Aquaculture 468 (2017) 307313

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Benecial effect and potential molecular mechanism of chloroquine on

sperm motility and fertilizing ability in yellow catsh
Jin Zhang a, Wenge Ma a, Binyue Xie a, Jian-Fang Gui a,b, Jie Mei a,
a
College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Huazhong Agricultural
University, Wuhan 430070, China
b
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of the Chinese Academy of Sciences, Wuhan 430072, China

a r t i c l e

i n f o

Article history:
Received 17 May 2016
Received in revised form 7 October 2016
Accepted 19 October 2016
Available online 20 October 2016
Keywords:
Chloroquine
Sperm motility
Fertilizing ability
Autophagy
Toll-like receptor

a b s t r a c t
Sperm motility is a critical determinant of male fertility in vertebrates including sh species. Chloroquine, an autophagy inhibitor, has been reported to own debated inuence on sperm quality of mammalians. The objectives
of this study were to determine the effect of chloroquine on sperm motility and fertilizing efciency in yellow catsh, an important freshwater sh species in China. Gradient doses of chloroquine were intraperitoneally injected
into yellow catsh that resulted in an increasing expression level of LC3-II and inhibition of autophagy. Subsequently, various sperm parameters were assessed with the computer-assisted sperm analysis (CASA) system.
Chloroquine injection signicantly increased straight line velocity (VSL), curvilinear velocity (VCL) and average
path velocity (VAP) of sperm compared with the control. Consequently, 1 M chloroquine/g body weight was revealed to be an optimal dose for in vivo treatment, which led to elevation of fertility rate, whereas no effect on the
hatching rate. The comparative transcriptome analysis between chloroquine-treated and control testes were
conducted in order to gure out the possible molecular mechanism of chloroquine to regulate sperm motility. Interestingly, Gene Ontology (GO) analysis indicated that chloroquine treatment not only inhibited autophagy
pathway but also signicantly reduced toll-like receptor signaling pathway, suggesting a possible trade-off between male reproduction and immunity. KEGG analysis showed up-regulation of many pathways including
PI3K-AKT signaling pathway which has been reported to be correlated with spermatogenesis and sperm maturation in yellow catsh. Our data reveals a potential trade-off between reproductive traits and immune function,
and suggests that the application of chloroquine could improve sperm quality and fertilization efciency in
broodstock sh.
Statement of relevance: Our research demonstrated that an optimal dose of chloroquine could efciently inhibit
autophagy and improve sperm motility and fertilization efciency. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to gure out the possible molecular
mechanism of chloroquine to regulate sperm mobility.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Over the past decades, the sh farming industry paid more attention
to the quality of eggs and larvae rather than to that of sperm, although
the quality of both gametes is associated with fertilization success,
growth and survival of the progeny (Rurangwa et al., 2004). Under the
pressure from sexual selection and sperm competition, interest for
sperm quality analysis has been aroused to increase the reproductive
success and maintain the genetic diversity of the lineages (Mehlis et
al., 2015; Galeotti et al., 2012). Multiple quality indexes including motility, spermatocrit, viability and fertilization success have been used to
dene the quality of the sperm. Sperm motility is a quantitative trait
Corresponding author.
E-mail address: jmei@mail.hzau.edu.cn (J. Mei).

http://dx.doi.org/10.1016/j.aquaculture.2016.10.028
0044-8486/ 2016 Elsevier B.V. All rights reserved.

and a critical determinant of fertility (Rurangwa et al., 2004), and it

can be evaluated by Computer-assisted sperm analysis (CASA) in
many vertebrates including sh species (Gallego et al., 2013).
A number of small peptides and compounds have been reported to
regulate sperm motility, mainly by increasing [Ca2+] and cAMP level
in mammalians. Also, many small molecules have been shown to be inducers of the acrosome reaction, including glycine, prostaglandin E, cholesterol sulfate, acetylcholine, g-aminobutyric acid (GABA), ATP and
other glycans (Yoshida et al., 2008). In sh species, sperm motility is induced when spermatozoa are released from the male genital tract into
the aqueous environment (Alavi and Cosson, 2006). External environmental factors, such as osmotic pressure, ionic and gaseous components
inuence sh spermatozoa motility and activation (Dzyuba and Cosson,
2014). Recent studies showed that dietary supplementation with n-3
polyunsaturated fatty acids, astaxanthin could improve sperm motility

