Effect of nonylphenol on male reproduction: Analysis of rat epididymal biochemical

markers and antioxidant defense enzymes

Hamdy A.A. Aly
a,
, scar Domnech
b
, Zainy M. Banjar
c
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
b
Department of Physical Chemistry, Faculty of Pharmacy, Barcelona University, Spain
c
Department of Medical Biology, School of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
a b s t r a c t a r t i c l e i n f o
Article history:
Received 7 December 2011
Revised 20 February 2012
Accepted 23 February 2012
Available online 7 March 2012
Keywords:
Nonylphenol
Epididymis
Sperms
Antioxidants
Lipid peroxidation
The mechanism by which nonylphenol (NP) interferes with male reproduction is not fully elucidated. There-
fore, the present study was conducted to evaluate the effect of NP on male reproductive organ's weight,
sperm characteristics, and to elucidate the nature and mechanism of action of NP on the epididymis. Adult
male Wistar rats were gavaged with NP, dissolved in corn oil, at 0, 100, 200 or 300 mg/kg/day for 30 consec-
utive days. Control rats were gavaged with vehicle (corn oil) alone. Body weight did not show any signicant
change while, absolute testes and epididymides weights were signicantly decreased. Sperm count in cauda
and caput/corpus epididymides, and sperm motility was signicantly decreased. Daily sperm production was
signicantly decreased in a dose-related manner. Sperm transit time in cauda epididymis was signicantly
decreased by 300 mg/kg, while in the caput/corpus epididymis it was signicantly decreased by 200 and
300 mg/kg of NP. Plasma LDH was signicantly increased while; plasma testosterone was signicantly
decreased in a dose-related pattern. In the epididymal sperm, NP decreased acrosome integrity, m and
5-nucleotidase activity. Hydrogen peroxide (H
2
O
2
) production and LPO were signicantly increased in a
dose-related pattern. The activities of SOD, CAT and GPx were signicantly decreased in the epididymal
sperm. In conclusion, this study revealed that NP treatment impairs spermatogenesis and has a cytotoxic
effect on epididymal sperm. It disrupts the prooxidant and antioxidant balance. This leads oxidative stress
in epididymal sperms of rat. Moreover, the reduction in sperm transit time may affect sperm quality and fer-
tility potential.
2012 Elsevier Inc. All rights reserved.
Introduction
In humans, the decline and well known regional differences in
semen quality (Jorgensen et al., 2001), the increasing incidence of
testicular cancers (Jacobsen et al., 2000), and increasing rates of
developmental abnormalities in male and female reproductive tracts
(Sultan et al., 2001) during the last ve decades, have been related
to environmental contaminants (Uguz et al., 2009).
Nonylphenol (NP) belongs to the group of alkylphenols, which
are degraded products of alkylphenolpolyethoxylates (APEs), a well
known class of environmental endocrine disrupters (Raecker et al.,
2011). Nonylphenol polyethoxylate (NPE) is a non-ionic surfactant
widely used as component of detergents, paints, herbicides, and
many other synthetic products (Gong and Han, 2006a, 2006b,
2006c, 2006d). Nonylphenol (NP) is a nal metabolite of NPE. NP is
more stable and persistent (Sone et al., 2004; Rivero et al., 2008).
With the development of industry, large amounts of NP have been
discharged into water (Cheng et al., 2006).
It was reported that NP treatment resulted in great damage to the
reproductive system, including altered steroidogenesis, disturbed
testicular structure and decreased sperm number in the epididymis
(McClusky et al., 2007). Numerous studies have shown that NP expo-
sure produces oxidative stress, enhancing reactive oxygen species
(ROS) generation in human blood neutrophils (Okai et al., 2004). In
addition, NP administration increased ROS level and depressed
the activity of antioxidant enzymes in rat epididymal sperms
(Chitra et al., 2002), testis (Chitra and Mather, 2004) and testicular
Sertoli cells (Gong and Han, 2006a, 2006b, 2006c, 2006d). Moreover,
it has been reported that NP induced disturbed intracellular Ca
2+
homeostasis (Gong et al., 2008), endoplasmic reticulum stress
(Gong et al., 2009) which lead to apoptosis in rat Sertoli cells (Li
et al., 2010). However, the cellular/biochemical mechanisms by
which NP causes reproductive toxicity has not been fully elucidated
(Wu et al., 2010).
Various environmental contaminants can induce oxidative stress
by generating ROS such as hydrogen peroxide (H
2
O
2
) and superoxide
anion (O
2

