic molecules,
enzyme inactivation, DNA damage, changes in biofilm permeability
and even death (Datta et al., 2012).
Spermatogenesis is a complex process of male germ cell prolifer-
ation and maturation from spermatogonia to spermatozoa (Santos
and Kim, 2010). However, irradiation can influence spermatogen-
esis, as doses as low as 0.1 Gy are known to cause damage to
spermatogenia (Gong et al., 2013). Less than 2% of men who have
received total-body irradiation are able to father a child later in
life (Otala et al., 2004). Pediatric and adolescent patients with
tumors treated with radiotherapy may have deficiencies in the
gonadotropins, follicle-stimulating hormone (FSH) and luteiniz-
ing hormone (LH), as well as testosterone (Zhang et al., 2006).
Therefore, with the advent of new radiotherapy modalities other
than heavy ions, there has been a considerable improvement in
the survival rate of cancer patients, and men of child-bearing age
undergoing radiotherapy are often concerned about the possibility
of future children. Thus, it is important to ascertain whether high
LET radiation exposure induced spermatogenesis dysfunction, and
pay more attention to the reproductive potential and the possible
genetic alteration in the spermatogenic cells of these patients.
The analysis of male and female reproductive functions has
gained impetus deepening our insight into the normal and patho-
logical complexities of infertility and its causes; however, much
still remains to be elucidated. To understand these biological com-
plexities, the physiological function of each protein at the tissue
level needs to be carefully addressed. Remarkable advances in
proteomics technology, especially in the protein/peptide resolu-
tion technique and mass spectrometry have offered unparalleled
opportunities for global and targeted analyses of the expres-
sion, regulation and modification of proteins in various biological
systems (Kolialexi et al., 2008). In recent years, the reference pro-
teomes of male reproductive cells/tissues/organs such as sperm
(Gilany et al., 2011), epididymis (Li et al., 2010) and testis (Li et al.,
2011) have been obtained and these have enriched our knowl-
edge on the proteins expressed in these cells/tissues/organs. Efforts
are also being made to identify the proteins which contribute to
the pathology of various reproductive health disorders such as
azoospermia and oligozoospermia in men (Upadhyay et al., 2012).
In this study, we used a proteomic approach based on a two-
dimensional electrophoresis (2-DE) reference map to determine
alterations in protein expression in the testes of pubertal Swiss-
Webster mice subjected to CIR. In order to further confirm the
proteomic results, the gene expression changes were analyzed.
Serum testosterone and serum FSH levels were measured to eval-
uate the toxic effects of CIR on pubertal mice testes. Superoxide
dismutase (SOD) and intracellular ROS were measured to confirm
mitochondrial disruption and oxidative stress induced by CIR in
pubertal mice testes. ATP levels were examined to estimate testis
energy balance in pubertal mice testes following CIR. Integration
of the proteome, gene analysis and biochemical data provided
evidence to help understand the underlying mechanisms of CIR
toxicity in male pubertal patients following CIR.
2. Materials and methods
2.1. Animals
Male mice about weighing 18–22 g (4 weeks) of Swiss-Webster mice provided
by Lanzhou Medical College (Lanzhou, China) were used under identical breeding
conditions, all animals were kept in 22 ± 2 ◦ C, 60 ± 10% humidity and light: dark
cycle 12 h:12 h. A total of 30 mice were randomly divided into three groups includ-
ing control (0 Gy), low dose group (1 Gy) and high dose group (4 Gy), each group
constituted 10 mice. The studies were approved by the Institutional Animal Care
Committee.
2.2. Irradiation procedure and animals treatment
Mouse was fixed in a chamber and whole-body irradiated with carbon ion beam
at 300 MeV/U and 31.3 keV/␮m of the beam entrance, with dose rate was approx-
imately 0.5 Gy/min at the Heavy Ion Research Facility in Lanzhou (HIRFL, Institute
of Modern Physics, Chinese Academy of Sciences, Lanzhou, China). The carbon ion
is equipped with a passive beam delivery system. Ten mice from each group were
weighed and killed by cervical dislocation at 14 days after irradiation. Blood was
collected and centrifuged at 2000g at 4 ◦ C for 15 min. Serum was stored at −80 ◦ C
until analysis. The testes were weighed and frozen immediately in liquid nitrogen
and stored at −80 ◦ C until analysis.
2.3. Serum testosterone and serum FSH levels
Concentrations of serum testosterone and serum FSH were measured using
ELISA kits (Elabscience Co., Wuhan, China).
2.4. Histopathologic quantification and evalution of testicular cells apoptosis
The testes were fixed in 4% paraformaldehyde solution (4 g paraformaldehyde
in 100 mL PBS) and then embedded in paraffin blocks. Testicular sections of 5 ␮m
were cut, stained with hematoxylin–eosin (H&E) (Heechul et al., 2008) and observed
under a light microscope (Zeiss, Germany). A score of 0–10 was used for each tubule
according to the number of damaged cells in the seminiferous tubules as described
by Trivedi et al. (2010). Histological quantification of spermatogenesis in the testi-
cular sections was performed by assigning a Johnsen score (Trivedi et al., 2010; Li
et al., 2013a).
The terminal dUTP nick end-labeling (TUNEL) assay was used to evaluate testi-
cular cells apoptosis. Testicular sections were deparaffinized, rehydrated, and then
incubated with 20 mg/mL proteinase K for 20 min and rinsed in Tris buffer saline
(TBS). The steps for TUNEL staining were carried out using the In Situ Cell Death
Detection, POD Kit (Roche, Mannheim, Germany). The sections were incubated with