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J. Zhang et al. / Aquaculture 468 (2017) 307313

and fertilization rate (Koprucu et al., 2015; Tizkar et al., 2015). However,
few reports existed that explain the molecular mechanism corresponding to the improvement of sperm motility.
Chloroquine is an anti-malaria drug and well known as an autophagy inhibitor (Fang et al., 2013). The effects of chloroquine on sperm are
time and dose-dependent, as short-term or low-dose treatment
resulted in an increase in sperm motility and fertility in mammalians
(Norman and Gombe, 1975; Egbunike, 1982), whereas long-term or
high-dose treatment resulted in a dose dependent decrease in sperm
motility and fertility (Adeeko and Dada, 1998; Okanlawon et al.,
1993). However, molecular mechanisms of chloroquine for increasing
sperm motility and successful fertilization remain unclear. All-male
yellow catsh has been successfully produced since males grow faster
than females (Mei and Gui, 2015). To investigate whether autophagy
affects sperm motility and fertilization efciency in male yellow
catsh, chloroquine was administered intraperitoneally to adult
sh, and the sperm motility was evaluated by CASA. In addition, we
utilized comparative transcriptome analysis to identify differently
expressed genes and pathways between chloroquine-treated and
control testis. Hopefully, our ndings would reveal the potential
effect and molecular mechanism of chloroquine on sperm motility
and fertilizing ability in sh species.
2. Materials and methods
2.1. Experimental animals and chloroquine treatment
One-year-old sexually mature yellow catsh with similar size were
collected from the farming place at Jiangxia, Wuhan, Hubei province,
China. All sh were acclimatized in the laboratory facility for one
week as previously described (Xiong et al., 2015). The experimental operations were conducted as the requirement of the institution animal
care and use committee of Huazhong Agricultural University.
Chloroquine (sigmaAldrich) was dissolved in 1 PBS and injected
into healthy male individuals (100 2.5 g) behind the pectoral n, at a

dose of 50, 100, 500 and 1000 M per 100 g, respectively. The control
groups were injected only with 1 PBS solution. For each of the independent experiments, four male yellow catsh per group were used.

2.2. Evaluation of sperm motility and in vitro fertilization efciency

Sperm samples were collected from control and treated groups 24 h
after the administration of 1 M chloroquine/g body weight. Sperm concentration was assessed by conventional hemocytometry using a phasecontrast microscope (400 magnication) according to a previous
study (Gennotte et al., 2012). In each case, sperm samples were diluted
with sperm preservative uid (63 mM NaCl, 19 mM KCl, 1.3 mM CaCl2,
4.7 mM MgSO47H2O, 2.5 mM NaHCO3, pH 7.4) to a concentration of
6 108 sperms/mL. Total sperm number of each individual was calculated by multiplying sperm concentration and sample volume. The
nal concentration of sperm is about 550 cells/l activation solution.
Spermatozoa motility and kinematic parameters were quantied by
CASA II using Animal Motility Software Manual Version 1.4 (Hamilton-Thorne Research, Beverly, USA) in which camera speed is
60 frames/s as previously described (Kasimanickam et al., 2007; Kwon
et al., 2013). The 10 phase-contrast objective was chosen to analyze
the spermatozoa, and the movement of sperms in each sample (1 mL
activation solution) was recorded from at least three randomly selected
elds for three times. By convention, all sperm present or entering the
eld in the rst 10 frames are identied and counted during the
acquisition.
The ratio of egg/sperm has been optimized by preliminary experiments to investigate fertilization efciency of sperm when treated
with 100 M chloroquine or not. In particular, each group containing
2.5 g eggs (about 400 eggs per gram) was respectively articially inseminated with 0, 10, 20, 30, 40 and 50 L sperm at a concentration of
8 106 sperms/mL. Embryos were incubated with aerated water and
at room temperature. The percentage of fertilized and unfertilized embryos were recorded and calculated at 16 h post fertilization.

