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African Journal of Pig Farming ISSN 2375-0731 Vol. 3 (9), pp. 001-006, September, 2015. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

DNA damage and decrease of cellular oxidase activity 
in piglet sertoli cells exposed to gossypol 

 
Ming Zhang1,2, Hui Yuan1*, Zuping He3, Liyun Yuan1, Jine Yi1, Sijun Deng1, 

Li Zhu1, Chengzhi Guo1, Yin Lu1, Jing Wu1, LixinWen1, Qiang Wei1 and Liqun Xue1
 

 
1
College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan 410128, People’s Republic 

of China. 
2
Jiangxi Biotech Vocational College, Nanchang, Jiangxi 330200, People’s Republic of China. 

3
Department of Biochemistry and Molecular & Cellular Biology, Georgetown. University Medical Center, 3900 

Reservoir Road NW, Washington, DC 20057, USA. 
 

Accepted 5 January, 2015 
 
The study was designated to explore the toxic effects of gossypol on piglet sertoli cells. Sertoli cells were isolated 
from piglet testes using a two-step enzyme digestion and followed by differential plating. Piglet sertoli cells were 
cultured and classified into five groups, that is, group A, the control without gossypol, group B with 2.5 μg/ml 
gossypol, group C with 5 μg/ml gossypol, group D with 10 μg/ml gossypol and group E with 20 μg/ml gossypol. We 
found that sertoli cells’ growth was inhibited by gossypol at dose 2.5 μg/ml when compared with the control group. 
The oxidase activity of sertoli cell also decreased at 2.5 μg/m gossypol. Moreover, DNA damage of sertoli cells was 
observed at 5 μg/ml gossypol. Putting this into consideration, our study suggests that exposure of gossypol to 
sertoli cells leads to an inhibition of sertoli cell growth and oxidase activity of sertoli cells at a low concentration, 
whereas gossypol results in DNA damage of sertoli cells at a higher concentration. 

 

Key words: Gossypol, sertoli cells, oxidase, DNA damage. 

 
INTRODUCTION 

 
Gossypol is a yellow polyphenolic compound isolated 
from the seed of cotton plants of the Malvaceae family. It 
exists in two forms, free and bound. Free gossypol is the 
major toxic ingredient of cottonseed meal, which is a 
source of protein for animal feed and its protein content is 
as high as 33.21 to 45.09%. However, the toxicity of gos-
sypol has limited the use of cottonseed meal. Gossypol is 
generally known as a male anti-fertility by suppressing 
spermatogenesis (Dodou et al., 2005) and potential 
anticancer agent, which can induce the apoptosis of 
human breast cancer cells (Ye et al. 2010). Cytotoxic 
gossypol and its derivatives have been used for male 
contraception in humans (Coutinho, 2002). Ten micro-  
 
 

 
*Corresponding author. E-mail: yuanhui7269@yahoo.com.cn. 

 
Abbreviations: SOD, Superoxide dismutase; GSH-Px, 
glutathione peroxidase; MDA, malondialdehyde; FCS, fetal calf 
serum; DNTB, 5,5'-dithio-bis (2-nitrobenzoic acid) or Ellman’s 
reagent. 

 
 
 

 
grams per day dose of gossypol, causes azoosper-mia 
and deleterious changes in monkey’s testicular 
morphology, especially in semi-iniferous tubules (Sharma 
et al., 1999). In bulls, gossypol (8 mg/kg per day) induces 
testicular degeneration and significantly increases in 
primary and secondary sperm abnormalities and signifi-
cant degeneration of germ cells between type B sperma-
togonia and pachytene primary spermatocytes (Cerelli 
and Johnson, 1999). In rats, gossypol with 15 mg/kg per 
day significantly decreases sperm concentration and in-
creases the number of abnormal sperms (Romualdo et 
al., 2002). This effect maybe caused by gossypol, which 
disrupts the important cell functions (Ojha et al., 2006). 
Recently, gossypol has been shown to impair the gap 
junctional intercellular communication in the cultured 
Sertoli cells (Zhou et al., 2008).  