the diluted TUNEL reaction mixture (TdT-enzyme was diluted 1:10 with sterile
water) for 60 min at 37 ◦ C. The sections were blocked with 3% BSA for 25 min at
room temperature, incubated with the secondary antifluorescein-POD-conjugate
for 30 min, and then labeled with diaminobenzidine (DAB). After each step, the sec-
tions were thoroughly washed with TBS. The sections were then counterstained with
hematoxylin, dehydrated, cleaned and mounted. Positive cells were stained brown.
Quantitative analysis of testicular cell apoptosis was estimated according to Lysiak
et al. (2000). Apoptotic cells were TUNEL-positive and the positively stained nuclei
in 30 circular seminiferous tubule cross-sections per testis section were counted.
2.5. Protein preparation, two-dimensional gel electrophoresis (2-DE) and image
analysis
Frozen testis tissues were prepared for extraction of proteins and mRNA. The
testicular tissue was treated with a lysis buffer containing 7 M urea, 2 M thiourea, 4%
(W/V) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (Chaps),
2% (W/V) dithiothreitol (DTT) in the presence of 1% (V/W) protease inhibitor cocktail
(Sigma Chemical, St. Louis, MO, USA). Protein concentration was measured by the
Bio-Rad Bradford protein assay with bovine serum albumin (Sigma) as a standard.
Protein samples (600 ␮g) from three groups were dissolved in 350 ␮L IEF sample
buffer [8 M urea, 2% CHAPS, 65 mM DTT, 0.2% (w/v) Bio-Lyte 3-10 ampholytes, and
Bromophenol Blue (trace)] and loaded onto an IPG strip (Immobiline 17 cm DryS-
trip pH 3–10, Bio-Rad, USA), then covered with 1 mL of mineral oil for IEF using a
Protean IEF cell (Bio-Rad Laboratories, USA). Procedure for IEF was accordied to the
following program: 14 h at 50 V; 250 V for 1 h; 1000 V for 1 h; 9000 V for 6 h; 9000 V
for 80,000 V h. After being focused, the IPG strips were equilibrated for 15 min in
equilibration solution [6 M Urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris–HCl,
pH 8.8, 1% (w/v) DTT] and then alkylated for a further 15 min in this solution without
DTT but with 2.5% iodoacetamide. Electrophoresis of reduced and alkylated samples
was carried out on Protean II xi Cell (Bio-Rad Laboratories, USA) using 12% polyac-
rylamide SDS-PAGE gels [30% acrylamide solution, 1.5 M Tris (pH 8.8), 10% SDS, 10%
ammonium persulfate, TEMED and ultrapure water]. The second dimension elec-
trophoresis, SDS-PAGE, was carried out to following program: at 25 mA/gel until
bromophenol blue eluted from the gel at a room temperature with water cooling (Li
 H. Li et al. / Toxicology Letters 225 (2014) 433–444
Table 1
The primers and PCR reaction parameters of each target gene.
Gene Accession number (NCBI) Primer forward/reverse
␤-Actin NM 007393.3 5 -CCACCATGTACCCAGGCATT-3 /
5 -AGGGTGTAAAACGCAGCTCA-3
GRP78 D78645.1 5 -AGAAACTCCGGCGTGAGGTAGA-3 /
5 -TTCCTGGACAGGCTTCATGGTAG-3
VCP BC043053.1 5 -GCGCTCTTTAAGGCGATTGG-3 /
5 -TAGGCCATCCATGAGGGTCA-3
ACO NM 080633.2 5 -CATCAAGGTCAAAGGGAA-3 /
5 -GTAGCGAGCAGTGTCAGG-3
PKM NM 011099.3 5 -ATTACCAGCGACCCCACAGA-3 /
5 -AAGGTACAGGCACTACACGC-3
GSTA3 NM 010356.3 5 -GGAGTTCAACCAGGGCAATA-3 /
5 -GGCGGATCTGGAGATAATGA-3
GSP1 NM 013541.1 5 -GGAAGGAGGAGGTGGTTA-3 /
5 -CATAGTTGGTGTAGATGAGGG-3
SOD1 NM 011434.1 5 -TGCAGGGAACCATCCACTTC-3 /
5 -ACATGCCTCTCTTCATCCGC-3
et al., 2013b; Li and Zhang, 2013). After electrophoresis, the gels were stained with
colloidal Coomassie Blue G 250 (CCB) procedure for overnight. Following staining,
the gel was neutralized with 0.1 M Tris/phosphoric acid (pH 6.5) for 1–3 min, then
destained with 25% methanol. Three 2-DE gels were prepared for each sample and
were scanned with UMAX scanner and stored as TIF files. Spot detection and match-
ing were performed using the PDQuest 8.0 software (Bio-Rad Laboratories, USA). The
spots detected automatically by the software were visually inspected. Spot filtering
and editing were performed manually to remove artifacts and to correct for spots
that did not split correctly. Each group consisted of three independent replicates
that were used to produce 3 gels/group. The gel replicates were subsequently com-
bined into average gels, which represented spots that were reproducibly present on
all of the replicate gels. The criteria used to select spots for the analysis were more
than a one-fold difference in protein quantities (normalized spot volume) between
control and irradiation groups.
2.6. In-gel trypsin digestion of proteins and MALDI-TOF/TOF MS analysis
Spots were manually cut from 2-DE gels, destained for 20 min in 30 mM potas-
sium ferricyanide/100 mM sodium thiosulfate (1:1 v/v) and washed with Milli-Q
water until the gels were destained. The spots were incubated in 0.2 M NH4 HCO3
for 20 min and then lyophilized. Each spot was digested overnight in 12.5 ng/mL
trypsin in 25 mM NH4 HCO3 . The peptides were extracted three times with 60% ace-
tonitrile (ACN)/0.1% trifluoroacetic acid (TFA). The extracts were pooled and dried
completely by a vacuum centrifuge.
MS and MS/MS data for protein identification were obtained by using a MALDI-
TOF-TOF instrument (4800 proteomics analyzer; Applied Biosystems, Foster City,
CA, USA). Instrument parameters were set using the 4000 Series Explorer software
(Applied Biosystems). The MS spectra were recorded in reflector mode in a mass
range from 800 to 4000 with a focus mass of 2000. MS was used a CalMix5 standard to
calibrate the instrument (ABI 4700 Calibration Mixture). For one main MS spectrum
25 subspectra with 125 shots per subspectrum were accumulated using a random
search pattern. For MS calibration, autolysis peaks of trypsin ([M + H] + 842.5100 and
2211.1046) were used as internal calibrates, and up to 10 of the most intense ion
signals were selected as precursors for MS/MS acquisition, excluding the trypsin
