ORIGINAL ARTICLE
Antioxidant effect of manganese on the testis structure
and sperm parameters of formalin-treated mice
S. Tajaddini1, S. Ebrahimi2, B. Behnam3,4, M. Bakhtiyari3,5, M. T. Joghataei3,5, M. Abbasi5, M. Amini3,
S. Amanpour6 & M. Koruji3,5
1
2
3
4
5
6
Department of Basic Sciences, Payame Noor University, Iran;
Department of Basic Sciences, Payame Noor University, Iran;
Cellular and Molecular Research Center, Tehran University of Medical Sciences, Tehran, Iran;
Department of Medical Genetics and Molecular Biology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran;
Department of Anatomical Science, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran;
Vali-Asr Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran
Keywords
Formaldehyde—manganese—sperm parameters—testicular structure
Correspondence
Morteza Koruji, Cellular and Molecular
Research Center and Department of
Anatomical Sciences, School of Medicine,
Tehran University of Medical Sciences,
Hemmat Highway, PO Box 14155-5983,
Tehran, Iran.
Tel.: +98 21 86704603;
Fax: +98 21 88058689;
E-mail: [email protected]
Accepted: December 12, 2012
doi: 10.1111/and.12069
Summary
Manganese inhibits oxidative stress damage. The aim of this study was to
investigate the protective role of manganese on testis structure and sperm
parameters in adult mice exposed to formaldehyde (FA). Twenty adult male
NMRI mice were selected and randomly divided into four groups: (i) control;
(ii) sham; (iii) ‘FA’-exposed group; and (iv) ‘FA and manganese chloride’exposed group. The FA-exposed groups received 10 mg kg1 FA daily for
14 days, and manganese chloride was just injected intraperitoneally 5 mg kg1
on 2nd weeks. Mice were sacrificed, and spermatozoa were collected from the
cauda of the right epididymis and analysed for count, motility, morphology
and viability. The other testicular tissues were weighed and prepared for histological examination upon removal. Seminiferous tubules, lumen diameters and
epithelium thickness were also measured. The findings revealed that FA significantly reduced the testicular weight, sperm count, motility, viability and normal morphology compared with control group (P 0.05). In addition,
seminiferous tubules atrophied and seminiferous epithelial cells disintegrated in
the FA group in comparison with the control group (P 0.05). However,
manganese improved the testicular structure and sperm parameters in FA-treated mice testes (P 0.05). According to the results, manganese may improve
and protect mice epididymal sperm parameters and testis structure treated with
FA respectively.
Introduction
Spermatogenesis is a complex process of cellular changes
by which the spermatogonial stem cells begin to differentiate to mature spermatozoa. It also includes mitotic,
meiotic and post-meiotic phases (Kolasa et al., 2012).
Spermatogenesis begins 5–7 days and 10–13 years after
birth in rodents and humans respectively (Dym et al.,
2009), and continues throughout adult lifetime (McLean,
2005).
One-tenth of couples worldwide are affected by
reduced fertility, and infertile men account for about half
of these cases (Okabe et al., 1998). Among numerous factors that influence male fertility, oxidative stress has been
246
reported as one of the most effective factors on semen
quality (Tuncer et al., 2010) that has an important
impact on the motility and fertilising ability of spermatozoa (Aydemir et al., 2006).
Formaldehyde (FA) (CH2O) is an important chemical
for the global economy and is considered as a ubiquitous
environmental pollutant. It is widely used in domestic
and work environments including cosmetics, heating and
cooking emissions, hospitals, industrial settings, dissecting
room for fixing cadavers and routine histology and histopathology techniques (Ye et al., 2005; Gules & Eren,
2010).