). ROS may initiate a series of reactions that damage

Toxicology and Applied Pharmacology 261 (2012) 134141
Corresponding author.
E-mail address: hamdyaali@yahoo.com (H.A.A. Aly).
0041-008X/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2012.02.015
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j our nal homepage: www. el sevi er . com/ l ocat e/ yt aap
cellular components resulting in cell death (Wang et al., 2003).
When produced in excessive amounts, the ROS stimulate DNA frag-
mentation and a loss of sperm function associated with peroxidative
damage to the mitochondria and plasma membrane. Moreover,
sperm plasma membrane, being rich in polyunsaturated fatty acids,
is highly susceptible to ROS attack. In the same time, epididymis
being enriched with antioxidant defense system protects the sperm
during its transit through caput to cauda region of epididymis and
facilitates their maturation process (Vernet et al., 2004).
The epididymis is known to play an important role in providing
the microenvironment for sperm maturation and storage of sperm
(Cornwall, 2009; Raymond et al., 2010). Numerous proteins synthe-
sized and secreted by the epididymal epithelia cells are thought
to be considerable members of epididymal microenvironment and
be involved in male reproductive activities including the initiation
of sperm maturation, spermoocyte recognition, the acrosome reac-
tion directly or indirectly (Yenuqu et al., 2006), interaction with the
zona pellucida, and binding to and fuse with plasma membrane of
the oocyte (Cuasnicu et al., 2002). All these modications necessitate
a minimum period in which they can occur, a time in which the sper-
matozoa must stay in the epididymal caput and corpus (Franca et al.,
2005). During transit through the epididymis, many morphological,
physiological and biochemical characteristics of the spermatozoa are
modied, as part of the maturation process (Orgebin-Crist, 1969).
Sperm transit time through the epididymis has an important role in
the maturation of spermatozoa. Thus an alteration in this time can
provoke problems in such maturation as well as alter the number of
gametes available for ejaculation (Klinefelter, 2002).
However, studies concerning the effect of nonylphenol on the
reproductive organs and fertility of male rats are limited. Moreover,
the nature and mechanism of action of NP on the epididymis is not
fully elucidated. Therefore, it is important to investigate whether
environmental estrogens can have direct effects on mammalian
sperm function. The present study was conducted to evaluate the
effect of NP on male reproductive organs weight, sperm characteris-
tics, and to elucidate the nature and mechanism of action of NP on
the epididymis.
Materials and methods
Chemicals. Nonylphenol (4-nonylphenol) with 98% analytical
standard, pyrogallol, sodium azide, Alexa Fluor-488-PNA, Rh123 and
glutathione reductase were purchased from Sigma-Aldrich Chemical
Company, St. Louis, MO, USA. All other chemicals are of analytical
grade.
Animals and treatments. Adult male Wistar rats (90 days) were
housed in clean polypropylene cages and maintained on a 12 h
light:dark cycle and a temperature of 2025 C with ad libitum access
to food and water. For 7 days before the experiment, rats were
handled daily for 5 min to acclimate themto human contact and min-
imize their physiological responses to handling for subsequent proto-
cols (Ma and Lightman, 1998). NP was dissolved in corn oil and given
to rats by gavage at 0, 100, 200 or 300 mg/kg/day for 30 consecutive
days. Gavage volume was adjusted according to the weight of each
rat. The doses and duration were selected as per previous publica-
tions (de Jager et al., 1999; Hossaini et al., 2001; Han et al., 2004a,
2004b; Gong and Han, 2006a, 2006b, 2006c, 2006d; Baek et al.,
2007; Yon et al., 2007). The control group of animals was maintained
and gavaged corn oil vehicle alone. Body weight was recorded before
dosing and euthanasia.
Necropsy. The animals were fasted overnight. Twenty four hours
after the last dose, the animals were weighed and blood samples
were collected from the retro-orbital sinus, under ether anesthesia,
in heparinized tubes. Samples were centrifuged and supernatant
plasma was separated from the clot as soon as possible and stored
at 80 C until analysis. Animals were euthanized, testes, caput/
corpus and cauda epididymides were removed and cleaned from
adhering fat and connective tissues. The weights of testes and cauda
epididymides were recorded in grams. The caput/corpus epididymi-
des were used for sperm count while cauda epididymides from each
animal were used for sperm count and motility and biochemical
parameters. One testis from each rat was used for evaluation of
daily sperm production (DSP).
Sperm count and motility. Cauda epididymides were dissected out,
weighed, immediately minced in 5 ml of physiological saline and
then incubated at 37 C for 30 min to allow spermatozoa to leave
the epididymal tubules. The percentage of motile sperms was
recorded using a phase contrast microscope at a magnication of
400. Total sperm number was determined by using a Neubauer
hemocytometer as previously described (Yokoi et al., 2003). To deter-
mine sperm motility, 100 sperms each were observed in 3 different
elds, and classied into motile and non-motile sperms, and the mo-
tility was expressed as percentage incidence. Aliquots of this sperm
suspension were used to assess the acrosomal integrity, mitochon-
drial membrane potential (m) and 5-nucleotidase activity of
sperm and the others were used for following biochemical studies.
In the same manner, caput/corpus portion of the epididymis was
cut into mall fragments with scissors, and spermcounted as described
for the cauda epididymis.
Daily sperm production (DSP) per testis and sperm transit time in the
epididymis. Daily sperm production was determined in adult rats
as previously described (Blazak et al., 1993). One testis was weighed,
decapsulated and homogenized in 50 ml of ice-cold 0.9% sodium
chloride solution containing 0.01% Triton X-100 using a Polytron
homogenizer (Sharpe et al., 1995). The homogenate was allowed to
settle for 1 min and then was gently mixed, and a 10 ml aliquot was
transferred to a glass vial and stored on ice. After thorough mixing
of each sample, the number of sperm heads (step 19 spermatid
head) in four chambers of Neubauer type hemocytometer was coun-
ted under a light microscope with 40 objective. To calculate daily
sperm production (DSP), the number of spermatids at stage 19 was
divided by 6.1, which is the number of days of the seminiferous
cycle in which these spermatids are present in the seminiferous epi-
thelium. The sperm transit time through the caput/corpus epididymis
or cauda epididymis was calculated by dividing the number of sperm
within each of these regions by the DSP (Robb et al., 1978).
Plasma lactate dehydrogensae (LDH) enzyme and testosterone. Plas-
ma lactate dehydrogensae (LDH) enzyme was estimated as pre-
viously described (Jaiswal et al., 2005). Testosterone was measured
in heparinized plasma using Pathozyme Testosterone ELISA kit.
Briey, standards, specimens and controls were dispensed into ap-
propriate wells, followed by testosterone HRP reagent and anti-
testosterone reagent, before mixing thoroughly and incubating at
37 C for 90 min. Wells were then rinsed with de-ionized water and
substrate solution was dispensed into each well, gently mixed and
incubated for 20 min. The reaction was stopped with the stop
reagent and the absorbance recorded at 450 nm (Chen et al., 1991).
Acrosomal integrity. Epiuorescence microscopy was used to assess
sperm acrosome integrity after staining with Alexa Fluor-488-PNA
(peanut agglutinin) conjugate (Molecular Probes, Eugene, OR, USA).
Alexa Fluor-488-PNA is formed by the coupling of the lectin Trypsin
inhibitor from soybean (SBTI) and Alexa Fluor-488. Excitation of
Fluor-488-PNA at 490 nm results in green uorescence of 519 nm.
The lectin SBTI binds to the acrosin and also inhibits the catalytic ac-
tivity of serine proteases. Briey, sperm samples from each treatment
were smeared on microscopic slides and air-dried. The samples were
135 H.A.A. Aly et al. / Toxicology and Applied Pharmacology 261 (2012) 134141
then xed with 99% methanol and kept at room temperature until
staining. For staining, slides were incubated with 10 g/ml Alexa
Fluor-488-PNA at 37 C for 30 min, washed with PBS and then ana-
lyzed under epiuorescence microscope (Zeiss, Axiophot, Germany)
using an appropriate lter. In each replicate, about 100 sperms were
counted to determine the proportion of sperm with intact acrosomes.
While spermatozoa displaying intensively and moderately bright
uorescence in the acrosomal region were considered as intact acro-
somes, spermatozoa displaying weak, patchy or no uorescence in
the acrosomal region were considered as damaged acrosomes. In
each replicate, about 100 sperms were counted to determine the pro-
portion of sperm with intact acrosomes (Varisli et al., 2009).
Mitochondrial membrane potential (m). The m of the sperm
after treatment with nonylphenol was measured using a uorescent
dye rhodamine 123 (Rh123), a cell permeable cationic dye that can
accumulate in energized mitochondria based on the highly negative
m (Hong and Liu, 2004). Depolarization of m results in leakage
of Rh123 from mitochondria, and a decrease in intracellular uo-
rescence. The sperm samples in different groups were diluted
(~5010
6
sperm/ml) and 300 l of the diluted samples were loaded
with 10 M of Rh123 at 37 C in the dark for 30 min. After being load-