autolysis peaks and the matrix ion signals. In MS/MS positive ion mode, for one
main MS spectrum 50 subspectra with 50 shots per subspectrum were accumulated
using a random search pattern. Collision energy was 2 kV, collision gas was air, and
default calibration was set by using the Glu1-Fibrino-peptide B ([M + H] + 1570.6696)
spotted onto Cal 7 positions of the MALDI target. Combined peptide mass finger-
printing PMF and MS/MS queries were performed by using the MASCOT search
engine 2.2 (Matrix Science, Ltd, Boston, MA, USA) embedded into GPS-Explorer Soft-
ware 3.6 (Applied Biosystems) on the NCBI database with the following parameter
settings: 100 ppm mass accuracy, trypsin cleavage one missed cleavage allowed, car-
bamidomethylation set as fixed modification, oxidation of methionine was allowed
as variable modification, MS/MS fragment tolerance was set to 0.4 Da. A GPS Explorer
protein confidence index ≥ 95% were used for further manual validation.
2.7. Immunoblotting and Real-time quantitative RT-PCR
60 ␮g of proteins was subjected to 12% SDS-PAGE, and transferred to a PVDF
membrane. The membranes were blocked with 5% skimmed milk in TBS, and
immunoblotted with the rabbit polyclonal IgG anti-GRP78 (Cat. no. BS1154),
anti-VCP (Cat. no. BS3773) (Bioworld technology, co, Ltd, Nanjing, China), anti-
GSTA3 (Cat. no. bs-5163R), anti-GSTP1 (Cat. no. bs-2735R), PKM1/M2 (Cat. no.
bs-0101R), anti-SOD1 (Cat. no. bs-1079R) (Biosynthesis Biotechnology, co, Ltd, Bei-
jing, China), anti-␤-actin (Cat. no. 13E5) (Cell signal, Boston, USA), and a horseradish
peroxidase-labeled secondary antibody (Cat. no. ZB2301) (Zhongshanjinqiao
435
Products length (bp) Cycles Annealing Denaturation
temperature (◦ C) temperature (◦ C)
253 35 60 94
176 40 60 95
324 40 60 95
193 40 60 95
385 40 60 95
194 40 60 95
252 35 60 94
202 40 60 95
Biotechnology Co., Beijing, China). Immunoreactivity was detected using an
enhanced chemiluminescent HRP substrate kit (Millipore, Billerrica, USA) and the
images were captured by a FluorChem 2 imaging system (Alpha Innotech, San Lean-
dro, CA, USA). Quantitative analysis of the relative density of the bands in Western
blots was performed using the Quantity One 4.5.2 image analysis software (Bio-Rad).
Data were corrected for background and expressed as optical density (OD/mm2 ).
Total testis RNA was extracted using the TRIZOL reagent (Invitrogen Corp.,
California, USA) according to the manufacturer’s instructions. The cDNA was then
synthesized via reverse transcription (RT) using an oligo-(dT)15 primer and the
M-MuLV reverse transcriptase (Invitrogen) in accordance with the manufacturers’
recommendations. PCR reactions were performed with the eppendorf mastercycler
S PCR system (Eppendorf, Wesseling-Berzdorf, Germany). The ␤-actin used as a ref-
erence gene to analyze the target gene quantitatively. The target gene primers were
designed by Invitrogen (Invitrogen) and list in Table 1. Real-time PCR reactions were
performed with the FTC-3000 qPCR system (Funglyn Biotech Inc., Scarborough, ON,
Canada). SYBR Green PCR Master Mix reagent kits (Takara, Dalian, China) were used
according to the manufacturer’s instructions for quantification of gene expression.
The Ctvalue was obtained in the amplification reaction. Ct represents the cycle at
which the fluorescence signal is first significantly different from background. The
relative expression ratio (R) of a target gene was calculated based on the description
of Barlow et al. (Shi et al., 2010).
2.8. Detection of ATP
The amount of ATP was measured by the protocol of ATP detection kit (Beyotime,
China). 20 mg testis tissues was ground with 200 ␮L of lysis from the ATP detection
kit, then with glass homogenizer. After centrifuged at 12,000g for 5 min at 4 ◦ C, the
supernatant was transferred to a new tube for ATP test. The luminescence from a
100 ␮L sample was assayed in a luminometer together with 100 ␮L ATP detection
buffer from the ATP detection kit. The standard curve of ATP concentration was
prepared from a know amount (1 nM–1 ␮M) (Peng et al., 2009).
2.9. Measurement of the activity of SOD and Intracellular ROS
The activity of SOD were measured in tissue homogenates of the testis using the
commercial reagent kits (Jiancheng Bioengineering Ltd., Nanjing, China). In brief,
frozen testes tissues from control and irradiated mice were homogenized fully in
chilled NaCl solution (0.86%) on ice to obtain 10% (w/v) testis tissues homogenate.
The homogenate was centrifuged at 3000g/min for 15 min at 4 ◦ C. Supernatants
were collected and divided to aliquots for the measurements of SOD activity accord-
ing to the instructions of reagent kits. Total protein level in the supernatants was
determined by Bradford method.
The fat and connective tissues adhering to testis were removed, then testis was
cut with a scalpel blade. The macerated tissue was transferred to a 2 mL Eppen-
dorf tube (containing 1.5 mL trypsin solution, 8 mg/mL trypsin in PBS/BSA 0.1%) for
enzymatic digestion at 34 ◦ C for 30 min. After centrifugation at 1800g for 5 min, the
supernatant was discarded, and the pellet was resuspended in a 1.5 mL Eppendorf
tube (containing 3 mg of hyaluronidase and 0.9 mg of collagenase) for a second diges-
tion at 34 ◦ C and 90 cycles/min agitation for 30 min. After centrifugation at 1000g
for 5 min, the supernatant was discarded, and the pellet was resuspended at a final
concentration of 1 × 106 cells/mL (Sarabia et al., 2009). Cells (1 × 106 ) loaded with
5 ␮M 2 ,7 -dichlorofluorescin diacetate (DCFH-DA). After incubation for 30 min in
the dark, then, cells were detached, and resuspended in 1 mL PBS. Cellular ROS as
a result of the oxidation of DCFH-DA was measured (excitation, 470 nm; emission,
530 nm, Tecan M200, Switzerland) (Matás et al., 2010).
436 H. Li et al. / Toxicology Letters 225 (2014) 433–444