In summary, animal experiments showed that gossypol 
damages animal spermatogenic epithelium, leads to 
abnormality and death of sperm, or even depletion of no 
sperm. Thus, gossypol causes reduced reproductive 
capacity or male infertility. This study was designated to 
probe the toxic effects of gossypol on animal male repro- 



 
 
 

 

duction using piglet sertoli cells in vitro as experimental 
model. 
 

 
MATERIALS AND METHODS 
 
Piglet testes 
 

Testes were obtained from 3 to 4 weeks old piglets (Changsha, 
Hunan, China), placed in ice-cold phosphate-buffered saline (PBS) 
with 600 IU/ml penicillin-streptomycin and sent to the laboratory 
within 2 h. 
 
 
Isolation, culture, and identification of testicular sertoli cells of 
piglets 
 

Testicular capsule was removed under sterile conditions and 
semi-niferous tubules were isolated from piglet testis using 
mechanical dissociation and a one step enzymatic digestion with 1 
g/l collagenase and 2.5 g/ trypsin, pursuant to the procedure as 
described previously (Dym et al., 1995; He et al., 2007) with minor 
modifications. Cell mixture containing male germ cells and sertoli 
cells were obtained using the second enzymatic digestion, colla-
genase IV, hyaluronidase and trypsin and sertoli cells were further 
separated from germ cells by differential plating according to the 
procedure described previously (He et al., 2007), Dirami et al., 
1999). For differential plating, germ cells and sertoli cells were 
placed into tissue culture dish in the Dulbecco’s modified Eagle’s 
medium (DMEM)/F12 medium supplemented with 10% fetal calf 
serum (FCS) for 3 h at 34°C. Sertoli cells were attached to the 
culture plates, whereas male germ cells remained in suspension 
and were removed. Cell viability of sertoli cells was determined with 
0.4% trypan blue exclusion assay. 

Freshly isolated piglet sertoli cells were plated at a density of 2 × 

10
6
 cells/ml in DMEM/F12 supplemented with 10% FCS in a 

humidified incubator with 5% CO2 and 100% humidity for 24 h. 
Sertoli cells were identified by oil red O staining and Fas ligand 
(FasL) expression as assayed by immunocytochemistry when 80 to 
90% of the dish was confluent with cells. 

 

Experimental groups and MTT assay 
 

Piglet sertoli cells were classified into five groups, which are: 
group A, the control without gossypol; group B with 2.5 μg/ml 
gossypol; group C with 5 μg/ml gossypol; group D with 10 μg/ml 
gossypol; group E with 20 μg/ml gossypol. After culture for 24 h, the 
prolife-ration of sertoli cells were determined using MTT assays with 
quadruplicate, according to the procedure as previously described 
(Mosmann, 1983). 

 

Determination of malondialdehyde (MDA) level, superoxide 
dismutase (SOD) and glutathione peroxidase (GSH-Px) activity 
of sertoli cells 
 

Malondialdehyde (MDA) level in the sonicated sertoli cells was 
measured by the thiobarbutiric acid method (Heath and Packer, 
1968) and was presented as nmol per mg of protein. The activity of 
superoxide dismutase (SOD) in the sonicated sertoli cells was 
measured by the xanthine oxidase method (McCord and Fridovich, 
1969) and presented as units per mg of protein. Activity of gluta-
thione peroxidase (GSH-Px) in the sonicated sertoli cells was 
measured by the 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) 
reaction test (Flohe and Gunzler, 1984) and presented as units per 
mg of protein. All the procedures were followed with the instruction 

 
 
 
 

 
on the kits from Nanjing Jiancheng Company (China). 

 

Determination of DNA damage of sertoli cells by gossypol 
 
Quantitation of DNA damage in sertoli cells were performed using a 

single-cell microgel electrophoresis technique under alkaline conditions 

(Singh et al., 1988) with certain modifications: the time of lysis, denature 

and electrophoresis were changed to 2 h, 1 h and 40 min, respectively. 

Sertoli cells were observed for epifluorescence under Fluophot 

microscope and 400 cells were counted in each group (4 parallel glass 

slides per group). Being excited by ultraviolet light, the nuclear DNA and 

the migration of DNA in orange-red DNA image (the comet tail) was 

clearly observed. 

 

Statistical analysis 
 

All the data were presented as mean ± SEM, statistically 
significant differences (p < 0.05) and extremely significant 
differences (p < 0.01) were determined among various groups by 
analysis of variance (ANOVA) and Tukey post-test using SPSS 
12.0 statistical software. 
 

