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Reproductive Toxicology 37 (2013) 3139

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Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox

Resveratrol improves sperm motility, prevents lipid peroxidation and enhances


antioxidant defences in the testes of hyperthyroid rats
Giovana M. Ourique a , Isabela A. Finamor a , Etiane M.H. Saccol a , Ana P.K. Riffel a , Tanise S. Ps a ,
Karina Gutierrez b , Paulo B.D. Goncalves b , Bernardo Baldisserotto a , Maria A. Pavanato a ,
Ktia P. Barreto a,
a
Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil
b
Laboratrio de Biotecnologia e Reproduco Animal BioRep, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil

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

Article history: Hyperthyroidism may lead to a loss of sperm motility and an increase in oxidative stress (OS) in testes
Received 28 June 2012 and may cause male reproductive disorders. Thus, the use of compounds with antioxidant properties
Received in revised form 8 January 2013 may be a strategy for preventing these disorders. The effect of resveratrol (RSV) on sperm motility and on
Accepted 15 January 2013
variables of the antioxidant status in the testes of rats with triiodothyronine-induced hyperthyroidism
Available online 4 February 2013
(100 g/kg) was investigated. Hyperthyroid rats presented lower sperm motility, higher levels of lipid
hydroperoxides and thiobarbituric reactive substances, lower catalase and glutathione peroxidase activ-
Keywords:
ities and higher glutathione-S-transferase activity in their testes than control animals. RSV treatment
Hyperthyroidism
Polyphenol
(1 mg/kg and 10 mg/kg) was able to prevent these effects in the hyperthyroid rats and had no effect in the
Oxidative stress control animals. In conclusion, RSV might be a strategy for therapeutic intervention to preserve sperm
Antioxidant enzymes motility and to prevent OS in testes, preserving testicular function in those with hyperthyroidism.
Sperm quality 2013 Elsevier Inc. All rights reserved.
Testicular dysfunction

1. Introduction include several enzymatic (such as superoxide dismutase (SOD),


catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-
Thyroid hormones (THs) are known to play a fundamental role transferase (GST)) and non-enzymatic systems (such as glutathione
in the regulation of energy metabolism in almost all mammalian (GSH)) [6]. ROS are highly reactive and can lead to oxidative dam-
tissues. TH receptors are present in sperm; developing germ cells; age in macromolecules such as lipids, proteins and DNA [2,5,7,8]. In
and Sertoli, Leydig, and peritubular cells in neonatal, prepubertal hyperthyroid rats, tissues, such as the testis, that are rich in polyun-
and adult rats [1]. Although THs are not the primary regulators saturated fatty acids and exhibit low amounts of antioxidants show
of testicular function, THs have an early and strong inuence on more susceptibility to oxidative damage, which may result in male
the proliferation and maturation of germ cells and promote testis infertility [3,8,9]. This decreased fertility could also be caused by
growth [2,3]. One of the most important effects of THs is the trigger- the lipid peroxidation (LPO) of sperm cell membranes, injury to the
ing of a hypermetabolic state with an excess generation of reactive midpiece and axonemal structures, malfunctioning of capacitation
oxygen species (ROS) caused by enhanced mitochondrial respira- and acrosomal reactions and, nally, the loss of motility [10].
tion. This increased metabolic rate leads to oxidative stress (OS), The use of compounds with antioxidant properties, such as
which has been implicated in the pathophysiology of hyperthy- melatonin [11], curcumin and vitamin E [9], have shown efciency
roidism [25]. in protecting testes from the OS generated during experimental
OS occurs when there is an increased generation of ROS, hyperthyroidism. However, no studies have found data on the
decreased antioxidant defences, or a combination of both. The con- effects of resveratrol (3,5,4 -trihydroxystilbene, RSV) as an auxil-
centration of ROS is controlled by antioxidant defences, which iary tool in the treatment of this experimental condition. RSV is a
natural polyphenol found mainly in grapes and wines and plays an
important role as an antioxidant, acting as an effective scavenger
of superoxide (O2 ), hydroxyl (OH ) and metal-induced radicals
Corresponding author at: Departamento de Fisiologia e Farmacologia, Univer-
[1217]. RSV also exhibits a protective effect against LPO in cell
sidade Federal de Santa Maria, 1000, Avenida Roraima, Camobi 97105-900, Santa
Maria, Rio Grande do Sul, Brazil. Tel.: +55 55 32209381; fax: +55 55 32208241. membranes and against the DNA damage caused by ROS [14,15,17].
E-mail address: barreto.kp@gmail.com (K.P. Barreto). RSV treatment has been shown to prevent ethanol-induced LPO