Table 2 (P < 0.001) and serum FSH levels were significantly increased (1 Gy
Effects of body weight, testes weight, and testes ratio in pubertal male mice at 14
group, P < 0.01; 4 Gy group, P < 0.001).
day after CIR.

Control 1 Gy 4 Gy 3.2. Histopathologic changes

Body weight (g) 28.97 ± 3.085 28.62 ± 3.925 24.89 ± 1.132
Testes weight (g) 0.1695 ± 0.0526 0.1077 ± 0.0236* 0.0588 ± 0.0109** Fig. 2 shows the histological changes in pubertal mice testicular
Testes ratio 0.0058 ± 0.0013 0.0044 ± 0.0011* 0.0023 ± 0.0006**
tissue. In normal testes, the seminiferous tubules were smooth,
Values represent the average ± S.E.M. from 10 mice per group. Asterisks indicate the tubular lumen contained a large number of spermatozoa, and
a statistically significant difference from control: * P < 0.05, ** P < 0.01 on one-way the spermatogenic cells were regular (Fig. 2A). At 14 days after
ANOVA with Duncan’s post hoc analysis.
CIR, only a few spermatozoa were observed, and a few sperma-
tocytes and spermatogonia remained in the epithelium together
3. Results with Sertoli cells (Fig. 2B). Only a few Sertoli cells were seen
in the seminiferous epithelium. Large prominent vacuolizations
The body weight, testes weight and testes ratios in pubertal male were found in damaged tubules, with almost no spermatozoa in
mice at 14 days after CIR are presented in Table 2. A significant tubular lumen (Fig. 2C). The Johnsen score reflected the quantitative
decrease in the pubertal mouse testes weight and testes ratio was assessment of seminiferous tubules (Fig. 2D). Significant damage
observed in the irradiated groups. to the seminiferous tubule was observed in the irradiated groups
(P < 0.001).
3.1. Serum testosterone and serum FSH levels
3.3. Evaluation of testicular cells apoptosis
The effects of CIR on serum testosterone and serum FSH lev-
els in pubertal male mice were analyzed (Fig. 1). At 14 days after Apoptotic testicular cells were examined by TUNEL assay (Fig. 2).
irradiation, serum testosterone levels were significantly decreased Almost no TUNEL-positive cells were observed in the control group

Fig. 1. The effects of CIR on serum testosterone (A) and serum FSH (B) levels in pubertal male mice. Values represent the average ± S.E.M. from 10 mice per group. Asterisks
indicate a statistically significant difference from control: ** P < 0.01, *** P < 0.001 on one-way ANOVA with Duncan’s post hoc analysis.

Fig. 2. Testes histopathology in pubertal male mice at 14 days after CIR: Control (A), 1 Gy (B), 4 Gy (C). Photomicrographs of sections stained with H&E (magnification 200×):
sg, spermatogonia; sc, spermatocytes; esd, early spermatids; lsd, late spermatids; lc, Leydig cell. Severe degeneration of spermatogenic cells in the seminiferous tubule (* ),
arrows indicate Sertoli cells in the seminiferous tubule (C). Representative photomicrographs of TUNEL staining of apoptotic cells in pubertal mouse testes at 14 days after
CIR: Control (E), 1 Gy (F), 4 Gy (G). Arrows indicate TUNEL-positive cells in the seminiferous tubule (magnification 400×): sg, spermatogonia; sc, spermatocytes; esd, early
spermatids; lsd, late spermatids. Testicular damage as evaluated by the Johnsen score (D). Histogram of apoptotic-positive spermatogenic cells from mouse testis (H). Values
represent the average ± S.E.M. Asterisks indicate a statistically significant difference from control: *** P < 0.001 on one-way ANOVA with Duncan’s post hoc analysis.
 H. Li et al. / Toxicology Letters 225 (2014) 433–444
Fig. 3. Representative 2-DE image for protein expression maps. Proteins (600 ␮g loaded) were separated by IEF of pH 3–10, followed by 12% SDS-PAGE and visualized by
colloidal Coomassie blue staining. Arrows represent differential protein spots and the spot numbers of representative proteins with high expression factors refer to Table 3
were indicated.
(Fig. 2E), however, the number and signal density of TUNEL-positive
spermatogenic cells significantly increased in the 1 Gy group
(Fig. 2F). An increased number of TUNEL-positive cells and severe
damage were found in the 4 Gy group (Fig. 2G). The TUNEL-positive
spermatogenic cells were mainly spermatogonia, spermatocytes,
early spermatids and late spermatids, and these cells seemed to be
initially more susceptible to CIR toxicity. Quantitative analysis of
testicular apoptosis is shown in Fig. 2H. A significant increase in
TUNEL-positive cells was found in the seminiferous tubule in the
irradiated groups (P < 0.001).
3.4. Profiles of testis protein
Fig. 3 illustrates the representative testes protein profiles. A dif-
ference of more than one-fold in protein quantity (normalized spot
volume) is the standard in detecting differentially expressed pro-
tein spots by PDQuest 8.0 software. A significant difference in the
proteome was observed between the control and irradiated groups.
A total of 8 differentially expressed proteins were found between
the control and irradiated groups, of which spot 1, 3, 5, 6 and 7 were
down-regulated, and spot 2, 4 and 8 were up-regulated in the 2-DE
gels of the irradiated groups.
3.5. Identification of differential proteins
The 8 differentially expressed proteins were identified using
MALDI-TOF-TOF. Protein identification was carried out via peptide
sequencing using the Mascot search engine 2.2 (Matrix Science)
embedded into GPS-Explorer Software 3.6 (Applied Biosystems)
on the National Center for Biotechnology Information database.
Among the eight protein spots, seven spots are the known
proteins (Table 3). The results of %Adj. volume are shown in
Fig. 4. These proteins were 78 kDa glucose-regulated protein
(GRP78), valosin-containing protein, isoform CRA a (VCP), aconi-
tate hydratase–mitochondrial precursor (ACO), pyruvate kinase
isozymes M1/M2 isoform 1 (PKM1/M2), glutathione-S-transferase
A3 (GSTA3), glutathione S-transferase P 1 (GSTP1), and Cu/Zn super-
oxide dismutase (SOD1). The functions of these proteins included
energy supply, the endoplasmic reticulum, cell proliferation, cell
cycle, antioxidant capacity and mitochondrial respiration.
3.6. Differential protein expression detected by immunoblotting
In order to confirm the 2-DE results, we identified the pro-
teins expressed using immunoblotting. The expression of GRP78
(Fig. 5B), ACO (Fig. 5F), GSTA3 (Fig. 5J), GSTP1 (Fig. 5M) and SOD1
(Fig. 5O) in testes was significantly decreased in the irradiated
groups. The expression of PKM1/M2 (Fig. 5H) and VCP (Fig. 5D) was
437
significantly increased in the irradiated groups. The levels of pro-
tein expression detected by immunoblotting were consistent with
the 2-DE results.
3.7. Gene expression
Based on the proteomic results, we examined the expression
of genes to compare the correlation between protein expression
and gene expression (Fig. 6). The expression of GRP78, ACO, GSTA3,
GSTP1 and SOD1 in testes was significantly decreased in the irradi-
ated groups. The expression of PKM1/M2 and VCP was significantly
increased in the irradiated groups. The mRNA levels of these pro-
teins were consistent with the immunoblotting results.
3.8. ATP content
The effects of CIR exposure on ATP content in pubertal male mice
testes were analyzed (Fig. 7). CIR exposure significantly decreased
ATP content in irradiated mice testes (P < 0.05 in 1 Gy group;
P < 0.001 in 4 Gy group). These results suggest that CIR disturbed
energy balance by reducing ATP content.
3.9. The ROS and activity of SOD
To further confirm that antioxidant capacity was involved in
the mitochondrial respiratory chain and oxidative stress identified
via our proteomic method, the ROS in testicular cells and activ-
ity of SOD were measured. A significant increase in the DCF was
observed in the irradiated groups (1 Gy, P < 0.05; 4 Gy, P < 0.001)
(Fig. 8). SOD activity in testes tissues was also markedly decreased
in the irradiated groups (P < 0.001). These results suggest that CIR
affects the mitochondrial respiratory chain and oxidative stress in
testicular cells by increasing ROS and decreasing antioxidant capac-
ity in testes.
4. Discussion
In the present study, decreased serum testosterone, ATP levels
and SOD activity, and increased serum FSH and ROS were observed.
Furthermore, eight differentially expressed proteins which showed
more than a one-fold difference in protein quantities (normalized
spot volume) were identified in the testes of mice exposed to CIR
using 2-DE methods. These proteins were primarily involved in
endoplasmic reticulum chaperones, the mitochondrial respiratory
chain, oxidative stress and glycolysis enzymes. Alterations in mRNA
levels of the genes involved in these proteins were also detected.
These results demonstrate that high doses of CIR caused testicular
toxicity in pubertal male mice which involved multiple protein
pathway changes as shown by proteomic analysis.
 438
Table 3
Differentially expressed protein spots (%Adj. Volume) in testes after CIR.