 

RESULTS 

 

Isolation, identification and culture of piglet sertoli 
cells 

 

Piglet sertoli cells were isolated by a 2-step enzymatic 
digestion with trypsin and collagenase and followed by 
differential plating. Cell viability was up to 90% as 
assayed by trypan blue exclusion. After 24 h of culture, 
sertoli cells were attached to dish and formed a large 
columnar or irregular appearance with an elongated 
cellular body. Sertoli cells were connected with each 
other and showed mosaic-like arrangement and inter-
cellular irregular protrusions. On both sides of cells, there 
were several prominences and strong refraction. Phago-
cytosed objects, varying sizes of vacuoles and the cell 
nucleus were observed in the cytoplasm of the cells. Oil 
red O staining showed that, red lipid droplets occurred 
near the nucleus or at the two poles of cytoplasm (Figure 
1a). After 72 h of culture, high expression of FasL in 
sertoli cells was observed by immunocytochemistry 
(Figure 1b). 
 

 

Effects of gossypol on proliferation of piglets sertoli 
cells 

 

Piglets’ sertoli cells were treated with different concen-
trations of gossypol for 24 h and the effect of gossypol on 
the proliferation of sertoli cells was assessed by MTT 
assay (Table 1). Table 1 showed that, with the increased 
concentration of gossypol, the sertoli cell proliferation 
decreased gradually. Compared with the control group A, 
cell growth inhibition in group B was significant difference 
(P < 0.05), while groups C, D and E showed extremely 
significant difference (P < 0.01) when compared with 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. Identification of the isolated piglet sertoli cells: (A) The oil red O staining showed that red lipid droplets (arrows) 
were presented near the nucleus or at the two poles of cytoplasm of the isolated piglet cells, which confirmed the identity 
of piglet sertoli cells. Cell nuclei were counterstained with hematoxylin; (B) Immunocytochemistry revealed that the 
isolated piglet cells were positive for FasL (arrows), further verifying the identity of piglet sertoli cells. Magnification: (A) 
and (B) 400 ×. 

 

 
Table 1. Cell proliferation of piglet sertoli cells by MTT assay.  

 
Group OD value at 490 nm Inhibition of proliferation (%) 

Control (A) 0.66 ± 0.06 0 

B 0.46 ± 0.03* 30.59 

C 0.40 ± 0.05** 39.27 

D 0.39 ± 0.01** 41.01 

E 0.30 ± 0.02** 54.95 
 

Compared to the control group, “*” indicates significant difference (p < 0.05), “**” indicates 
extremely significant difference (p < 0.01). 

 

 

group A. The results suggest that, the effect of gossypol 
on growth inhibition of sertoli cells is in a dose-dependent 
manner. 

 

Effects of gossypol on antioxidant enzymes activities 

 

Superoxide dismutase (SOD) and glutathione 
peroxidase (GSH-Px) are two important enzymes in the 
antioxidant defense system. Malondialdehyde (MDA) is 
regarded as a major marker of lipid peroxidation in tissue. 
After expo-sure to gossypol, MDA content, SOD and 
GSH-Px activities of sertoli cells were measured and the 
data was shown in Table 2. Compared with the control 
group A, the increase of malondialdehyde content in  
group B was significantly different (P < 0.05）.And the 
 
increase of MDA content in groups C, D and E was 
extremely significant (P < 0.01). These results indicate 
that, the increase of MDA content of sertoli cells by 
gossypol is also in a dose-dependent manner.  

The decrease of SOD and GSH-Px activity was obser- 

 
 

 

ved in group B significant (P < 0.05) when compared with 
the control group A, but extremely significant decrease 
was observed in groups C, D and E (P < 0.01). Notably, 
the decreases of SOD and GSH-Px activities were 
correlated with the concentration of gossypol. 
 