0890-6238/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.reprotox.2013.01.006
32 G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139

in testes [14] and the LPO induced by tert-butyl hydroperoxide in method, with a commercially available kit specic for rats (Amer-
sperm, protecting sperm chromatin and membranes [13]. However, sham Pharmacia Biotech Inc., Arlington Heights, IL, USA).
to our knowledge, there are no reports demonstrating the effects of
this compound in the testes and sperm of hyperthyroid rats. There- 2.5. Removal of epididymis and retrieval of spermatozoa
fore, the present study aimed to investigate the effects of RSV on
sperm quality and on variables of antioxidant status in the testes The epididymides were excised and freed from the adherent
of rats with triiodothyronine (T3)-induced hyperthyroidism. and fat tissues. The cauda epididymis was collected, transferred
to a petri plate and immersed in sterile silicone oil, nearby a drop
2. Materials and methods of 200 L of Ferts medium (114 mM NaCl, 3.22 mM KCl, 0.34 mM
NaH2 PO4 , 25 mM NaHCO3 , 16 mM C3 H5 O3 Na, 0.6% BSA, 0.1% phe-
2.1. Chemicals nol red, 2 mM CaCl2 2H2 O, 0.5 mM MgCl2 6H2 O, and Milli-Q water).
All reagents were adequately preheated. Longitudinal incisions
RSV was obtained from Chengdu Hawk Bio-Engineering Co., were made in the cauda epididymis with a ne needle and a scalpel
Ltd. (Chengdu, Jintang County, China), and its purity and struc- blade to release the spermatozoa. The sperm motility was evaluated
ture were previously conrmed using chromatography and nuclear by placing a 4 l drop of Ferts medium containing the spermatozoa
magnetic resonance, respectively. T3 (3,3 ,5-triiodo-l-thyronine) on a slide and then examining the drop using a light microscope
and all other reagent-grade chemicals were obtained from Sigma (Olympus CX40) at 100. The motility was estimated from three
(St Louis, Missouri, USA). different elds in each sample, and the mean was used as the nal
value of motility.
2.2. Animals To analyse the morphology, the sperm were kept in a
formaldehyde-citrate solution, and 200 spermatozoa were ana-
Adult male Wistar rats (7090 days; 130200 g) were obtained lysed from each animal. The analysis was performed using a Leica
from the Central Animal Breeding Facility of the Federal University DMI 4000B inverted microscope with differential interference con-
of Santa Maria, RS, Brazil. The animals were kept in polypropylene trast. Morphological abnormalities of the head (small, amorphous,
cages with controlled temperature (23 2 C) and a light-dark cycle pinhead or isolated form, i.e., no tail attached), midpiece (with-
of 12 h with access to water ad libitum and to approximately 30 g out characteristic curvature or broken) and tail (doubled, broken or
daily per animal of rodent laboratory chow (Supra, So Leopoldo, rolled into a spiral) were examined, and the percentage of normal
RS, Brazil). High performance liquid chromatography (HPLC-DAD) or abnormal sperm was determined.
was performed to verify that RSV was not present in the feed, even
in trace amounts. All animal procedures were approved by the 2.6. Tissue homogenate preparation
Animal Ethics Committee of the Federal University of Santa Maria
(process 111/2011). The testes were immediately excised and frozen at 20 C. This
tissue was homogenised in 0.1 M phosphate buffer (pH 7.4) using a
2.3. Experimental protocol glass-Teon grinder. The homogenate was centrifuged at 100 g
for 10 min at 4 C to discard the nuclei and cell debris, and the
Rats (n = 48) were randomly divided into six groups with eight resulting supernatant fraction was frozen at 80 C for further mea-
animals each: the control group (C), control group treated with surements. The protein concentrations in the supernatant were
1 mg/kg of RSV (CR1), control group treated with 10 mg/kg of determined using the method of Lowry et al. [18] with bovine
RSV (CR10), hyperthyroid group (H), hyperthyroid group treated albumin as the standard.
with 1 mg/kg of RSV (HR1), and hyperthyroid group treated with
10 mg/kg of RSV (HR10). 2.7. Oxidative damage measurements
Hyperthyroidism was induced by daily intraperitoneal injec-
tions (i.p.) of T3 at a dose of 100 g/kg body weight [5] for six LPO, as indicated by the amount of lipid hydroperoxides, was
weeks. The T3 was dissolved using 2.5 mM sodium hydroxide, and measured with a modied version of the xylenol orange method
the nal solution was prepared with 0.9% saline. The rats in the [19]. This technique can detect the primary products of peroxida-
control groups (C, CR1 and CR10) received only an equal volume of tion using the oxidation of Fe2+ by lipid hydroperoxides in an acidic
vehicle. medium with xylenol orange dye, which forms a complex with
After two weeks, the animals received daily i.p. injections of RSV, Fe3+ . The amount of dye was measured in a spectrophotometer at
freshly prepared in 25% ethanol, at a dose of 1 mg/kg or 10 mg/kg 560 nm, and the results were reported as nmol/mg protein.
concomitant to the T3 treatment for four weeks with six total exper- LPO was also estimated based on the formation of thiobarbituric
imental weeks. Groups C and H received only i.p. injections of acid reactive substances (TBARS) [20]. The amount of malondialde-
vehicle solution in the same conditions. At the end of the exper- hyde (MDA) produced was used as an index of LPO. Briey, the
imental period (six weeks) and 24 h after the last injections, the sample was mixed with 20% TCA and 0.67% TBA and heated for 1 h
animals were weighed, anesthetised with xylazine and ketamine at 100 C. After cooling, the precipitate was removed by centrifuga-
and euthanised for the removal of their heart, testis, epididymis tion. The absorbance of the organic phase was measured at 535 nm,
and prostate, which were immediately weighed. and the results were expressed as nmol MDA/mg protein.