ed with Rh123, sperm samples were washed and the uorescence
intensity was assessed using a spectrouorimeter with excitation
wavelength at 488 nm and emission wavelength at 530 nm. The
m was expressed as uorescence intensity of Rh123.
5-Nucleotidase assay. The activity of 5-nucleotidase was deter-
mined by measuring the rate of release of inorganic phosphate from
adenosine 5-monophosphate according to the method of Heppel
and Hilmoe (1955) with minor modication in substrate concentra-
tion (Chakrabarti et al., 2003). Briey, the control and nonylphenol-
treated rat sperm pellets (~20 million sperm/ml) were collected by
centrifugation at 3000 g at 37 C, washed twice in 0.9% saline, and
suspended in 0.1 mol/l TrisHCl buffer (pH 8.5) with each reaction
tube containing 3 mmol/l cAMP and 50 mmol/l MgCl
2
dissolved in
0.1 mol/l TrisHCl buffer. The tubes were incubated at 37 C for
30 min followed by addition of 0.5 ml 20% trichloroacetic acid to
terminate the reaction. The mixture was then centrifuged at 10
000 g at 4 C. The supernatant was evaluated for phosphate estima-
tion (Chen et al., 1956). Because the activity of 5-nucleotidase is
directly proportional to absorbance, the comparative activity was
expressed in terms of absorbance at 820 nm.
Biochemical analysis. The cauda epididymal sperm suspension
was centrifuged at 800g for 10 min at 4 C and the pellet was
resuspended in 0.01 M TrisHCl buffer (pH 7.4). The sperm suspen-
sion was homogenized with the help of a glass-Teon homogenizer
and the aliquots of the homogenate were suitably processed for the
assessment of following biochemical parameters. Protein concentra-
tions were determined using a BCA kit (Pierce, Rockford, USA) that
employed bovine serum albumin as a standard.
Hydrogen peroxide generation. Hydrogen peroxide generation was
assayed by the method of Pick and Keisari (1981). Briey, the incuba-
tion mixture contained 1.641 ml phosphate buffer (50 mM, pH 7.6),
54 l horseradish peroxidase (8.5 units/ml), 30 l of 0.28 nM phenol
red, 165 l of 5.5 nM dextrose, and 600 l of enzyme source, incubat-
ed at 35 C for 30 min. The reaction was terminated by the addition of
60 l of 10 N sodium hydroxide. The absorbance was read at 610 nm
against a reagent blank on a Spectrophotometer. For standard curve,
known amounts of hydrogen peroxide and all the above reagents
except enzyme source were incubated for 30 min at 35 C, followed
by addition of 60 l of 10 N sodium hydroxide, and reading of optical
density at 610 nm. The quantity of H
2
O
2
produced was expressed as
nmol of H
2
O
2
generated/min/mg protein at 35 C.
Lipid peroxidation (LPO). Malondialdehyde (MDA), formed as an end-
product of the peroxidation of lipids, served as an index of the inten-
sity of oxidative stress. MDA reacts with thiobarbituric acid to
generate a colored product that can be measured optically at
532 nm. A break down product of LPO, thiobarbituric acid reactive
substance was measured by the method of Buege and Aust (1976).
Briey, the stock solution contained equal volumes of trichloroacetic
acid 15% (w/v) in 0.25 N HCl and 2-thiobarbituric acid 0.37% (w/v)
in 0.25 N HCl. One volume of the test sample (sperm suspension)
and two volumes of stock reagent were mixed in a screw-capped cen-
trifuge tube, vortexed and heated for 15 min on a boiling water bath.
After cooling on ice the precipitate was removed by centrifugation at
1000 g for 15 min and absorbance of the supernatant was measured
at 532 nm against blank containing all the reagents except test
sample. The value is expressed as mol of malondialdehyde equiva-
lent formed/min per mg protein.
Assessment of enzymic antioxidants. Superoxide dismutase (SOD) was
assayed by the method of Marklund and Marklund (1974). Briey, the
assay mixture contained 2.4 ml of 50 mM TrisHCl buffer containing
1 mM EDTA (pH 7.6), 300 l of 0.2 mM pyrogallol and 300 l enzyme
source. The decrease in absorbance was measured immediately at
420 nm against blank at 10 s intervals for 3 min on a spectrophotom-
eter. The activity of enzyme was expressed in nmol pyrogallol oxi-
dized/min/mg protein. Catalase (CAT) was assayed as previously
mentioned (Claiborne, 1985). Briey, the assay mixture contained
2.40 ml of phosphate buffer (50 mM, pH 7.0), 10 l of 19 mM hydro-
gen peroxide and 50 l enzyme source (sperm suspension). The de-
crease in absorbance was measured immediately at 240 nm against
blank at 10 s intervals for 3 min on a spectrophotometer. The activity
of enzyme was expressed in mol of hydrogen peroxide consumed/
min/mg protein. Glutathione peroxidase (GPx) was assayed by the
method of Paglia and Valentine (1967). Briey, the assay mixture
contained 1.59 ml of phosphate buffer (100 mM, pH 7.6), 100 l of
10 mM EDTA, 100 l of sodium azide, 50 l of glutathione reductase,
100 l of reduced glutathione, 100 l of 200 mM NADPH, 10 l of
hydrogen peroxide and 10 l enzyme source. The oxidation of
NADPH was measured immediately at 340 nm against blank at 10 s
intervals for 3 min on a spectrophotometer. The activity of enzyme
was expressed in nmol of NADPH oxidized/min/mg protein.
Statistical analysis. Differences between obtained values (mean
SD, n=6) were compared by one way analysis of variance (ANOVA)
followed by the TukeyKramer multiple comparison test. A P value
less than 0.05 was taken as a criterion for a statistically signicant
difference.
Results
Body weight and organ weights
No signicant differences in body weight occurred among the
groups during the experimental period, but terminal body weight of
animals from the high-dose group was reduced by approximately
4% from the control value (Table 1). NP caused a signicant depres-
sion in spermatogenesis due to cytotoxicity, which resulted in death
of immature germ cells in the seminiferous epithelium. As a result,
the total number of cells available for spermatogenesis reduced and
this led to a signicant decrease in absolute weights of testes
(Pb0.05, Pb0.001 and Pb0.001) and cauda epididymides (Pb0.05,
Pb0.01 and Pb0.01) of animals treated with 100, 200 and 300 mg/
kg/day respectively as compared to the corresponding control
(Table 1).
136 H.A.A. Aly et al. / Toxicology and Applied Pharmacology 261 (2012) 134141
Epididymal sperm characteristics
Sperm count in caput/corpus (Pb0.01, Pb0.001 and Pb0.001 re-
spectively) and cauda epididymides (Pb0.05, Pb0.001 and Pb0.001
respectively) and sperm motility (Pb0.05, Pb0.01 and Pb0.001
respectively) were signicantly decreased in animals treated with
100, 200 and 300 mg/kg/day of NP in comparison to corresponding
control groups (Table 1).
Daily sperm production and sperm transit time
Daily sperm production was signicantly decreased (Pb0.05,
Pb0.01 and Pb0.001 respectively) in animals treated with 100, 200
and 200 mg/kg/day of NP in a dose dependent- manner in relation
to the control group (Table 1). Animals treated with 200 and
300 mg/kg/day of NP showed a signicant reduction (Pb0.01) in
sperm transit time (acceleration of transit time) in caput/corpus
epididymis as compared to the corresponding control. A dose of
100 mg/kg/day did not show any signicant change. Sperm transit
time in cauda epididymides of animals treated with 100 and
200 mg/kg/day of NP did not show any signicant change, while
300 mg/kg/day of NP showed a signicant reduction (Pb0.05) as com-
pared to the untreated animals (Table 1).
Measurement of plasma LDH and testosterone
Treatment with 100, 200 and 300 mg/kg/day of NP showed a sig-
nicant increase in plasma LDH activity (24.72%, 31.57% and 54.64%
respectively) (Fig. 1A), while plasma testosterone content was signif-
icantly decreased (6.42%, 9.22% and 12.57% respectively) in a dose-
related manner as compared to the corresponding controls (Fig. 1B).
Sperm acrosomal integrity
Fig. 2A shows the acrosomal status of rat sperms following treat-
ment with 100, 200 and 300 mg/kg/day of NP. Percent acrosomal
integrity of sperms in all NP-treated animals showed a signicant
reduction (Pb0.01, Pb0.001 and Pb0.001 respectively) as compared
to the corresponding control.
Mitochondrial membrane potential (m)
Mitochondrial membrane potential of rat sperm after treatment
with various doses of NP was shown in Fig. 2B. m was signicantly
decreased (Pb0.05, Pb0.001 and Pb0.001 respectively) after treat-
ment with 100, 200 and 300 mg/kg/day of NP as compared to the
corresponding untreated animals.
5-Nucleotidase
As compared to the corresponding control, the release of inorganic
phosphate was signicantly lower (Pb0.05, Pb0.01 and Pb0.001
respectively) in all the NP-treated groups (Fig. 3) as reected by re-
duced absorbance of phosphomolybdate complex, which is directly
proportional to the activity of 5-nucleotidase. The reduction in the
activity showed a dose-related pattern.
Oxidative status
NP at doses of 100, 200 and 300 mg/kg/day signicantly increased
H
2
O
2
production (20.98%, 30.8% and 41.29% respectively) and LPO
(15.97%, 24.71% and 31.94% respectively) in a dose-related manner
in rat epididymal sperm as compared to the corresponding controls
(Figs. 4A and B respectively).
Antioxidant status
Treatment with 100, 200 and 300 mg/kg/day of NP showed a sig-
nicant reduction in SOD (13.8%, 29.27% and 38.2% respectively)
(Fig. 5), CAT (15.46%, 33.33% and 49.28% respectively) and GPx
(9.16%, 19.6% and 28.28% respectively) activities in rat epididymal
Table 1
Effect of nonylpenol on body weight, absolute weights of testes and epididymides and sperm characteristics.
Parameter Control Nonylphenol (mg/kg b.wt.)
100 mg 200 mg 300 mg
Body weight (g) 195.54.64 192.334.13 190.334.76 188.55
Absolute weights (g)
Testes 2.570.2 2.280.12