Various studies indicated the harmful effects of FA on
the reproduction, respiratory and haematological systems
© 2013 Blackwell Verlag GmbH
Andrologia 2014, 46, 246–253
S. Tajaddini et al.
(Collins, 2004; Ye et al., 2005; Zhou et al., 2006b). Some
studies on mice demonstrated that formaldehyde usage
can lead to testicular atrophy and decreases testes weight,
diameter of seminiferous tubules and seminiferous epithelial height (Ye et al., 2005; Golalipour et al., 2007; Gules
& Eren, 2010). It decreases the motility and number of
spermatozoids (Golalipour et al., 2007). FA exposure can
also inhibit spermatogenesis and induce apoptosis of
spermatogenetic cells in testicular tissue (Ozen et al.,
2002; Cheng et al., 2003; Tang et al., 2003; Zhou et al.,
2006b,c). FA induces cellular injury and oxidative damage
in many tissues by increasing the production of reactive
oxygen species (ROS) (Zhou et al., 2006b).
To date, antioxidants have been used to reduce the
oxidative stress that breaks the oxidative chain reaction
(Miller et al., 1993; Chen et al., 2006; Bilaspuri & Bansal,
2008; Elbetieha et al., 2011). Manganese chloride, a
well-known antioxidant, plays an important role in scavenging free oxygen radicals and stabilising the cell membranes (Chen et al., 2006; Elbetieha et al., 2011).
Manganese is able to quench the peroxyl radicals and
acts as a chain breaking antioxidant. Some studies indicated that manganese – as a potent inhibitor of oxidative
stress – can reduce the oxidative stress and improve
sperm motility or semen quality under in vitro conditions (Lapointe et al., 1996; Bansal & Bilaspuri, 2008;
Bilaspuri & Bansal, 2008; Cheema et al., 2009). Eybl &
Kotyzova (2010) showed that Mn2+ pre-treatment in
acute cadmium intoxication significantly protects testes
against oxidative damage in vivo. Although in vivo antioxidant effects of Mn2+ (including lipid peroxidation,
antioxidant defence system and cadmium distribution in
the tissues of mice) have been indicated, its impact on
sperm parameters and testes structure remained to be
clarified. Therefore, this study was designed to investigate
effects of manganese pre-treatment on the mouse sperm
profile and testes structure in a model of formalin treated and to answer the question whether Mn2+ can
reverse the effects of formalin.
Material and methods
Animals
Fifty adult male NMRI mice (ages of 6–8 weeks, weight
30–35 g) were purchased from Pasture’s Institute (Tehran, Iran) and kept at the animal house of Tehran University of Medical Sciences (Tehran, Iran). The animals
were housed in plastic cages at 12-h light/dark cycle,
22 °C and fed with standard commercial laboratory chew
and water. Five animals were housed in a cage to prevent
overcrowding. The research was conducted in accordance
with the National Research Council guidelines.
© 2013 Blackwell Verlag GmbH
Andrologia 2014, 46, 246–253
Manganese treatment in formalin-treated mice
Selection of optimal dose for manganese chloride
To determine optimal and toxic amounts of manganese
chloride and formaldehyde, different doses were injected.
The animals were segregated into six groups each consisting of five male mice (n = 30, 6–8 weeks old). Formaldehyde and manganese chloride were injected to the
animals intraperitoneally (10 and 15 mg kg1) for
14 days and 5, 10, 20 and 30 mg kg1 respectively.
Experimental design
The animals (n = 20, 6–8 weeks old) were randomly
divided into four groups each consisting of five male
mice including: (i) control group: mice have normal
mode, (ii) sham group: mice intraperitoneally received
physiological saline (Samen Pharmaceutical Co., Mashhad, Iran) for 2 weeks (one spermiogenesis period in
mice), (iii) single FA treatment group (FAt): mice intraperitoneally received 10 mg kg1 FA for 14 days (Merck,
Darmstadt, Germany) (twice per day) and (iv) combined
manganese chloride and FA-treated group (FAt-Mn2+):
mice intraperitoneally received manganese chloride
(Merck) at the dose of 5 mg kg1 per day in 2nd weeks
and exposed to FA by administration at a dose of
10 mg kg1 (twice per day) for 2 weeks. Results from five
separate experiments were used for all groups.
Epididymal sperm parameters
The mice were sacrificed by cervical dislocation following
treatments. The caudae of the right epididymes were
excised and minced and incubated in a pre-warmed Petri
dish containing 1 ml of phosphate-buffered saline (PBS,
pH = 7.4) at 37 °C. The spermatozoon was allowed to
disperse (20–25 min), and sperm suspension was analysed
under light microscope at a magnification of 9400. The
sperm profile was assessed according to criteria of the
World Health Organization (fifth edition) with modification (Khaki et al., 2008, 2009; Awodele et al., 2010; Eybl
& Kotyzova, 2010).