Protein Protein name Accession pI/Mw Peptides Protein Protein score Sequence Function Molecular function/biological
spot (NCBI) Matched score C.I. (%) coverage (%) process

Spot 1 78 kDa glucose- gi|1304157 5 21 836 100 20% Probably plays a role in facilitating the assembly of ATP binding/cysteine-type
regulated .09/72,526.5 multimeric protein complexes inside the ER endopeptidase inhibitor
protein [Mus activity involved in apoptotic
musculus] process/ribosome binding
Spot 2 Valosin gi|148670553 5 24 496 100 25% In cooperation with other chaperones, Hsp70s stabilize ATP binding/nucleoside-
containing .14/90,868.4 preexistent proteins against aggregation and mediate the triphosphatase
protein, folding of newly translated polypeptides in the cytosol as activity
isoform CRA a well as within organelles. These chaperones participate in
[Mus all these processes through their ability to recognize
musculus] nonnative conformations of other proteins. They bind
extended peptide segments with a net hydrophobic

H. Li et al. / Toxicology Letters 225 (2014) 433–444

character exposed by polypeptides during translation and
membrane translocation, or following stress-induced
damage
Spot 3 Aconitate gi|18079339 8 21 771 100 20% Implicated as a critical step in numerous cellular pathways, Aconitate hydratase
hydratase, .08/86,151.3 including signal transduction, membrane trafficking, and activity/citrate hydro-lyase
mitochondrial the regulation of mitosis. May be involved in the (cis-aconitate-forming)
precursor [Mus regulation of perinuclear intravesicular membrane traffic activity/
musculus]
Spot 4 Pyruvate gi|31981562 7 9 280 100 28% Glycolytic enzyme that catalyzes the transfer of a ATP binding/potassium ion
kinase .18/58,378.2 phosphoryl group from phosphoenolpyruvate (PEP) to binding/pyruvate kinase
isozymes ADP, generating ATP. Stimulates POU5F1-mediated activity
M1/M2 isoform transcriptional activation
1 [Mus
musculus]
Spot 5 Unnamed gi|74187644 5 6 151 100 26%
protein .23/42,052.9
product [Mus
musculus]
Spot 6 Glutathione-S- gi|31981724 8 10 312 100 50% Conjugation of reduced glutathione to a wide number of Glutathione transferase
transferase A3 .76/25,401.3 exogenous and endogenous hydrophobic electrophiles. activity
[Mus This GST has a high catalytic activity for aflatoxin B1-8,9
musculus] epoxide
Spot 7 Glutathione gi|10092608 7 8 418 100 Conjugation of reduced glutathione to a wide number of Glutathione transferase
S-transferase P .68/23,765.2 exogenous and endogenous hydrophobic electrophiles. activity/kinase regulator
1 [Mus Regulates negatively CDK5 activity via p25/p35 activity/nitric oxide binding
musculus] translocation to prevent neurodegeneration
Spot 8 Cu/Zn gi|226471 6 8 434 100 Destroys radicals which are normally produced within the Copper ion binding/superoxide
superoxide .03/15,922.8 cells and which are toxic to biological systems dismutase activity/zinc ion
dismutase binding
[Mus
musculus]
 H. Li et al. / Toxicology Letters 225 (2014) 433–444 439

Fig. 4. The %Adj. Volume of differentially expressed protein spots in testes after CIR. The differential protein spots are in black circles. GRP78, 78 kDa glucose-regulated
protein; VCP, valosin-containing protein, isoform CRA a; ACO, aconitate hydratase–mitochondrial precursor; PKM1/M2, pyruvate kinase isozymes M1/M2 isoform 1; GSTA3,
glutathione-S-transferase A3; GSTP1, glutathione S-transferase P 1; SOD1, Cu/Zn superoxide dismutase. Values represent the average ± S.E.M. from 3 gels per group. Asterisks
denote values that are significantly different from control. * P < 0.05, ** P < 0.01, *** P < 0.001 on one-way ANOVA with Duncan’s post hoc analysis.

Fig. 5. Expression of differential protein by Western blot analysis ((A), (C), (E), (G), (I), (L), (N)). Relative expression of differential protein ((B), (D), (F), (H), (J), (M), (O)). GRP78,
78 kDa glucose-regulated protein; VCP, valosin-containing protein, isoform CRA a; ACO, aconitate hydratase–mitochondrial precursor; PKM1/M2, pyruvate kinase isozymes
M1/M2 isoform 1; GSTA3, glutathione-S-transferase A3; GSTP1, glutathione S-transferase P 1; SOD1, Cu/Zn superoxide dismutase. Values represent the average ± S.E.M. from
3 gels per group. Asterisks denote values that are significantly different from control. * P < 0.05, ** P < 0.01, *** P < 0.001 on one-way ANOVA with Duncan’s post hoc analysis.
440 H. Li et al. / Toxicology Letters 225 (2014) 433–444

Fig. 6. The mRNA expression of each differential protein by Real-time quantitative RT-PCR. GRP78, 78 kDa glucose-regulated protein; VCP, valosin-containing protein, isoform
CRA a; ACO, aconitate hydratase–mitochondrial precursor; PKM1/M2, pyruvate kinase isozymes M1/M2 isoform 1; GSTA3, glutathione-S-transferase A3; GSTP1, glutathione
S-transferase P 1; SOD1, Cu/Zn superoxide dismutase. Values represent the average ± S.E.M. from 3 replications per group. Asterisks denote values that are significantly
different from control. * P < 0.05, ** P < 0.01, *** P < 0.001 on one-way ANOVA with Duncan’s post hoc analysis.