 

DNA damage of gossypol on piglet sertoli cells 
 
Comet tail length formed in comet assay is an important 
parameter in evaluating the DNA damage. After UV 
excitation, sertoli cell DNA from gossypol treated groups 
showed an orange-red color. DNA fragments migration 
were observed in gossypol treated groups (Figure 2a, b), 
suggesting that, gossypol induces DNA damage in sertoli 
cells. Table 3 showed that, 90.43% control cell were 
categorized at grade 0, no cells were categorized at 
grade 3 and 4. The increased gossypol concentration 
caused more severe DNA damage. The rate of DNA tail 
was used to show the degree of DNA damage. An incre-
ased DNA tailing rate was observed along with the 



 
 
 

 
Table 2. Effects of gossypol on malondialdehyde (MDA) content, superoxide dismutase 
(SOD) and glutathione peroxidase (GSH-Px) activities of piglet sertoli cells.  

 
Group MDA (nmol/mgprot) SOD (U/mgprot) GSH-Px (U/mgprot) 

Control (A) 2.67 ± 0.26 210.37 ± 24.63 18.87 ± 3.43 

B 3.13 ± 0.47* 198.58 ± 21.32* 16.68 ± 3.26* 

C 5.19 ± 1.60** 178.86 ± 22.64** 13.96 ± 2.97** 

D 5.46 ± 0.88** 137.57 ± 21.57** 12.05 ± 2.18** 

E 11.24 ± 1.98** 105.13 ± 19.48** 9.63 ± 2.27** 
 

Compared to the control group, “*” indicates significant difference (p < 0.05), “**” indicates 
extremely significant difference (p < 0.01).  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 2. The single-cell gel electrophoresis (comet assay) showed the DNA damage of gossypol on piglet 
sertoli cells: (A) DNA image of control group; (B) comet assay revealed that damaged DNA of gossypol-treated 
group B contained strand breaks migrates farther in the gel than intact DNA, creating an image resembling a 
celestial comet. Magnification: 400×. 

 

 

Table 3.  Effect of gossypol on DNA damage of piglet sertoli cells.  
 
 Total  Cell number of different degree  

Rate of DNA  

Group cell Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 
 

tail (%)  

 

number 
 

      
 

       
 

Control (A) 100 90.43 ± 4.34 5.17 ± 1.02 4.40 ± 1.23 0.00 ± 0.00 0.00 ± 0.00 9.57 ± 1.25 
 

B 100 86.33 ± 4.51 6.29 ± 2.03 4.61 ± 1.62 1.65 ± 1.14 1.12 ± 0.53 13.67 ± 1.46 
 

C 100 68.94 ± 5.73** 18.28 ± 3.79** 5.39 ± 0.97* 4.82 ± 1.13* 2.57 ± 0.46* 31.06 ± 3.13** 
 

D 100 60. 52 ± 3-48** 20.05 ± 2.68** 8.76 ± 1.55** 6.13 ± 1.35** 4.41 ± 1.3** 39.35 ± 2.57** 
 

E 100 40.12 ± 2.53** 24.06 ± 2.85** 12.24 ± 2.32** 13.56 ± 1.75** 10.02 ± 2.41** 59.88 ± 4.31** 
 

 
Compared to the control group, “ 

 

 

increased gossypol concentration. These data indicates 
that, the overall level of DNA damage gradually increased 
in an obvious dose-response to gossypol (Table 3). 

 

 

DISCUSSION 

 

Breeding domestic livestock and aquatic animals with 

 
 

 

cottonseed cake has significant economic benefits. How-
ever, gossypol has been found toxicity in male repro-
ductive system as a feedstuff additive. This study showed 
that, gossypol inhibited the growth of piglet sertoli cells in 
a dose-dependent manner. Thus, the current may shed 
insight into male reproductive toxicity of gossypol in clinic. 
We used 10% fetal calf serum (FCS) for the growth and 
survival of piglet sertoli cells. Recently, we demonstrated 



 
 
 

 

that 10% FCS promotes proliferation of piglet sertoli cells 
(Zhang et al. 2010). The control group and gossypol-
treated groups were cultured with FCS and thus fetal calf 
serum has no effect for us to investigate the toxicity of 
gossypol on piglet sertoli cells.  