Fig. 1. Comparisons of semen kinematic parameters between chloroquine-treated groups and control group in yellow catsh. Different doses (0, 50, 100, 500 and 1000 M/100 g body
weight) of chloroquine were used. Four kinematics parameters including average path velocity (A), straight line velocity (B), curvilinear velocity (C), and linearity (LIN) were assessed
with the computer-assisted sperm analysis (CASA) system.

J. Zhang et al. / Aquaculture 468 (2017) 307313

309

Premier 5.0 software (Supplementary Table S1). Data are reported as

the mean SEM from independent experiments. P b 0.05 was dened
as statistically signicant.
2.5. Western blot analysis
At 24 h post chloroquine treatment, total proteins of testis were extracted from yellow catsh using One step animal tissue active protein
extraction kit (Sangon, Shanghai, China). Equal amount of protein
samples were separated by electrophoresis on 15% SDS-PAGE and
transferred onto a nitrocellulose membrane (Merck Millipore). After
blocking with 5% non-fat milk in TBS, the membranes were rstly
incubated with primary antibodies, rabbit polyclonal anti-LC3A/B (Cell
Signaling Technology, 4108) and mouse monoclonal anti--actin (Cell
signaling, 4967S), then incubated with HRP-conjugated secondary antibodies and visualized using ECL Western blotting detection reagents
(GE Healthcare), according to previously described (Jing et al., 2015).
3. Results
3.1. Effects of chloroquine on sperm motility

Fig. 2. Comparisons of fertility rate (A) and hatching rate (B) between chloroquine-treated
groups and control group in a combination of 1000 eggs with different numbers of sperms.
The data represents mean SEM of more than three separate experiments. *P b 0.05 and
**P b 0.01.

2.3. Solexa library construction, sequence assembly and functional analysis

of differentially expressed genes

To investigate whether chloroquine inuences sperm motility of yellow catsh, chloroquine was administered intraperitoneally into adult
sh, and the sperm motility was evaluated by CASA. The progressive
motility, curvilinear velocity (VCL), average path velocity (VAP),
straight line velocity (VSL) and linearity (LIN, the ratio of net distance
moved to total path distance (VSL/VCL)) were detected. In the process
of sperm motility, seven time points were selected and calculated to
evaluate sperm activity. The values of kinetic parameters (VAP, VSL
and VCL) related to the vigour of spermatozoa goes up before 6 s and
then come down. Compared with the control, all the values of VAP,
VSL and VCL obviously increased when the dose of chloroquine is
100 M, whereas they decreased as the dose is 1000 M. However,
the values of three kinetic parameters displayed no consistent
change after treating with 50 M or 500 M chloroquine. After 20 s,
the values of these parameters in yellow catsh kept stable low levels
until sperm death because of power consumption (Fig. 1AC). The

The total RNAs were extracted from testes of each control (3 individuals) and chloroquine-treated (3 individuals) yellow catsh, treated by
Turbo Rnase-free DNase (Ambion) and analyzed by the Agilent 2100
Bioanalyzer (Agilent Technologies). Using the Illumina RNA Sample
Preparation Kit, solexa library of control or chloroquine-treated group
was constructed with an amount of 3 g mixed total RNA following
the previous description (Wu et al., 2015). All contigs from these two libraries were further assembled to obtain non-redundant UniGenes by
TGICL software and used for further study. The raw reads of these two
libraries have been deposited to the NCBI database (accession no:
SRP070973).
The unigenes were searched against databases of NCBI nr, SWISSPROT, TrEMBL, Cdd, pfam and KOG by BlastX with E-value of less than
10 e 5. Differential expression proles between control and chloroquine-treated testes were calculated using FPKM method. Gene Ontology (GO) term and Kyoto Encyclopedia Genes and Genomes (KEGG)
analysis were performed as previously (Wu et al., 2015). Gene-set enrichment analysis (GSEA) was conducted with Genomica software to
compare general biochemical and cell biological categories from GO
and KEGG, with a P value threshold of 0.05 for signicant enrichment.