1.950.14

1.670.22

Cauda epididymides 0.450.03 0.390.008

0.370.04

0.360.05

Epididymal spermatozoa
Cauda sperm count (10
6
/rat) 65.334.32 58.52.43

54.673.33

46.174.83

Sperm motility % 813.74 73.52.88

70.175.23

60.334.32

Caput/corpus sperm count (10

6
/rat) 34.833.19 28.831.17

26.332.07

23.53.27

DSP (10
6
/testis/day) 26.672.34 241.1

231.41

20.671.37

Sperm transit time in the cauda (days) 2.460.17 2.440.1 2.380.07 2.230.17

Sperm transit time in the caput/corpus (days) 1.310.05 1.20.04 1.150.1

1.130.09

Data are expressed as meanS.D. (n=6). The symbol represents statistical signicance (ANOVA) from control: *pb0.05; **pb0.01; ***pb0.001. DSP: daily sperm production.
Fig. 1. Effect of nonylphenol on plasma LDH (A) and testosterone (B) in adult rat.
Values are expressed as meanSD, n=6. The symbol represents statistical signi-
cance (ANOVA) from control: *, Pb0.05. **, Pb0.01, ***, Pb0.001.
137 H.A.A. Aly et al. / Toxicology and Applied Pharmacology 261 (2012) 134141
sperm in comparison to the corresponding controls (Figs. 6A and B
respectively).
Discussion
Despite the wide distribution of NP in the environment and its
great hazard to the reproductive health of human and animals, the
detailed mechanism of NP toxicity has not been fully elucidated
(Gong et al., 2009). In this study, the body weight of rats did not
show any signicant change due to treatment with NP indicating
that the general metabolic condition of the animals was within nor-
mal range. According to Clegg et al. (2001), among other parameters
that can be utilized in evaluation of risks from toxic effects on male
reproductive apparatus, is the determination of absolute weights of
testes and epididymides. Organ weight is a fundamental benchmark
for the toxicological studies (Yavasoglu et al., 2008). The weight of
the testes and epididymides is basically dependent on the mass of
the differentiated spermatogenic cells and spermatozoa (Takahashi
and Oishi, 2001). It is reasonable to consider that organ weights
reduction is not always related to body weight reduction (Clegg
et al., 2001). The reduction in testes and epididymides weights in
the current study may be due to inhibition of spermatogenesis, there-
fore decreased daily sperm production and decreased number of
sperms in the epididymal lumen. The reduction in spermatogenesis
may be due to inhibition of steroidogenic enzyme activity
(Takahashi and Oishi, 2001). Previous studies reported signicant
decrease in absolute weight of the epididymis in rat exposed to 3 or
15 or 75 mg/kg/day of NP (Hossaini et al., 2001).
Our data showed a signicant reduction in the sperm number in
the caput/corpus and cauda epididymis and decreased sperm motility
in all doses. The reduction in sperm count was consistent with
changes in epididymal and testes weights. These changes in sperm
count may be due to an adverse effect of NP on spermatogenesis.
The harmful effect of NP on spermatogenesis could be ascribed to
either the reduction in plasma testosterone level (Gong and Han,
2006a) or NP induced LPO and H
2
O
2
production (Chitra and Mathur,
2004; Gong and Han, 2006b). In order to better understand the actual
effects of any chemical exposure on sperm viability, it is important to
analyze the effects of exposure of the chemical on vital structures of
the spermatozoa which play important roles in sperm function.
After sperm production in testes, these cells are transported, stored
and protected by the epididymis (Sullivan et al., 2007). During transit
through the epididymal sperm undergo maturation and acquire pro-
gressive motility and ability to fertilize ova (Cornwall, 2009). In most
cases; motility should closely correlate with m.
The reduction in sperm count and motility was associated with
acceleration of transit time (reduced sperm transit time), which
may be associated to decreased plasma testosterone level. Androgens
Fig. 2. Effect of nonylphenol on acrosomal integrity (A) and mitochondrial membrane
potential (m) (B) of epididymal rat sperm. Values are expressed as meanSD,
n=6. The symbol represents statistical signicance (ANOVA) from control: *,
Pb0.05. **, Pb0.01, ***, Pb0.001.
Fig. 3. Effect of nonylphenol on 5-nucleotidase activity in epididymal rat sperm. Values
are expressed as meanSD, n=6. The symbol represents statistical signicance
(ANOVA) from control: *, Pb0.05. **, Pb0.01, ***, Pb0.001.
Fig. 4. Effect of nonylphenol on H
2
O
2
production (A) and LPO (B) in epididymal rat
sperm. Values are expressed as meanSD, n=6. The symbol represents statistical sig-
nicance (ANOVA) from control: *, Pb0.05. **, Pb0.01, ***, Pb0.001.
Fig. 5. Effect of nonylphenol on SOD activity in epididymal rat sperm. Values are
expressed as meanSD, n=6. The symbol represents statistical signicance
(ANOVA) from control: *, Pb0.05. **, Pb0.01, ***, Pb0.001.SOD: superoxide dismutase.
138 H.A.A. Aly et al. / Toxicology and Applied Pharmacology 261 (2012) 134141
act on the spermtransit time by controlling the luminal uid viscosity
and epididymal duct contractility, ensuring an optimal transit rate
(Sujarit and Pholpramool, 1985). Studies revealed that various toxic
compounds accelerated sperm transit time and this was associated
with low concentrations of testosterone (Sullivan et al., 2007), as
seen in the current study. The caput secretes 75% of the proteins
that participate in epididymis sperm maturation (Arrotia et al.,
2004). The reduction of sperm transit time in both the caput/corpus
and cauda epididymis can strongly harm the normal maturation of
sperm in the epididymis, interfere with sperm quality, and conse-
quently, diminish the fertility index (Garcia et al., 2011). The number
of spermatids present in the testis and the total DSP are important
indicators of male fertility potential (Fernandez et al., 2008).
Decreased daily testicular sperm production could be a direct impact
of decreased testosterone concentration. Further, decrease in DSP
is correlated with decrease in the testis weight indicating that
the germ cell death or cell loss from the epithelium may be due to
tubular atrophy which is a main reason for decreased testis weight
(Narayana, 2008).
LDH, as the main enzyme for glucose metabolism in testis sper-
matogenic cells, its activity is related to the mature of seminiferous
epithelium and sperm activity and could be used as an important in-
dicator of spermatogenesis (Qiong et al., 2011). The activity of LDH is
a biomarker of cell integrity (Aly and Khafagy, 2011). Elevated plasma
levels of LDH have been markedly correlated with chronic oxidative
stress, cardiotoxicity, testis carcinoma and many inammatory condi-
tions (Jaiswal et al., 2005). The increase in activity of plasma LDH
observed in treated animals suggests that NP exposure causes deteri-