4.1. CIR disrupts spermatogenesis and steroidogenesis in pubertal

male mice

In the present study, a significant decrease in testes weight

Fig. 7. The ATP content of each experimental groups. Values represent the aver-
age ± S.E.M. from 6 mice per group. Asterisks denote values that are significantly
different from control. * P < 0.05, *** P < 0.001 on one-way ANOVA with Duncan’s post
hoc analysis.
and testes ratios was observed in the irradiated groups 14 days
after irradiation. These decreases in testes weight and testes ratios
revealed that the significant toxic effect of CIR on pubertal mice
testes was prolonged, indicating that CIR can have a serious impact
on the development of pubertal mouse testes. The significant
decline in the Johnsen score and significant increase in TUNEL-
positive cells demonstrated that CIR caused dysfunction of pubertal
mouse spermatogenesis. A significant reduction in SOD and ATP
levels, and a significant increase in ROS level were also found, which
demonstrated that CIR disrupted the antioxidative and energy sys-
tem in testicular tissue. These results clearly demonstrate that CIR
disrupts spermatogenesis in pubertal male mice.
The two main functions of the mammalian testis are production
of germ cells for spermatogenesis and steroidogenesis (de Kretser,
2004; Carreau et al., 2011). The primary hormonal controls on sper-
matogenesis involve the action of testosterone and FSH on germ
cells (Walker and Cheng, 2005). FSH is synthesized and secreted