2.4. Quantitative real-time PCR (qRT-PCR) analysis

To validate solexa sequencing results, the expression of selected
genes was veried by qRT-PCR. The cDNA synthesis and qRT-PCR
were accomplished as described (Chen et al., 2015) using -actin as
the internal control. The primer sequences were designed using Primer

Fig. 3. Autophagy was inhibited by chloroquine in testes. Representative western blot (top
panel) and quantitative analysis normalized to -actin (bottom panel) for LC3-II protein
expression were indicated. *P b 0.05, **P b 0.01 and ***P b 0.001.

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J. Zhang et al. / Aquaculture 468 (2017) 307313

percentage of LIN was signicantly improved in sperm samples obtained from sh treated with 50 M and 100 M chloroquine, and subsequently reduce with 500 M and 1000 M chloroquine compared with
control group (Fig. 1D).
3.2. Effects of chloroquine on fertilizing efciency and hatching rate in yellow catsh
Generally, fertilization capacity of sperm is a potential trait to measure sperm quality. In channel catsh, 5.0 105 to 1.2 108 sperm
per egg produced the highest rate of fertilization (67 to 87%) (Bart and
Dunham, 1996). Based on our experience and experiments, the optimal
ratio for high fertilization is a combination of 1000 eggs per batch and
1.83.0 107 sperm. When the numbers of sperm were 6 106,
1.2 107, 1.8 107, 2.4 107 and 3.0 107 in the control groups, the
percentages of fertilization were 24.7%, 42.6%, 66.2%, 72.3% and 74.0%,
respectively. Interestingly, in the chloroquine-treated group, the corresponding fertilization rates were 41.1%, 60.2%, 82.4%, 81.6% and 84.1%
when the sperm numbers were ranged from 6 106 to 3.0 107,
with 16.4%, 17.6%, 10.1%, 9.3% and 10.1% higher values in selected treatment groups compared with the control groups (Fig. 2A). However,
there is no signicant difference in hatching rate between control
group and chloroquine-treated (Fig. 2B).

3.3. Chloroquine inhibits autophagy in yellow catsh

To determine whether autophagy could be inhibited in testis by
chloroquine, the expression of autophagy-associated molecules LC3
was checked by western blot after treating with different concentration
of chloroquine (control, 50 M, 100 M, 500 M and 1000 M). In comparison with control testes, the expression of LC3-II increased following
treated with different doses of chloroquine (Fig. 3). 100 M chloroquine
treatment resulted in a signicant upregulation of LC3-II expression,
while the highest expression of LC3-II was observed in testes treated
with 1000 M chloroquine. These data suggest that autophagic response
was explicitly inhibited by chloroquine in testes of yellow catsh.
3.4. Gene ontology (GO) analysis of differentially expressed genes (DEGs) in
the comparative transcriptome between chloroquine-treated and control
testes
In order to identify genes and signaling pathways response to chloroquine treatment in testes of yellow catsh, two solexa libraries were
constructed by control and chloroquine-treated testes respectively.
After sequence assembly and functional annotation, the expression of
assembled unigenes were counted by FPKM method. Totally, 4686
DEGs were discovered in response to chloroquine treatment, including

Fig. 4. Gene ontology (GO) classication of the DEGs with lower expression in chloroquine-treated group compared with the control group: biological process (red), cellular component
(blue), molecular function (green).