To determine the percentage of motile spermatozoa,
the suspension was prepared by repipetting. One drop of
the suspension was placed on a clean glass slide for film
recording with a video microscope (Olympus, Tokyo,
Japan). At least five fields from each slide were recorded
with camera for sperm motility assessment via analysing
the recorded films and counting progressive, nonprogressive and immotile spermatozoa graded from 0 to II.
Viability was assessed by eosin B staining (0.5% in saline). Twenty microlitre sample of the sperm suspension
was placed on a glass slide, mixed with 7-ll eosin and
observed under a light microscope (9400 magnification).
247
Manganese treatment in formalin-treated mice
S. Tajaddini et al.
Live spermatozoa remained colourless following staining,
whereas red sperm heads were considered and classified
as dead. Morphology of 200 sperm heads was counted
from each sample in five fields of vision randomly, and
the percentage of live spermatozoa was recorded. For
sperm counting, 1 ml of the sperm suspension was
diluted with 1 ml of formaldehyde fixative (10% formalin
in PBS). Ten microlitre from the diluted solution was
transferred into a haemocytometer (Thoma, assistant
Sondheim/Rhön, Germany), and sperm count was evaluated per 250 small squares of a haemocytometer.
To assay sperm morphology, smears were prepared
from the sperm suspension (10 ll) and stained with the
Papanicolaou method. The morphology of 200 spermatozoa was observed under 91000 oil immersion lens. With
the staining, the nuclei turn blue, and acrosome and tail
become pink. Abnormal morphology was counted from
each sample in five fields of vision randomly, and percentage of abnormal morphology was recorded categorised into three groups including head, neck and tail
abnormality.
Morphological analysis of testes
For histological study, the right-side testes were dissected,
weighed, fixed in a fresh Bouin’s solution for 24 h and
finally dehydrated and embedded in paraffin. Then, 5-lm
sections were cut and stained with haematoxylin–eosin
(H&E) protocol. The pictures of the specimens were
taken, and the diameters of seminiferous tubules and
lumen in addition to the height of seminiferous epithelium were measured utilising the camera microscope
(Olympus) and Software Image J (version 1.240; National
Institutes of Health, USA). For each testis, above-mentioned parameters were measured in 100 randomly
selected round or nearly round tubular profiles.
Measuring the change in body weight, testis weight and
gonadosomatic index (GSI)
To determine the changes in body weight (at the beginning of the experiment), each of the male mice was
weighed before being anaesthetised. After washing the testis, their weight was measured. The ratio of the weight of
both testicles to the body weight was calculated, and the
percentage was determined and recorded as GSI.
Statistical analysis
Differences between groups were determined using
one-way analysis of variance (ANOVA) followed
Tukey’s tests. The results are presented as mean and were regarded as being significant at P 0.05.
248
an
by
SD
All
calculations were performed using the software
version 16.0 (SPSS Inc., Chicago, IL, USA).
SPSS,
Results
Optimal dose for manganese chloride
In these studies, the doses were selected based on the
mortality rate of mice. A comparison among FA-exposed
(10 and 15 mg kg1) and control groups indicated that
mortality rates were increased at 15 mg kg1, statistically
and significantly. Most deaths occurred during 1st week
of the study. Overall survival rates at the end of 14th day
were 80% and 20% for the doses 10 and 15 mg kg1
compared to the control group (100%) respectively. So,
the dose of 10 mg kg1 FA was picked up for the study.
Also, at the several high doses of MnCl2 (10, 20,
30 mg kg1), many mice died in 1st week (7 days of
treatment). Survival rates at the end of 14th day were
100%, 25%, 25% and 20%, at 5, 10, 20 and 30 mg kg1
(MnCl2) and control groups respectively. At the adequate
doses (10 mg kg1 FA and 5 mg kg1 MnCl2), all
animals were survived. Therefore, assessments were just
performed in low optimal doses.