Previous research of gossypol on male reproductive 
toxicity is mainly focused on the structure of testis, 
epididymis and spermatogenic cells. Gossypol causes 
sparse array of testis seminiferous tubule, ambiguous or 
self-dissolving nucleus of epitheliums and reduces the 
amount of sperm, unclear sperm structure and swelling 
mitochondrial. Gossypol inhibits testicular lactate dehy-
drogenase activity. Thereby it undermines the animal 
spermatogenic epithelium, results in abnormality and 
death of sperm, or even no sperm. Finally, it causes sire 
infertility, reduces mitochondrial function and results in 
infertility (Monsees et al., 1998). Many researches 
suggests that, apoptosis plays an important role in testis 
regression (Furuta et al., 1994). Apoptotic spermatogenic 
cells can be found at all levels, especially at sperma-
tocytes and sperm cells. The apoptotic cells occasionally 
aggregate chain-like or piles. Andrade et al. (2006), 
utilized hemicastration and electron microscopy to con-
firm that the epididymis is a definitive target of gossypol 
(de Andrade et al., 2006). Sertoli cell junctional protein 
maybe the early target of toxins such as gossypol (Fiorini 
et al., 2004). The effects of gossypol on sertoli cells are 
still unclear.  

Oxidative stress can cause potential harm to animal 
organism. When oxidative stress is over generated or the 
enzymatic and non-enzymatic antioxidant defense sys-
tems are inefficient, it can stimulate some chain reactions 
causing cytotoxic effect. Superoxide dismutase (SOD) 
and glutathione peroxidase (GSH-Px) are the two major 
part of oxidase defense system. Superoxide dismutase is 
closely related to the cellular oxidative metabolism which 
widely exists in vivo. It is a natural scavenger of reactive 
oxygen species (ROS) and superoxide anion radicals. In 
the antioxidant enzymes, SOD is the first enzyme to 
combine with active oxygen free radicals. SOD specifi-
cally combines with superoxide anions and acts synergis-
tically with GSH-Px in preventing cell membrane lipid 
peroxidation and damaging metabolites’ formation. They 
directly capture and eliminate free radicals (Alul et al., 
2003). Through hydrogen atom donors’ effect of blocking 
or terminating radical consecutive reaction chain, super-
oxide dismutase prevents the action of free radicals, 
suppress cell membrane lipid peroxidation during patho-
logical lesion and protect biological macromolecules and 
membranes from damage. Glutathione peroxidase 

converts hydrogen peroxide (H2O2) into H2O. GSH-Px 

has a strong ability of scavenging lipid peroxide and 
hydrogen peroxide which were induced by active oxygen 
species and hydroxyl radicals. Thus, SOD and GSH-Px 
can protect biological macromolecules and membranes 
from damages. The values of SOD, GSH-Px and malon-
dialdehyde (MDA) content of oxidative metabolites 

  
  

 
 

 

Malondialdehyde is often considered as the major 
hallmark of lipid peroxidation damage, which causes 
secondary damage to cell functions, genotoxicity and 
carcinogenesis (Korchazhkina et al., 2003). DNA damage 
is the major effect of gossypol in piglet sertoli cells. The 
tail ratio in gossypol treated groups from 5 μg/ml was 
remarkably different from the control, as detected by 
single cell gel electrophoresis. This result suggests that, 
gossypol can cause DNA damage in piglet sertoli cells at 
high concentration. Activities of superoxide dismutase, 
glutathione peroxidase and malondialdehyde content in 
gossypol treated groups B to E were significantly different 
from the control, whereas DNA damage was observed in 
groups exposed to gossypol at concentrations above 5 
μg/ml. The DNA damage induced by gossypol could be 
as a result of its stimulation of hydroxyl radicals produc-
tion (El-Sharaky et al., 2009); gossypol also causes DNA-
breaking (Li et al., 2000). Thus, gossypol has a marked 
effect on the superoxide dismutase, glutathione pero-
xidase activities, malondialdehyde content and DNA 
damage of sertoli cells. 
 
 

Conclusion 
 

The present revealed that gossypol has obvious toxic 
effects on piglet sertoli cells, as evidenced by the inhibi-
tion of sertoli cell proliferation and DNA damage, 
increased malondialdehyde level, as well as reduced 
superoxide dismutase and glutathione peroxidase activi-
ties of piglet sertoli cells, which may lead to decrease in 
reproduction of male pigs and as well decreased their 
reproductive capacity. 
 

 

ACKNOWLEDGEMENT 
 
This work was supported by the Department of Education 
of Hunan Province, China (Grant No. 03A020). 
 
 
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