J. Zhang et al. / Aquaculture 468 (2017) 307313

2144 up-regulated genes and 2542 down-regulated genes. The Gene

Ontology database (www.geneontology.org) was used to determine
which kinds of GO term DEGs were assigned to, based on three categories: biological process, cellular component and molecular function
(Fig. 4). In the biological process category, a majority of 2542 down-regulated genes were enriched in DNA integration (GO:0015074), transposition, DNA-mediated (GO:0006313) and cellular glucuronidation
(GO:0052695). Interestingly, most toll-like receptor signaling pathways
including TLR1, TLR2, TLR4, TLR5, TLR6, TLR9, TLR10 and MyD88-dependent TLR signaling pathways were in the top lists. In the cellular
component category, cytolytic granule (GO:0044194) was the most
prominent term followed by nuclear chromosome (GO:0000228)
and nuclear pore (GO:0005643). In the molecular function category,
RNA-directed DNA polymerase activity (GO:0003964), endonuclease
activity (GO:0004519) and nucleic acid binding (GO:0003676) were
the most represented terms. Moreover, 2144 up-regulated genes were
also performed with GO analysis and assigned to different terms (Supplementary Fig. S1). Gamete generation (GO:0007276) and MHC class
II protein complex (GO:0042613) were enriched in the up-regulated
DEGs.
3.5. KEGG analysis of differentially expressed genes (DEGs)
To identify differentially biological pathways, the DEGs were
mapped to reference canonical pathways in KEGG. Enrichment analysis
shows that most of the up-regulated DEGs were involved in pathways
of antigen processing and presentation (ko04612), rheumatoid
arthritis (ko05323) and intestinal immune network for IgA production
(ko04672). PI3K-Akt signaling pathway (ko04151) was also up-regulated after chloroquine treatment. The down-regulated DEGs were enriched
in drug metabolism-cytochrome P450 (ko00982), chemical carcinogenesis (ko05204), arrhythmogenic right ventricular cardiomyopathy

311

(ko05412) and metabolism of xenobiotics by cytochrome P450

(ko00980) (Fig. 5).
3.6. qRT-PCR conrmation of DEGs involved in toll-like receptor signaling
pathways between chloroquine-treated and control testes
To verify the accuracy of the sequencing data, nine DEGs related to
toll-like receptor and Myd88 signaling pathway were selected and validated by qRT-PCR. In the qRT-PCR result, except TLR1, expression of the
other eight TLR genes were signicantly reduced in chloroquine-treated
group compared to the control group (Fig. 6A). Except TLR3 and TLR7,
the other seven TLR genes had similar relative expression between
data of qRT-PCR and comparative transcriptome (Fig. 6B). The inconsistent expression of TLR3 and TLR7 might be caused by their low expression value in the transcriptome calculated by RPKM method.
4. Discussion
Known as an anti-malaria drug and inhibitor of autophagy, chloroquine has been shown to have potential inuence on spermatozoa
and sperm functions in mammalians. However, there is no report
about the effects of chloroquine on sperm of sh species. Different
doses of chloroquine were administered intraperitoneally into adult
yellow catsh, and the sperm motility was evaluated by CASA system.
Our results demonstrated that an optimal dose of chloroquine could efciently inhibit autophagy and improve sperm motility and fertilization
efciency, by regulating multiple pathways including PI3K-Akt and TLR
signaling pathways. Hence, chloroquine will be a suitable cost-effective
medicine to improve sperm quality and fertilization for aquaculture
practices.
The effects of chloroquine on sperm are time and dose-dependent in
mammalians. In livestock animals such as porcine and bovine, short-

Fig. 5. KEGG classication of the DEGs between testis transcriptomes of chloroquine-treated group and control group. The red and green columns represent the up-regulated and downregulated signaling pathways in response to chloroquine treatment, respectively.

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J. Zhang et al. / Aquaculture 468 (2017) 307313