oration of germinal epithelium (Pant et al., 2004). Testosterone has
been shown to be essential for spermatogenesis completion, because
it stimulates the conversion of round spermatids into elongated sper-
matids between stage VII and stage VIII of the spermatogenetic cycle.
Androgen deciency disturbs the spermiation process (Saito et al.,
2000) by altering spermatid-Sertoli cell junctions, which results in
premature detachment of round spermatids from Sertoli cells and
seminal epithelium (Beardsley and O'Donnell, 2003), along with apo-
ptosis and activation of caspases (Tesarik et al., 2002). Administration
of NP resulted in decreased plasma testosterone level, suggesting that
NP has an inhibitory effect on testosterone production. The decrease
in plasma testosterone concentration may be due to decrease in plas-
ma LH concentration through negative feedback inhibition in the
anterior pituitary, thus altering the normal secretion of testosterone
by the testis (Fernandez et al., 2008). NP, a weak estrogenic and
endocrine disrupting compound, can regulate the secretion of gonad-
otrophins and interfering in the normal balance of hypothalamus-
pituitary-gonad axis (Lu et al., 2007). This environmental contami-
nant has toxic effects on male reproductive system (Murono and
Derk, 2002). Han et al. (2004a, 2004b) reported that NP signicantly
decreased serum testosterone at a dose of 250 mg/kg/day. On the
other hand, Wu et al. (2010) reported that NP has a differential effect
on testosterone synthesis. The mechanism by which NP reduces the
level of plasma testosterone needs further investigation.
Among the various viability characteristics of the rat sperm which
include motility, m, LDH, is the acrosome integrity. NP reduced the
acrosome integrity which may be due to the elevated level of ROS.
ROS are important mediators of normal function and are involved in
the induction and development of sperm hyperactivation, capacita-
tion and acrosome reaction (Aitken, 1995). Excessive production of
ROS results in alterations of mitochondrial oxidative phosphorylation,
depletion of ATP, increase in intracellular calcium, and activation of
degenerative enzymes, leading to loss of cellular function/integrity
and, ultimately, germ cell apoptosis (Salmasi et al., 2005). Moreover,
elevated level of ROS leads to inactivation of glycolytic enzymes,
damage to the acrosomal membranes (Alvarez and Storey, 1984)
and DNA oxidation, which render the sperm cell unable to fertilize
the oocyte, or produce a viable pregnancy (Gil-Guzman et al., 2001).
Mitochondria have been described as the sensor of oxidative
stress (Gao et al., 2003; Das et al., 2009). Alteration in the m is
known to be a major cause of precipitation of apoptosis in many cell
types (Luo et al., 1998). As was expected, signicant dissipation of
m was observed in the current study, indicative of mitochondrial
dysfunction as a result of increased H
2
O
2
production. A recent study
by Uguz et al. (2009) on rat sperms is consistent with the current
ndings involving acrosome integrity and m.
The plasma membrane plays vital roles in the process of sperm
migration and fertilization (Flesch and Gadella, 2000). In the current
study, NP was tested to execute its effect by structural and functional
modulation of the sperm plasma membrane (Sharma et al., 1996).
The damage to the membrane architecture was evidenced by a
marked decrease in 5-nucleotidase activity which may be due to
elevated level of ROS. The 5-nucleotidase belongs to a large group
of surface-located ectoenzyme, which is anchored to the plasma
membrane via GPI and C-terminus (Starter, 2006).
As has been previously shown, oxidative stress (enhanced ROS
and LPO as well as altered antioxidant enzymes) is considered as
one of the underlying mechanisms of toxicity (Ikediobi et al.,
2004) and antioxidants have been successful in abating some of
these deleterious effects (Yadav and Khandelwal, 2008). Oxidative
stress is a result of the imbalance between ROS and antioxidants in
the body, which can lead to spermatozoa damage, deformity and
eventually male infertility (Agarwal et al., 2008). In the current
study, the level of LPO and H
2
O
2
were increased in the epididymal
sperm of NP-treated rats. It has been reported that ROS such as
H
2
O
2
appear to be key agents causing cytotoxicity in spermatozoa
to produce oxidative stress by decreasing the enzymatic defenses
(Rao and Gangadharan, 2008). The degenerative pathway of mem-
brane components (LPO), reported here, may be the result of in-
creased production of free radicals and/or a decrease in antioxidant
status (Sudharsan et al., 2005). The sperm cell membrane contains
high levels of polyunsarurated fatty acids that bestow considerable
uidity, necessary to allow the cell to fuse the oocyte (Sikka,
2004). This, however, renders the cell more susceptible to ROS at-
tack and generation of lipid peroxides (de Lamirande and Gagnon,
1992). LPO in spermatozoa results in decreased membrane uidity
Fig. 6. Effect of nonylphenol on CAT (A) and GPx (B) activities in epididymal rat sperm.
Values are expressed as meanSD, n=6. The symbol represents statistical signi-
cance (ANOVA) from control: *, Pb0.05. **, Pb0.01, ***, Pb0.001.CAT: catalse; GPx:
glutathione peroxidase.
139 H.A.A. Aly et al. / Toxicology and Applied Pharmacology 261 (2012) 134141
and sperm motility, and a reduced capacity for fertilization (Sener et
al., 2003; Selvakumar et al., 2006). The epididymis contains an elab-
orate array of antioxidant enzymes and free radical scavengers to
ensure that the twin spermatogenic and steroidogenic functions of
the testes are not impacted by oxidative stress (Aitken and Roman,
2008). The antioxidant defense system protects the sperm during
its transit through caput to cauda region of epididymis and facilitates
their maturation process (Vernet et al., 2004). The present work also
shows that the change in LPO is accompanied by concomitant de-
crease in the activities of antioxidant enzymes namely SOD, CAT
and GPx. SOD acts as the rst line of defense against deleterious ef-
fects of oxyradicals in cells by catalyzing the dismutation of superox-
ide radicals to H
2
O
2
and molecular oxygen (Johnson and Giulivi,
2005). The decreased activities of SOD in NP-treated rats may lead
to the continuous production of superoxide anions which is known
to inactivate GPx. Similarly, when GPx fails to eliminate H
2
O
2
from
the cell, the accumulated H
2
O
2
has been shown to cause inactivation
of SOD. Thus, the balance of this enzyme system is essential to
dispose the superoxide anion and peroxides generated in the epidid-
ymal sperms (Selvakumar et al., 2005). The reduction in the activi-
ties of these enzymes and increase in LPO and H
2
O
2
could reect