Fig. 8. The SOD activity (A) and intracellular ROS (B) of each experimental groups. Values represent the average ± S.E.M. from 6 mice per group. Asterisks denote values that
are significantly different from control. * P < 0.05, *** P < 0.001 on one-way ANOVA with Duncan’s post hoc analysis.
 H. Li et al. / Toxicology Letters 225 (2014) 433–444
by the pituitary gland in the male (Baratta et al., 2001), and the
function of normal FSH level is to promote testicular seminifer-
ous tubule maturation (Walczak-Jedrzejowska et al., 2011) and
sperm production (Gordetsky et al., 2012). FSH is required for the
initiation of development and fertility during the pubertal stage
(Layman, 2000), and plays an important role in maintenance of
adult spermatogenesis (Plant and Marshall, 2001). A higher FSH
level is indicative of abnormal spermatogenesis and may indicate
primary testicular failure (Gordetsky et al., 2012). However, CIR
may directly impact testis tissue or the pituitary gland which could
cause changes in FSH. Increased serum FSH may affect seminiferous
tubular function and delay the cyclical restitution of the hemo-
testicular barrier and reduce the number of meiotic spermatocytes
in the seminiferous epithelium, which consequently leads to male
infertility. At 14 days after CIR, the results of the Johnsen score
clearly showed that high serum FSH disrupted spermatogenesis
and reduced the number of meiotic spermatocytes. Leydig cells pro-
duce the primary male steroid hormone, testosterone (Akingbemi
et al., 2003), which supports spermatogenesis and fertility, testi-
cular development is wholly dependent on testosterone (Walker,
2011). The role of testosterone in the testis seminiferous epithelium
is to promote sperm production, and testis size has a positive associ-
ation with serum testosterone level (Garamszegi et al., 2005), with
low serum testosterone levels resulting in spermatogenesis disor-
ders (de Kretser, 2004). At 14 days after CIR, the decrease in testes
weight and testes ratios clearly reflected the impact of low serum
testosterone levels on the development of pubertal mouse testes.
At 14 days after CIR, the decline in serum testosterone levels clearly
demonstrated that CIR disrupts steroidogenesis in male mice.
4.2. Carbon ion radiation disrupts the testis energy balance
The results of this study suggest that testes energy metabolism
in whole-body irradiated mice changed, mainly due to down-
regulation of ACO expression and up-regulation of PKI-M1/M2
expression. Aconitase catalyzes citric acid into isocitrate in the
Krebs cycle. Citrate is easily oxidized, under the action of aconi-
tase by dehydration, and when water was added to the reaction,
the hydroxyl group was transferred to the ␣-carbon atom by
the ␤-carbon atom, and isocitrate was generated by oxidative
dehydrogenation for further oxidative decarboxylation reactions.
Down-regulation of ACO, and mitochondrial precursor expres-
sion suggested that CIR inhibited testicular aerobic respiration
and reduced testicular energy supply through inhibition of aero-
bic respiration. Pyruvate kinase, a final-stage enzyme in glycolysis,
catalyzes the transfer of a phosphoryl group from phospho-
enolpyruvate (PEP) to adenosine diphosphate (ADP), generating the
substrates ATP and pyruvate for anaerobic and aerobic metabolism
(Echigoya et al., 2008). In mammals, there are four isozymes of
pyruvate kinase known as M1-, M2-, L (liver), and R (red blood
cell), the expression of which differ from one tissue to another.
The M1- and M2-type are produced from the same gene by alter-
native splicing (Dabrowska et al., 1998). PKI-M1/M2 up-regulation
compensated energy supply by increasing the glycolytic pathway
in the radiation group testes. After irradiation, metabolic pathways
aerobic oxidation was reduced and glycolytic enhanced in testis.
The capacity for aerobic respiration diminished and glycolysis
was enhanced by CIR after 14 days and testicular pathology showed
a severe energy supply imbalance, when the body only through
increased glycolysis supplemented the energy supply. The mech-
anism of this phenomenon caused by CIR is unclear and requires
further research. In this study, a significant decrease in ATP levels
was observed in the CIR groups at 14 days. This decrease in ATP
levels clearly demonstrated that high doses of CIR can disrupt tes-
ticular tissue energy balance by decreasing ATP levels. Decreased
serum testosterone level may be associated with decreased ATP
441
levels. There is evidence to show that mitochondria have a key role
in Leydig cell steroidogenesis. Maintenance of mitochondrial ATP
synthesis is one of the requirements for acute steroid biosynthesis,
and decreased ATP level was correlated with decreases in cAMP and
testosterone synthesis (Haider, 2007). Therefore, we speculate that
the reduction in ACO protein decreased ATP levels and may be one
of the factors which induced the decrease in serum testosterone
levels.
4.3. CIR changes endoplasmic reticulum function
The endoplasmic reticulum is the main site of intracellular
protein synthesis and is an intracellular Ca2 + repository. The
relationship between the endoplasmic reticulum and apoptosis is
manifested by two aspects: Endoplasmic reticulum Ca2+ regulation,
and apoptosis net activation of the enzyme in the endoplasmic reti-
culum. The endoplasmic reticulum is a cellular protein processing
and Ca2+ storage organelle. Misfolded proteins in the endoplas-
mic reticulum and unfolded proteins due to various causes, can
lead to cavity aggregation and Ca2+ balance disorders in the
endoplasmic reticulum, through the unfolded protein response
pathway and cell damage resistance and adaptability (Gonzalez-
Gronow et al., 2009). GRP78, also known as BiP (immunoglobulin
heavy chain binding protein) is an endoplasmic reticulum chap-
erone with a relative molecular mass of 78,000, and belongs to
the HSP70 family. An increase in GRP78 is an important sign of
endoplasmic reticulum stress, and a recent study on endoplasmic
reticulum homeostasis regulation found that GRP78 and multi-
important factors of various diseases were closely related (Miskovic
et al., 1997). GRP78 is mainly localized in the endoplasmic reti-
culum with lower levels of expression in normal tissues (Zheng
et al., 2009). The function of GRP78 is related to protein synthesis,
and misfolded proteins in the endoplasmic reticulum which accu-
mulate in the cavity can induce GRP78 up-regulation (Kaufman,
1999; Paschen, 2004). In the radiation group, GRP78 protein and
mRNA down-regulation, gradually decreased with increased radi-
ation dose, and a positive correlation with the change in height
between the two prompted the low expression of GRP78 and was
closely related to radiation dose. In the present study, the increase
in TUNEL-positive spermatogenic cells after CIR may be associ-
ated with net activation of apoptosis enzyme in the endoplasmic
reticulum. Our results demonstrated that CIR induced endoplas-
mic reticulum stress in the testes which led to spermatogenesis
disorders.
4.4. CIR affected cell proliferation and the cell cycle
VCP/p97 is a proliferation factor and a multi-functional ATPase
of the AAA+ family (Kang and Yang, 2011). VCP is a glyco-
syl phosphatidylinositol connected transferrin homolog, and the
macromolecular complexes formed by its combination with vari-
ous cofactors act as molecular chaperones, such as p47, Ufd1/Np14
binding complex formed by mitosis in the endoplasmic reticu-
lum and Golgi apparatus, and nuclear envelope membrane fusion
is closely related (Wang et al., 2004). VCP is highly expressed in
hyperplastic cells, and plays a major role in molecular chaperones
involved in membrane fusion, regulation of transcriptional activ-
ity, regulation of the cell cycle, endoplasmic reticulum-associated
protein degradation and apoptosis, regulation of physiological and
DNA damage response and pathological processes (Yamamoto
et al., 2003; Wang et al., 2004). VCP expression level changes after
CIR and may affect the expression of FAF1, p47, SAKS1, UBXD7
and other proteins (Kang and Yang, 2011), thereby affecting cell
proliferation, the cell cycle, DNA damage and other events.
In the radiation group, VCP protein and mRNA were
up-regulated, which may be an anti-apoptosis mechanism.
442 H. Li et al. / Toxicology Letters 225 (2014) 433–444
In toxic circumstances, increased apoptosis and proliferation
(more apoptosis than proliferation) is an anti-apoptosis mech-
anism in testicular tissue, thus regulating spermatogenesis and
spermatogenic cells (Lin et al., 1997). In the present study, the
number and signal density of TUNEL-positive spermatogenic cells
significantly increased in the irradiated groups. Meanwhile, the
increase in VCP may only play a role in anti-apoptosis, as it cannot
prevent CIR-induced apoptosis and other events. Therefore, in the
present study, the results of VCP may provide some useful informa-
tion on the way in which CIR affected cell proliferation, the cell cycle
and DNA damage in testes leading to spermatogenesis disorders.
4.5. CIR diminished testicular antioxidant capacity
Radiation damage to the reproductive system is mainly caused
by the oxidative stress (Adaramoye et al., 2012), an excessive
number of oxygen free radicals causes lipid peroxidation and
interferes with normal metabolism causing reproductive system
disorders and diseases (Agarwal and Allamaneni, 2004; Agarwal
et al., 2006; Reinhardt and Ribou, 2013). In the proteomic anal-
ysis, we found that three antioxidative stress proteins including
GSTA3, GSTP1 and SOD1 were significantly altered by CIR. The
glutathione S-transferases (GSTs) are a family of enzymes that cat-
alyze the conjugation of glutathione (GSH) with xenobiotics as