term or low-dose treatment resulted in an increase in sperm motility

and fertility (Norman and Gombe, 1975; Egbunike, 1982). Moreover,
chloroquine can be used in an articial insemination programme to
enhance conception of stored porcine sperm (Egbunike, 1989). However, long-term or high-dose treatment resulted in a dose dependent decrease in sperm motility and fertility in rat (Adeeko and Dada, 1998;
Okanlawon et al., 1993). In human patient semen, the percentage of
rapid-moving spermatozoa was signicantly enhanced by low concentrations of chloroquine, but signicantly inhibited at higher concentrations (Hargreaves et al., 1998). Our result demonstrated that low dose
of chloroquine (100 M) signicantly increased values of VAP, VCL,
VSL and LIN, whereas high dose of chloroquine (1000 M) decrease
values of VAP, VCL and VSL compared with the control (Fig. 1). Similar
to mammalians, the effects of chloroquine on sperm motility in yellow
catsh are also in a dose-dependent manner.
Autophagy has been shown to regulate early reproductive events including gamete development and maturation, fertilization and embryogenesis (Lim and Song, 2014; Kanninen et al., 2013). As shown in
Supplementary Fig. S1, chloroquine treatment activated multiple
specic signaling pathways that regulate gametogenesis and meiotic
mitosis, such as gamete generation, reciprocal meiotic recombination,
meiotic nuclear division, G2/M transition of mitotic cell cycle, mitotic
spindle assembly and mitotic cell cycle signaling pathways. The PI3KAKT signaling pathway plays important roles in testis development
and spermatogenesis, since deciency in p110beta subunit of
phosphoinositide 3-OH kinase impaired spermatogenesis and lead to
defective fertility (Ciraolo et al., 2010). Compared with XY testis of
yellow catsh, a higher level of PI3K-AKT signaling was observed
in YY testis and is correlated with a higher degree of testis maturity
(Wu et al., 2015). Meanwhile, PI3K-AKT pathway was induced by

chloroquine treatment (Fig. 5). These data suggest that PI3K-AKT pathway plays a very important role in spermatogenesis and maintenance of
high quality of sperm.
Chloroquine has been validated as an inhibitor of toll-like receptor in
mouse (Yasuda et al., 2008) and tongue sole (Cynoglossus semilaevis)
(Li and Sun, 2015). In yellow catsh, a number of toll-like receptor
signaling pathways including TLR 1/2/4/5/6/9/10 and MyD88-dependent TLR signaling pathways were signicantly reduced by
chloroquine treatment (Fig. 4). Interestingly, the activated TLRs
signicantly reduce sperm motility and suppressed fertilization in
human (Fujita et al., 2011; Zhu et al., 2016). LPS binds to its receptor,
TLR-4 that is expressed on the sperm surface, resulting in induction
of apoptosis (Okazaki and Shimada, 2012). These studies demonstrated
that stimulated TLR signalings inhibit sperm motility. Consequently,
reduction of TLRs plays essential roles in increasing sperm motility in
yellow catsh.
Both reproduction and immune responses are energetically costly,
and energetic trade-offs between reproduction and immunity have
been postulated in multiple organisms including insects (Schwenke et
al., 2015; McNamara et al., 2013; McNamara et al., 2014) and Japanese
quail (Coturnix coturnix) (Boughton et al., 2007). Chloroquine treatment
decreased TLR immune signaling and increased reproductive activity,
suggesting that energetic trade-offs between reproduction and immunity might also be existed in sh species.
In conclusion, 1 M chloroquine/g body weight was an optimal dose
for in vivo treatment to improve the sperm quality and fertility rate of
yellow catsh. Our ndings provide the rst evidence that chloroquine
can be used as an articial medicine to enhance sperm motility and
improve fertilization rate for aquaculture practices. Accordingly, chloroquine could also be applied in many other sh species. In addition, some

Fig. 6. qRT-PCR validation of TLR gene expression. (A) Prole of relative expression of selected genes by qRT-PCR. (B) Prole of normalized expression data in the transcriptome for selected
genes. *P b 0.05, **P b 0.01 and ***P b 0.001 indicate the signicant difference in gene expression between chloroquine-treated group and control group.

J. Zhang et al. / Aquaculture 468 (2017) 307313

other small molecules which can activate PI3K-AKT signaling pathway

may also be used to improve sperm quality.
Supplementary data to this article can be found online at doi:10.
1016/j.aquaculture.2016.10.028.
Funding
This work was supported by the Fundamental Research Funds for
the Central Universities (grant number 2662015PY101). The funders
had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Conict of interest
All authors declare that they have no conict of interest.
Ethical approval
Applicable international, national, and/or institutional guidelines for
the care and use of animals were followed.
Acknowledgments
We would like to thank all members of our research groups for providing feedback and suggestions.
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