the adverse effects of NP on this nely balanced antioxidant system.
In addition, SOD plays an important role in rat testicular maturation
and spermatogenesis (Jow et al., 1993). Any alteration in the activity
of testicular SOD may lead to growth arrest and impaired function of
the sperms. GPx and CAT have been considered the primary scaven-
gers of H
2
O
2
(Peltola et al., 1992; Zini and Schlegel, 1996). GPx is the
most important peroxidase for the detoxication of hydroperoxides.
It catalyzes the GSH-dependent reduction in hydroperoxides and
hydrogen peroxide. In the presence of inadequate GPx activity in
rat sperms to degrade H
2
O
2
, more H
2
O
2
would be converted to
toxic hydroxyl radicals that may contribute to severe oxidative dam-
age to sperm mitochondrial membranes. The antioxidant enzyme
CAT protects SOD against inactivation by hydrogen peroxide. Recip-
rocally, the SOD protects CAT against inhibition by superoxide anion
(Selvakumar et al., 2004). Thus, the balance of this enzyme system
may be essential to eliminate superoxide anion and peroxides gener-
ated in epididymal spermatozoa after NP treatment. The reduction in
CAT activity reects the inability of spermatozoa to eliminate H
2
O
2
produced by NP (Chitra et al., 2002).
In conclusion, these results demonstrate that exposure of NP elicit
decrease in testes and cauda epididymides weights, decreased sperm
count in cauda and caput/corpus epididymides, reduced sperm motil-
ity, decreased daily spermproduction and spermtransit time in cauda
and caput/corpus epididymides and reduced plasma LDH and testos-
terone. In addition it demonstrates reduction in acrosomal integrity,
m and 5-nucleotidase activity of cauda epididymal sperm. The re-
duction in the activities of antioxidant enzymes and increase in H
2
O
2
and LPO reveal that NP disrupts the prooxidant and antioxidant
balance. This leads to excessive generation of ROS, thereby causing
oxidative stress in epididymal sperm of rat. Moreover, the reduction
in sperm transit time may affect sperm quality and fertility potential.
This concludes that NP induces testicular toxicity and impairs sperm
functions, at least partly, by induction of oxidative stress in epididy-
mal sperm of rat.
Conict of interest statement
The authors declare that no conict of interest.
References
Agarwal, A., Makker, K., Sharma, R., 2008. Clinical relevance of oxidative stress in male
factor infertility: an update. Am. J. Reprod. Immunol. 59, 211.
Aitken, R.J., 1995. Free radicals, lipid peroxidation and sperm function. Reprod. Fertil.
Dev. 7, 659668.
Aitken, R.J., Roman, S.D., 2008. Antioxidant systems and oxidative stress in the testes.
Oxid. Med. Cell. Longev. 1, 1524.
Alvarez, J.G., Storey, B.T., 1984. Assessment of cell damage caused by spontaneous lipid
peroxidation in rabbit spermatozoa. Biol. Reprod. 30, 323331.
Aly, H.A., Khafagy, R.M., 2011. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced
cytotoxicity accompanied by oxidative stress in rat Sertoli cells: possible role of
mitochondrial fractions of Sertoli cells. Toxicol. Appl. Pharmacol. 252, 273280.
Arrotia, K.F., Joazeiro, P.P., Yamada, A.T., Tanaka, H., Nishimune, Y., Pereira, L.A., 2004.
Identication and characterization of an antigen recognized by monoclonal anti-
body TRA 54 in mouse epididymis and vas deferens. J. Androl. 25, 914921.
Baek, I.J., Yon, J.M., Lee, S.R., Jin, Y., Kim, M.R., Ahn, B., Hong, J.T., Choo, Y.K., Lee, B.J., Yun,
Y.W., Nam, S.Y., 2007. Effects of endocrine disrupting chemicals on expression of
phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes. J. Vet.
Sci. 8, 213218.
Beardsley, A., O'Donnell, L., 2003. Characterization of normal spermiation and spermiation
failure induced by hormone suppression in adult rats. Biol. Reprod. 68, 12991307.
Blazak, W.F., Treinen, K.A., Juniewicz, P.E., 1993. Application of testicular sperm
head counts in the assessment of male reproductive toxicity. Meth. Toxicol.
3A, 8694.
Buege, J.A., Aust, S.D., 1976. Lactoperoxidase catalyzed lipid peroxidation of micro-
somal and articial membranes. Biochim. Biophys. Acta 444, 192201.
Chakrabarti, K., Pal, S., Bhattacharyya, A.K., 2003. Sperm immobilization activity of
Allium sativum L. and other plant extracts. Asian J. Androl. 5, 131135.
Chen, P.S., Toribara, T.Y., Warner, H., 1956. Micro determination of phosphorous. Anal.
Chem. 28, 17561758.
Chen, A., Booksteinm, J.J., Meldrum, D.R., 1991. Diagnosis of the of the testosterone
secreting adrenal adenoma by selective venous catheterisation. Fertil. Steril. 55,
12021203.
Cheng, C.Y., Wu, C.Y., Wang, C.H., Ding, W.H., 2006. Determination and distribution
characteristics of degradation products of nonylphenol polyethoxylates in the riv-
ers of Taiwan. Chemosphere 65, 22752281.
Chitra, K.C., Mather, P.P., 2004. Vitamin E prevent nonylphenol-induced oxidative
stress in testis of rats. Indian J. Exp. Biol. 42, 220223.
Chitra, K.C., Mathur, P.P., 2004. Vitamin E prevents nonylphenol-induced oxidative
stress in testis of rats. Indian J. Exp. Biol. 42, 220223.
Chitra, K.C., Latchoumycandane, C., Mathur, P.P., 2002. Effect of nonylphenol on the
antioxidant system in epididymal sperm of rats. Arch. Toxicol. 76, 545551.
Claiborne, A., 1985. In: Greenwald, R. (Ed.), Handbook of Methods for Oxygen Radical
Research. CRC Press, Florida, pp. 283284.
Clegg, E.D., Perreault, D., Klinefelter, G.R., 2001. Assessment of male reproductive tox-
icity. In: Hayes, A.W. (Ed.), Principles and Methods of Toxicology. Taylor & Francis,
Philadelphia, PA, pp. 12631300.
Cornwall, G.A., 2009. New insights into epididymal biology and function. Hum. Reprod.
Update 15, 213227.
Cuasnicu, P.S., Cohen, D.J., Ellerman, D.A., Busso, D., Da Ros, V.G., Morgenfeld, M.M.,
2002. Changes in specic sperm proteins during epididymal maturation. In:
Robaire, B., Hinton, B.T. (Eds.), The Epididymis from Molecules to Clinical Prac-
tice. Kluwer AcademicPlenum Publisher, New York, pp. 389403.
Das, J., Ghosh, J., Manna, P., Sinha, M., Sil, P.C., 2009. Taurine protects rat testes against
NaAsO(2)-induced oxidative stress and apoptosis via mitochondrial dependent
and independent pathways. Toxicol. Lett. 187, 201210.
de Jager, C., Bornman, M.S., van der Horst, G., 1999. The effect of p-nonylphenol, an
environmental toxicant with oestrogenic properties, on fertility potential in adult
male rats. Andrologia 31, 99106.
de Lamirande, E., Gagnon, C., 1992. Reactive oxygen species and human spermatozoa. I.
Effects on the motility of intact spermatozoa and on sperm axonemes. J. Androl. 13,
368378.
Fernandez, C.D., Porto, E.M., Arena, A.C., Kempinas Wde, G., 2008. Effects of altered
epididymal sperm transit time on sperm quality. Int. J. Androl. 31, 427437.
Flesch, F.M., Gadella, B.M., 2000. Dynamics of the mammalian sperm plasma mem-