part of detoxification and drug-resistance pathways. The GSTs are
present in almost all eukaryotic species, and to date, six classes of
this enzyme have been described, Alpha, Mu, Pi, Sigma, Theta and
Zeta. In rats, each class consists of several subunits with various
nomenclatures in the literature. The rat GSTA3 subunit belongs to
the Alpha class, and is located on chromosome (Fotouhi-Ardakani
and Batist, 1999). GSTP1 codes for the enzyme, glutathione S-
transferase pi, and is located on chromosome 11q13 (Allan et al.,
2001).
A number of proteins play protective roles against ROS which
are produced under conditions of oxidative stress. The decline in
SOD1 protein in the irradiated groups indicated that CIR disrupts
SOD function and possibly led to the inhibition of testicular antiox-
idative ability, as SOD, which clears ROS superoxide radicals, is
considered the first line of cell defense against oxidative stress dam-
age (Aquilano et al., 2006). The significant decrease in SOD activity
in the irradiated groups further confirmed the reduction of SOD
protein and inhibition of its antioxidative capabilities following CIR
exposure. In addition, SOD is generally thought to play a central
role because it scavenges superoxide anions, the ROS initially gen-
erated from molecular oxygen in cells (Ishii et al., 2006). Thus, SOD
playing a role in the male reproductive system is a distinct possi-
bility (Mruk et al., 2002; Fujii et al., 2003). Although Cu–Zn–SOD
and Mn–SOD, encoded by the SOD1 and SOD2 genes, respectively,
represent a major intracellular superoxide-scavenging system, the
contribution of these proteins varies depending on the types of cells
(Ishii et al., 2006). Decreased GSTA3, GSTP1 and SOD1 protein in
this study may augment the testicular oxidative stress response to
radiation. These three proteins are antioxidants, which have been
shown to protect cells from damage due to oxidative stress. The
reduction of three protein and mRNA levels in this study may also
have contributed to the inhibition of antioxidative capability in
the testis by CIR. In this study, a significant increase in ROS lev-
els was observed in the CIR groups at 14 days. These increased ROS
levels clearly demonstrate that high doses of CIR can disrupt the
antioxidative system in testicular tissue.
The disruption of steroidogenesis by CIR in pubertal male
mice may be associated with oxidative stress, as oxidative stress
may disrupt testicular progesterone and testosterone synthesis
(Kostic et al., 2000). There is evidence that ROS may have
harmful effects on critical components of steroidogenesis (El-
Desoky et al., 2013). Decreased serum testosterone levels may
be the result of oxidative damage (Shi et al., 2009). There-
fore, CIR-induced testicular oxidative stress can decrease serum
testosterone, and the superfluous ROS originating from decreased
antioxidative ability and abnormal respiration in testis after CIR
may lead to reduced serum testosterone levels and disruption
of spermatogenesis. Consequently, GSTA3, GSTP1 and SOD1 pro-
teins have a causal role in the reduction in serum testosterone
levels. The reduction of these proteins may increase testicular
oxidative damage to induce a decline in serum testosterone lev-
els.
4.6. Spermatogenesis and steroidogenesis dysfunction have a
direct relationship with mitochondria following CIR
Under physiological conditions, mitochondria are the main site
of ROS production. Mitochondria use oxygen to produce ATP for the
body’s energy needs, but also generate ROS free radical damage in
mitochondria (Mujahid et al., 2005). Excess ROS can damage sper-
matogenic cells by attacking the cell membrane, lipoproteins and
polyunsaturated fatty acids, reduce membrane fluidity and lipid
peroxidation, and damage protein and DNA involved in the induc-
tion of spermatogenic cell apoptosis, which directly or indirectly
affect male reproductive function (Juránek and Bezek, 2005). Stud-
ies have shown that a very close relationship exists between ROS
and sperm deformity rate, as ROS produce excess numbers of sperm
morphological abnormalities, and activity increases (Zalata et al.,
2004).
In the proteomic analysis, we found that two proteins involved
in the mitochondrial respiratory chain were significantly altered by
CIR. ACO and GRP78, are involved in energy generation and regu-
lation, as well as mitochondrial respiration. Mitochondria oxidize
sugar, fat and amino acids and release energy to participate in the
oxidation reaction in the Krebs cycle and electron transport. Energy
conversion is the main function of mitochondria. ACO expression
significantly decreased with increased radiation dose, indicating
that in the radiation energy metabolic pathway, injury which led
to a decline in metabolic rate, was very important in testicular func-
tion after high-dose radiation. In the radiation group, ATP content
decreased which further validated the proteomic results.
Mitochondrial chaperones are involved in the arrangement of
functional proteins in the mitochondria, and the mitochondrial
outer protein transporter of the mitochondrial matrix promotes
mitochondrial protein folding and repair of misfolded proteins
(Garesse and Vallejo, 2001). In the radiation group, GRP78, which
is an important mitochondrial chaperone protein was down-
regulated, which may have affected the arrangement of proteins
in the mitochondrial respiratory complex, thereby affecting mito-
chondrial oxidative phosphorylation and ATP generation, and
affecting both Ca2+ storage and exchange, thereby affecting the
metabolism of small molecules, thus inhibiting the mitochondrial
transporter and affecting mitochondrial energy supply. This evi-
dence indicates that CIR affects mitochondrial function possibly by
disrupting the mitochondrial respiratory chain and energy forma-
tion in testes.
In the present study, almost all the results showed that
spermatogenesis and steroidogenesis dysfunction have a direct
relationship with mitochondria following CIR. Overall, CIR dis-
rupted mitochondrial antioxidant ability and energy balance, and
induced oxidative stress. Mitochondrial dysfunctional and oxida-
tive stress can disrupt steroidogenesis, as mitochondria are key
control points in steroid biosynthesis (Duarte et al., 2012). Mito-
chondrial disruption and oxidative stress simultaneously increased
spermatogenic cells apoptosis, as the mitochondrial signaling path-
way is important for spermatogenic cells apoptosis in testes (Nair
and Shaha, 2003). These results provide important clues that
 H. Li et al. / Toxicology Letters 225 (2014) 433–444
mitochondrial disruption and oxidative stress may play important
roles in pubertal testicular toxicity following CIR.
In biological systems, the genome is the genetic informa-
tion storage body (Frith et al., 2006), mRNA (transcriptome)
is a genetic intermediate for functional proteins (proteome) of
gene function execution, while the correlation between mRNA
and protein expression of gene products in the two different
stages were minimal (Greenbaum et al., 2003). The relation-
ship between transcription and translation was examined in the
present study, and the transcriptional levels of seven proteins
which were significantly altered by CIR exposure were exam-
ined. The mRNA levels of these proteins were consistent with
their protein levels in testes. These results also indicate that dif-
ferent protein expression was regulated by genes in the testes
after CIR. The concordance of proteomic/gene expression has been
shown in recent studies (Shi et al., 2010), and the relationship
between mRNA transcription and the abundance of a protein is
not always a direct relationship as many regulatory mechanisms
can affect these processes. Therefore, in the present study, mRNA
expression profiling, and proteomic analysis allowed us to begin
to link changes in protein levels with specific mechanisms of
transcriptional regulation. We compared the concordance of pro-
teomic/gene expression of 7 different proteins, and the results
demonstrated that protein expression is primarily regulated at the
level of transcription.
5. Conclusion
In conclusion, we successfully identified 7 proteins in the testes
of pubertal mice exposed to CIR using comprehensive proteomic
analysis. The results of transcriptome and proteome analyses of
these proteins demonstrated that testicular toxicity due to CIR
involved multiple protein functions and pathways. This work may
provide some useful information on the changes in testis physi-
ology and function induced by CIR in the field of pubertal male
reproductive toxicology. GSTA3, GSTP1, SOD1, ACO and GRP78 pro-
teins were associated with mitochondria, and both mitochondrial
disruption and oxidative stress may play important roles in testi-
cular toxicity following CIR.
Transparency document
We declare that we have no conflict of interest exits in the
submission of this manuscript, and manuscript is approved by all
authors for publication. There is no other personal interest of any
nature or kind in any product, service and/or company that could be
construed as influencing the position presented in, or the review of,
the manuscript entitled, “Differential proteome and gene expres-
sion reveal response to carbon ion irradiation in pubertal mice
testes”.
Acknowledgments
This research was supported by the National Basic Research Pro-
gram of China (2010CB834202), the National Natural Science Foun-
dation of China (10835011), the Scientific Technology Research
Projects of Gansu Province (0702NKDA045, 0806RJYA020) and the
Fostering Foundation for the Excellent Ph.D. Dissertation of Gansu
Agricultural University (2013002). We thank Professor Klaus-armin
Nave and Associate Professor Hauke B. Werner for their advice.
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