Testicular weight, body weight and gonadosomatic index
(GSI)
As shown in Table 1, upon sacrificing the mice, the mean
(testicular and body) weights were (0.19 0.01,
5.25 0.5), (0.16 0.01, 0.82 0.4), (0.19 0.01,
5.74 1.1) g in the ‘control’, ‘FA’ and ‘FA+Mn2+’groups
respectively. The testicular and body weights of mice were
significantly decreased in mice of FA groups in comparison with those in the control group (P 0.05). However, treatment with Mn2+ prevented this decline of
testicular weight (P 0.05) significantly. Meanwhile, the
testicular and body weights showed a significant increase
in the ‘FA+Mn2+’ group compared with FA group
Table 1 The effects of manganese on testis weight, body weight and
GSI (gonadosomatic index) in adult mice exposed to formaldehyde
Groups
Testis weight (g)
Control
Sham
FA
FA+Mn2+
0.38
0.37
0.33
0.38
0.02
0.01
0.03*
0.01
Body weight (g)
5.25
6.48
0.82
5.74
0.5
2.4
0.4*
1.1
GSI
1.08
1.06
0.10
1.03
0.09
0.07
0.11
0.07
Results from five separate experiments were used for all groups.
Values are the mean SD at different times.
*Significant difference versus other groups in the same column
(P 0.05).
© 2013 Blackwell Verlag GmbH
Andrologia 2014, 46, 246–253
S. Tajaddini et al.
Manganese treatment in formalin-treated mice
(P 0.05). In all groups, differences in GSI were not
significant.
Epididymal sperm analysis
Sperm count, motility, viability and rate of normal morphology showed a significant decrease in the FA-treated
group compared to the control group (Table 2)
(P 0.05). Although treatment with Mn2+ significantly
prevented the decline of sperm quantity and quality in
‘FA+Mn2+’ group (P 0.05), the sperm profiles were
not the same as in the control group. In the ‘FA+Mn2+’
group, sperm count, motility, viability and normal
morphology significantly increased compared to FA group
(P 0.05). The results showed a high level of abnormality in the sperm tail in mice exposed to FA compared to
the control group (Table 3, Fig. 1).
Morphological analysis of testes
Histological examinations of the testes revealed that
there were significant histological changes in mice testes
in the FA group (Table 4, Fig. 2). In this group, the
diameters of testicular seminiferous and epithelial
tubules significantly decreased compared to the control
group (P 0.05). However, in the ‘FA+Mn2+’ groups,
Table 2 The effects of manganese on sperm profile in adult mice exposed to FA
Groups
Sperm viability (%)
Control
Sham
FA
FA+Mn2+
86.02
82.76
35.63
66.73
1.2
6.6
1.7*
6.3**
Sperm motility (%)
64.47
62.98
23.22
43.48
2.6
4.7
7.7*
1.5**
Sperm count 9106
17.18
16.60
5.89
13.76
0.43
0.66
0.18*
0.36*,**
Normal morphology (%)
81.26
83.95
15.91
86.25
3.0
3.0
4.6*
4.6
Results from five separate experiments were used for all groups. Values are the mean SD at different times.
*Significant difference versus other groups in the same column (P 0.05).
**Significant difference versus control group in the same column (P 0.05).
Table 3 The effects of manganese on sperm morphology in adult mice exposed to FA
Groups
Normal (%)
Control
Sham
FA
FA+Mn2+
81.25
84.46
15.92
84.53
3.08
4.10
4.69*
3.57
Head abnormal (%)
0.33
0.51
2.99
0.69
0.22
0.38
1.26*
1.16
Neck abnormal (%)
1.20
2.11
2.78
1.03
0.82
2.11
2.70
0.94
Tail abnormal (%)
17.22
12.92
78.32
13.75
2.84
1.78
5.82*
3.10
Results from five separate experiments were used for all groups. Values are the mean SD at different times.
*Significant difference versus other groups in the same column (P 0.05).
Fig. 1 Different types of sperm shape abnormality. Sperm suspension was smeared onto glass slides and stained using the method of Papanicolaou. Spermatozoa were counted and categorised as normal, head abnormal, neck abnormal and tail abnormal spermatozoa.