brane in the process of fertilization. Biochim. Biophys. Acta 1469, 197235.
Franca, L.R., Avelar, G.F., Almeida, F.F.L., 2005. Spermatogenesis and transit through the
epididymis in mammals with emphasis on pigs. Theriogenology 63, 300318.
Gao, H.B., Tong, M.H., Hu, Y.Q., You, H.Y., Guo, Q.S., Ge, R.S., Hardy, M.P., 2003. Mechanisms
of glucocorticoid-induced Leydig cell apoptosis. Mol. Cell. Endocrinol. 199, 153163.
Garcia, P.V., Arrotia, K.F., Joazeiro, P.P., de Ftima Paccola Mesquita, S., de Grava
Kempinas, W., Pereira, L.A., 2011. Orchidopexy restores morphometric-
stereologic changes in the caput epididymis and daily sperm production in cryp-
torchidic mice, although sperm transit time and fertility parameters remain
impaired. Fertil. Steril. 96, 739744.
Gil-Guzman, E., Ollero, M., Lopez, M.C., Sharma, R.K., Alvarez, J.G., Thomas, A.J., Agarwal,
A., 2001. Differential production of reactive oxygen species by subsets of human
spermatozoa at different stages of maturation. Hum. Reprod. 16, 19221930.
Gong, Y., Han, X.D., 2006a. Effect of nonylphenol on steroidogenesis of rat Leydig cells.
J. Environ. Sci. Health B 41, 705715.
Gong, Y., Han, X.D., 2006b. Nonylphenol-induced oxidative stress and cytotoxicity in
testicular Sertoli cells. Reprod. Toxicol. 22, 623630.
Gong, Y., Han, X.D., 2006c. Effect of nonylphenol on steroidogenesis of rat Leydig cells.
J. Environ. Sci. Health B 41, 705715.
Gong, Y., Han, X.D., 2006d. Nonylphenol-induced oxidative stress and cytotoxicity in
testicular Sertoli cells. Reprod. Toxicol. 22, 623630.
Gong, Y., Pan, X., Huang, Y., Gao, Z., Yu, H., Han, X., 2008. NP-induced biophysical and
biochemical alterations of rat testicular Sertoli cell membranes related to disturbed
intracellular Ca(2+) homeostasis. Toxicol. Lett. 183, 1020.
Gong, Y., Wu, J., Huang, Y., Shen, S., Han, X., 2009. Nonylphenol induces apoptosis in rat
testicular Sertoli cells via endoplasmic reticulum stress. Toxicol. Lett. 186, 8495.
140 H.A.A. Aly et al. / Toxicology and Applied Pharmacology 261 (2012) 134141
Han, X.D., Tu, Z.G., Gong, Y., Shen, S.N., Wang, X.Y., Kang, L.N., Hou, Y.Y., Chen, J.X.,
2004a. The toxic effects of nonylphenol on the reproductive system of male rats.
Reprod. Toxicol. 19, 215221.
Han, X.D., Tu, Z.G., Gong, Y., Shen, S.N., Wang, X.Y., Kang, L.N., Hou, Y.Y., Chen, J.X.,
2004b. The toxic effects of nonylphenol on the reproductive system of male rats.
Reprod. Toxicol. 19, 215221.
Heppel, L.A., Hilmoe, R.J., 1955. In: Colowick, S.P., Kaplan, N.O. (Eds.), 5 nucleotidase
of seminal plasma. : Methods Enzymol., 2. Academic Press, NewYork, NY, pp. 547549.
Hong, H., Liu, G.Q., 2004. Protection against hydrogen peroxide-induced cytotoxicity in
PC12 cells by scutellarin. Life Sci. 74, 29592973.
Hossaini, A., Dalgaard, M., Vinggaard, A.M., Frandsen, H., Larsen, J.J., 2001. In utero
reproductive study in rats exposed to nonylphenol. Reprod. Toxicol. 15, 537543.
Ikediobi, C.O., Badisa, V.L., Ayuk-Takem, L.T., Latinwo, L.M., West, J., 2004. Response of
antioxidant enzymes and redox metabolites to cadmium-induced oxidative stress
in CRL-1439 normal rat liver cells. Int. J. Mol. Med. 14, 8792.
Jacobsen, R., Bostofte, E., Engholm, G., Hansen, J., Olsen, J.H., Skakkebaek, N.E., Moller,
H., 2000. Risk of testicular cancer in men with abnormal semen characteristics:
cohort study. BMJ 321, 789792.
Jaiswal, A., Parihar, V.K., Sudheer Kumar, M., Manjula, S.D., Krishnanand, B.R., Shanbhag, R.,
Unnikrishnan, M.K., 2005. 5-Aminosalicylic acid reverses endosulfan-induced testicu-
lar toxicity in male rats. Mutat. Res. 585, 5059.
Johnson, F., Giulivi, C., 2005. Superoxide dismutases and their impact upon human
health. Mol. Aspects Med. 26, 340352.
Jorgensen, N., Andersen, A.G., Eustache, F., Irvine, D.S., Suominen, J., Petersen, J.H.,
Andersen, A.N., Auger, J., Cawood, E.H., Horte, A., Jensen, T.K., Jouannet, P.,
Keiding, N., Vierula, M., Toppari, J., Skakkebaek, N.E., 2001. Regional differences
in semen quality in Europe. Hum. Reprod. 16, 10121019.
Jow, W.W., Schleger, P.N., Chichon, Z., Phillips, D., Goldstein, M., Bardin, W., 1993. Iden-
tication and localization of copperzinc superoxide dismutase gene expression in
rat testicular development. J. Androl. 14, 439447.
Klinefelter, G.R., 2002. Actions of toxicants on the structure and function of the epidid-
ymis. In: Robaire, B., Hinton, B.T. (Eds.), The Epididymis from Molecules to
Clinical Practice. Kluwer AcademicPlenum Publisher, New York, pp. 353369.
Li, D., Hu, Y., Shen, X., Dai, X., Han, X., 2010. Combined effects of two environmental
endocrine disruptors nonyl phenol and di-n-butyl phthalate on rat Sertoli cells in
vitro. Reprod. Toxicol. 30, 438445.
Lu, Y.Y., Chen, M.L., Sung, F.C., Wang, P.S., Mao, I.F., 2007. Daily intake of 4-nonylphenol
in Taiwanese. Environ. Int. 33, 903910.
Luo, X., Budihardjo, I., Zou, H., Slaughter, C., Wang, X., 1998. Bid, a Bcl2 interacting pro-
tein, mediates cytochrome c release from mitochondria in response to activation of
cell surface death receptors. Cell 94, 481490.
Ma, X.M., Lightman, S.L., 1998. The arginine vasopressin and corticotrophinreleasing
hormone gene transcription responses to varied frequencies of repeated stress in
rats. J. Physiol. 510, 605614.
Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in auto-
oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J.
Biochem. 47, 469474.
McClusky, L.M., de Jager, C., Bornman, M.S., 2007. Stage-related increase in the propor-
tion of apoptotic germ cells and altered frequencies of stages in the spermatogenic
cycle following gestational, lactational, and direct exposure of male rats to p-
nonylphenol. Toxicol. Sci. 95, 249256.
Murono, E.P., Derk, R.C., 2002. Exposure to octylphenol increases basal testosterone
formation by cultured adult rat Leydig cells. J. Steroid Biochem. Mol. Biol. 81,
181189.
Narayana, K., 2008. An aminoglycoside antibiotic gentamycin induces oxidative stress,
reduces antioxidant reserve and impairs spermatogenesis in rats. J. Toxicol. Sci. 33,
8596.
Okai, Y., Sato, E.F., Higashi-Okai, K., Inoue, M., 2004. Enhancing effect of the endocrine
disruptor para-nonylphenol on the generation of reactive oxygen species in human
blood neutrophils. Environ. Health Perspect. 112, 553556.
Orgebin-Crist, M.C., 1969. Studies onthe functionof the epididymis. Biol. Reprod. 1, 155175.
Paglia, D.E., Valentine, W.N., 1967. Studies on quantitative and qualitative characteriza-
tion of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158169.
Pant, N., Murty, R.C., Srivastava, S.P., 2004. Male reproductive toxicity of sodium arse-
nite in mice. Hum. Exp. Toxicol. 23, 399403.
Peltola, V., Huhtaniemi, I., Ahotupa, M., 1992. Antioxidant enzyme activity in the
maturing rat testis. J. Androl. 13, 450455.