© 2013 Blackwell Verlag GmbH
Andrologia 2014, 46, 246–253
249
Manganese treatment in formalin-treated mice
S. Tajaddini et al.
Table 4 The effect of manganese on testis structure in adult mice exposed to formaldehyde
Groups
Seminiferous tubule (lm)
Control
Sham
FA
FA+Mn2+
195.64
196.93
164.95
209.35
13.4
6.4
9.4*
20.3
Seminiferous lumen (lm)
86.57
76.17
112.46
74.74
Seminiferous epithelial (lm)
9.9
5.03
11.0*
11.6
109.08
120.74
52.49
134.61
9.4
6.2
16.7*
14.0
Results from five separate experiments were used for all groups. Values are the mean SD at different times.
*Significant difference versus other groups in the same column (P 0.05).
(a)
(b)
(c)
(d)
Fig. 2 Morphology of tubules in the testis of
(a) control, (b) sham and (c) FAt and (d)
FAt + Mncl2 groups. Depleted and abnormal
tubules without spermatozoa were observed
in germinal epithelium of formalin-treated
testis group (asterisk). Histological examinations of the testes FAt + Mncl2 group
revealed that administration of manganese
prevented from damage effects of FA (d).
Scale bar = 200 lm.
the diameters of seminiferous and epithelial tubules
significantly increased compared to the FA group
(P 0.05).
Discussion
The present study confirms previous findings that testicular weight, epididymal sperm parameters, diameter of
seminiferous tubules and epithelium height were
decreased in formalin-treated mice and that administration of manganese was associated with an increase in
sperm parameters and testicular changes.
During mouse spermatogenesis, round spermatids
mature in 16 distinct steps via elongated spermatids to
mature spermatozoa. Because it takes 13.5 days for
mature spermatozoa to appear in the lumen of the seminiferous tubules (Russell et al., 1990), the mice received
formaldehyde for 14 days intraperitoneally in this study.
In previous studies, to determine the sperm profiles, histopathological and morphometric changes in the testis,
experimental animals were exposed to formaldehyde for
250
5 days to 18 weeks (Tang et al., 2003; Zhou et al., 2006b;
Golalipour et al., 2007).
In the present study, epididymal sperm parameters
decreased in formalin-treated mice and administration of
manganese was associated with an increase in sperm
parameters. Previous study showed that administered
(Majumder & Kumar, 1995; Tang et al., 2003) and
inhaled (Zhou et al., 2006b) formaldehyde decreased the
motility, viability and number of spermatozoid cells in
experimental animals. Also, in vitro exposure of semen to
formaldehyde inhibited sperm motility and viability
(Majumder & Kumar, 1995). Various studies showed that
formaldehyde increases the production of ROS in many
tissues (Gurel et al., 2005; Saito et al., 2005; Zhou et al.,
2006b) including testicular tissue (Gules & Eren, 2010).
Excessive free radicals in testis increase germ cell apoptosis and inhibit the activity of spermatozoa (Fujii et al.,
2003; Ozen et al., 2005; Zhou et al., 2006b).
The mechanism by which formaldehyde deteriorates
the sperm profile has been elucidated. High levels of ROS
are linked with lipid peroxidation of the sperm outer
© 2013 Blackwell Verlag GmbH
Andrologia 2014, 46, 246–253
S. Tajaddini et al.
membrane, which in turn leads to loss of motility (Urata
et al., 2001), decreased sperm–oocyte fusion capacity
(Aitken, 1994) and increased chromatin damage (Aitken
& Krausz, 2001). Interference in processes of membrane
ion exchange and its enzymes decrease sperm motility
(Woo et al., 2000; Garcia et al., 2010). ROS also inhibits
intracellular enzyme; therefore, ATP cannot be available
for sperm motility (Woo et al., 2000; Bilodeau et al.,
2002; Arabi et al., 2003). On the other hand, the changes
inducing peroxidation of sperm membrane components
result in reduced enzymatic activity of Na/K-ATPase (as
an ion pump involved in the movement) and ultimately
declined sperm motility (Woo et al., 2000). Manganese
chloride as an antioxidant plays an important role in consuming free oxygen radicals and stabilising the cell membranes (Chen et al., 2006; Elbetieha et al., 2011). Previous
studies demonstrated that Mn2+ supplementation
increases the level of cAMP and improves movements or
flagellar beating of spermatozoa (Bilaspuri & Bansal,
2008). Increase in the level of cAMP can also stimulate
the Ca2+ uptake by the cell and increase the level of intracellular calcium (Ca2þ
i ) (Guraya, 2000). At a higher level,
increases membrane integrity and viability. In this
Ca2þ
i
study, the effect of manganese was similar to results
obtained by some other studies reporting a protection of
epididymal sperm characteristics by manganese (Lapointe
et al., 1996; Bilaspuri & Bansal, 2008; Eybl & Kotyzova,
2010; Elbetieha et al., 2011).