Pick, E., Keisari, Y., 1981. Superoxide anion and H2O2 production by chemically elicited
peritoneal macrophages-induction by multiple nonphagocytic stimuli. Cell. Immu-
nol. 59, 301318.
Qiong, L., Jun, L., Jun, Y., Yinzhu, Z., Xiaoyan, C., Mingliang, Y., 2011. The effect of Lam-
inaria japonica polysaccharides on the recovery of the male rat reproductive sys-
tem and mating function damaged by multiple mini-doses of ionizing radiations.
Environ. Toxicol. Pharmacol. 31, 286294.
Raecker, T., Thiele, B., Boehme, R.M., Guenther, K., 2011. Endocrine disrupting nonyl-
and octylphenol in infant food in Germany: considerable daily intake of nonylphe-
nol for babies. Chemosphere 82, 15331540.
Rao, M.V., Gangadharan, B., 2008. Antioxidative potential of melatonin against mercury
induced intoxication in spermatozoa in vitro. Toxicol. In Vitro 22, 935942.
Raymond, A.S., Elder, B., Ensslin, M., Shur, B.D., 2010. Loss of SED1/MFG-E8 results in
altered luminal physiology in the epididymis. Mol. Reprod. Dev. 77, 550563.
Rivero, C.L.G., Barbosa, A.C., Ferreira, M.N., Dorea, J.G., Grisolia, C.K., 2008. Evaluation of
genotoxicity and effects on reproduction of nonylphenol in Oreochromis niloticus
(Pisces: cichlidae). Ecotoxicol 17, 732737.
Robb, G.W., Amann, R.P., killian, G.J., 1978. Daily sperm production and epididymal
sperm reserves of pubertal and adult rats. J. Reprod. Fertil. 54, 103107.
Saito, K., O'Donnell, L., McLachlan, R.I., Robertson, D.M., 2000. Spermiation failure is a
major contributor to early spermatogenic suppression caused by hormone with-
drawal in adult rats. Endocrinology 141, 27792785.
Salmasi, A.H., Beheshtian, A., Payabvash, S., Demehri, S., Ebrahimkhani, M.R.,
Karimzadegan, M., Bahadori, M., Pasalar, P., Dehpour, A.R., 2005. Effect of morphine
on ischemia-reperfusion injury: experimental study in testicular torsion rat model.
Urology 66, 13381342.
Selvakumar, E., Prahalathan, C., Mythili, Y., Varalakshmi, P., 2004. Protective effect of
DL-alpha-lipoic acid in cyclophosphamide induced oxidative injury in rat testis.
Reprod. Toxicol. 19, 163167.
Selvakumar, E., Prahalathan, C., Mythili, Y., Varalakshmi, P., 2005. Benecial effects of
DL-alpha-lipoic acid on cyclophosphamide-induced oxidative stress in mitochon-
drial fractions of rat testis. Chem. Biol. Interact. 152, 5966.
Selvakumar, E., Prahalathan, C., Sudharsan, P.T., Varalakshmi, P., 2006. Chemoprotec-
tive effect of lipoic acid against cyclophosphamide-induced changes in the rat
sperm. Toxicology 217, 7178.
Sener, G., Sehirli, A.O., Ayanoglu-Dlger, G., 2003. Melatonin protects against mercur-
y(II)-induced oxidative tissue damage in rats. Pharmacol. Toxicol. 93, 290296.
Sharma, S.K., SaiRam, M., Ilavazhagan, G., Devendra, K., Shivaji, S.S., Selvamurthy, W.,
1996. Mechanism of action of NIM-76: a novel vaginal contraceptive from neem
oil. Contraception 54, 373378.
Sharpe, R.M., Fisher, J.S., Millar, M.M., Jobling, S., Sumpter, J.P., 1995. Gestational and
lactational exposure of rats to xenoestrogens results in reduced testicular size
and sperm production. Environ. Health Perspect. 103, 11361143.
Sikka, S.C., 2004. Role of oxidative stress and antioxidants in andrology and assisted
reproductive technology. J. Androl. 25, 518.
Sone, K., Hinago, M., Kitayama, A., Morokuma, J., Ueno, N., Watanabe, H., Iguchi, T.,
2004. Effects of 17-b-estradiol, nonylphenol, and bisphenol-A on developing
Xenopus laevis embryos. Gen. Comp. Endocrinol. 138, 228236.
Starter, N., 2006. Ecto-5-nucleotidase: structure function relationships. Purinergic
Signal 2, 343350.
Sudharsan, P.T., Mythili, Y., Selvakumar, E., Varalakshmi, P., 2005. Cardioprotective ef-
fect of pentacyclic triterpene, lupeol and its ester on cyclophosphamide-induced
oxidative stress. Hum. Exp. Toxicol. 24, 313318.
Sujarit, S., Pholpramool, C., 1985. Enhancement of sperm transport through the rat
epididymis after castration. J. Reprod. Fertil. 74, 497502.
Sullivan, R., Frenette, G., Girouard, J., 2007. Epididymosomes are involved inthe acquisition
of new sperm proteins during epididymal transit. Asian J. Androl. 9, 483491.
Sultan, C., Balaguer, P., Terouanne, B., Georget, V., Paris, F., Jeandel, C., Lumbroses, S.,
Nicolas, J., 2001. Environmental xenoestrogens, antiandrogens and disorders of
male sexual differentiation. Mol. Cell. Endocrinol. 178, 99105.
Takahashi, O., Oishi, S., 2001. Testicular toxicity of dietary 2,2-bis(4-hydroxyphenyl)
propane (bisphenol A) in F344 rat. Arch. Toxicol. 75, 4251.
Tesarik, J., Martinez, F., Rienzi, L., Iacobelli, M., Ubaldi, F., Mendoza, C., Greco, E., 2002.
In-vitro effects of FSH and testosterone withdrawal on caspase activation and
DNA fragmentation in different cell types of human seminiferous epithelium.
Hum. Reprod. 17, 18111819.
Uguz, C., Varisli, O., Agca, C., Agca, Y., 2009. Effects of nonylphenol on motility and
subcellular elements of epididymal rat sperm. Reprod. Toxicol. 28, 542549.
Varisli, O., Uguz, C., Agca, C., Agca, Y., 2009. Motility and acrosomal integrity compari-
sons between electro-ejaculated and epididymal ram sperm after exposure to a
range of anisosmotic solutions, cryoprotective agents and low temperatures.
Anim. Reprod. Sci. 110, 256268.
Vernet, P., Aitken, R.J., Drevet, J.R., 2004. Antioxidant strategies in the epididymis. Mol.
Cell. Endocrinol. 216, 3139.
Wang, H.T., Yang, X.L., Zhang, Z.H., Xu, H.B., 2003. Both calcium and ROS as common
signals mediate Na2
SeO
3
-induced apoptosis in SW480 human colonic carcinoma
cells. J. Inorg. Biochem. 97, 221230.
Wu, J.J., Wang, K.L., Wang, S.W., Hwang, G.S., Mao, I.F., Chen, M.L., Wang, P.S., 2010.
Differential effects of nonylphenol on testosterone secretion in rat Leydig cells.
Toxicology 268, 17.
Yadav, N., Khandelwal, S., 2008. Effect of Picroliv on cadmium induced testicular dam-
age in rat. Food Chem. Toxicol. 46, 494501.
Yavasoglu, A., Karaaslan, M.A., Uyanikgil, Y., Sayim, F., Ates, U., Yavasoglu, N.U., 2008.
Toxic effects of anatoxin-a on testes and sperm counts of male mice. Exp. Toxicol.
Pathol. 60, 391396.
Yenuqu, S., Hamil, K.G., French, F.S., Hall, F.H., 2006. Antimicrobial actions of human
and macaque sperm associated antigen 11 isoforms: inuence of the N-terminal
peptide. Mol. Cell. Biochem. 284, 2537.
Yokoi, K., Uthus, E.O., Nielsen, F.H., 2003. Nickel deciency diminishes sperm quantity
and movement in rats. Biol. Trace Elem. Res. 93, 141153.
Yon, J.M., Kwak, D.H., Cho, Y.K., Lee, S.R., Jin, Y., Baek, I.J., Lee, J.E., Nahm, S.S., Choo, Y.K., Lee,
B.J., Yun, Y.W., Nam, S.Y., 2007. Expression pattern of sulfated glycoprotein-2 (SGP-2)
mRNA in rat testes exposed to endocrine disruptors. J. Reprod. Dev. 53, 100710013.
Zini, A., Schlegel, P.N., 1996. Catalase mRNA expression in the male rat reproductive
tract. J. Androl. 17, 473480.
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