In our study, the sperm morphology assay showed that
sperm abnormality in general and abnormal sperm tail,
in particular, increased in formalin-treated mice. However, abnormal sperm rate decreased following the manganese treatment. Abnormal sperm tail may propose
some damage in proteins that is involved in the movements or flagellar/ciliary beating of spermatozoa (Lindemann & Goltz, 1988; Bilaspuri & Bansal, 2008). Also, the
rate of sperm head abnormality may indicate formaldehyde general toxicity on germ cells and their genetic
materials (Sikka, 1996; Tarin et al., 2000; Jauniaux et al.,
2003; Tang et al., 2003; Saito et al., 2005). Tramer et al.
(1998) showed that ROS cause lipid peroxidation of
sperm cell membranes, hence damaging the mid piece,
axonemal structure or disrupting the capacitation and
acrosomal reaction, which finally results in infertility. In
this study, manganese had more likely a protective effect
on spermatozoa against ROS in formaldehyde-treated
mice. There could be concordance between our finding
and those reported previously (de Lamirande & Gagnon,
1995; Zhou et al., 2006c).
In this study, it has also been demonstrated that formaldehyde exposure decreased diameter of seminiferous
tubules and epithelium height in the testis of mouse and
while manganese prevented these testicular deteriorations.
© 2013 Blackwell Verlag GmbH
Andrologia 2014, 46, 246–253
Manganese treatment in formalin-treated mice
Cytotoxic effect of formaldehyde has been shown in the
spermatogenesis process and seminiferous tubules (Zhou
et al., 2006b,c, 2011). Feldman (1973) reported that intraperitoneal administration of formaldehyde caused the
arrest of nucleic acid synthesis and proteins. Previous
investigators demonstrated that intraperitoneal administration (Tang et al., 2003) and inhalation (Zhou et al.,
2006a; Golalipour et al., 2007) of formaldehyde resulted
in some anatomical disturbances in the testes. These
abnormal changes include a decrease in the number or
degeneration of spermatogenic cells and Leydig cells, atrophy of the seminiferous tubules and disorganisation of
the seminiferous epithelial cells. These investigations are
supported and interpreted by the recent findings in this
study. Because formaldehyde decreases the effectiveness of
the testicular antioxidant system (Zhou et al., 2006b), we
conclude that administration of manganese chloride may
scavenge directly free radical and protect the spermatogenic cells and testicular structure.
In summary, the present study shows that manganese
chloride systemic administration improves epididymal
sperm profile and prevents testes morphological changes
in formalin-treated mice. It is also shown that systemic
administration of manganese chloride in low doses is
much more effective than its higher doses. Therefore, for
patients who fail to become fertile following working in
formalin environments, manganese chloride treatment or
vegetables containing manganese feeding might be a
promising therapeutic and/or a preventive option and a
more economical method. Of course, this is a preliminary
investigation and further studies are necessary to clarify
its effects on human testes and male fertility.
Acknowledgements
We appreciate the administrative contributions of Dr Z.
Mazaheri, M. Bakhshayesh, F. Esmaili, M. Hadadi, Z.
Fathi, S. Shabani and P. Hayat to this study. This study
was funded by a grant from Tehran University of Medical
Sciences (TUMS) (Number: 90-04-11-15933), and all
experiments have been performed in Cellular and Molecular Research Center, TUMS, Tehran, Iran. Authors
declare that there is no conflict of interest in this study.
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