2.4. Hormonal analysis 2.8. Activities of antioxidant enzyme

Blood samples were collected from the tail vein 8 h after fasting The activities of the antioxidant enzymes SOD, CAT, GPx and GST
and separated using centrifugation (500 g for 10 min). The serum were measured in the testicular homogenates.
was stored at 20 C for further analysis. The serum levels of T3 The total SOD activity was determined as the inhibition rate
were measured using a commercially available radioimmunoassay of autocatalytic adrenochrome generation at 480 nm in a reaction
kit (CIS Bio International, Bedford, MA, USA). Thyroid-stimulating medium containing 1 mM epinephrine and 50 mM glycine/NaOH
hormone (TSH) levels were measured using a radioimmunoassay (pH 10.2). The enzyme activity was expressed as USOD/mg protein.
G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139 33

Table 1
Assessment of thyroid state of rats.

Group Variable

T3 (ng/ml) TSH (ng/ml) Final body weight (g) Weight gain (%) HW/BW (mg/g)

C 0.206 0.023 1.759 0.034 332.3 12.4 54.1 5.0 3.09 0.12
CR1 0.214 0.015 1.804 0.028 336.2 12.0 55.9 3.2 3.05 0.05
CR10 0.194 0.014 1.696 0.023 319.5 12.8 55.5 3.7 3.13 0.008
H 0.940 0.105a 0.369 0.020a 282.2 10.9a 20.4 3.8a 4.18 0.27a
HR1 0.856 0.046a,b 0.310 0.029a,b 272.6 9.1a,b 21.7 2.0a,b 3.91 0.17a,b
HR10 0.982 0.059a,b 0.338 0.020a,b 277.7 9.1a,b 31.4 3.0a,b 3.83 0.11a,b

The data represent the mean SE of eight experiments.


T3 = triiodothyronine; TSH = thyroid-stimulating hormone; HW/BW = heart weight/body weight; C = control group; CR1 = control group treated with 1 mg/kg resveratrol;
CR10 = control group treated with 10 mg/kg resveratrol; H = hyperthyroid group; HR1 = hyperthyroid group treated with 1 mg/kg resveratrol; HR10 = hyperthyroid group
treated with 10 mg/kg resveratrol.
a
Denotes that the data are signicantly different from C at P < 0.05.
b
Denotes that the data are signicantly different from the corresponding control group, CR1 or CR10, at P < 0.05.

One SOD unit was dened as the amount of enzyme required for measured at 412 nm. The amount of GSH was expressed as nmol/mg
50% inhibition of the adrenochrome formation [21]. protein [25].
The CAT activity was evaluated by measuring the decrease in the
absorption at 240 nm in a reaction medium consisting of 50 mM 2.10. Statistical analysis
phosphate buffer (pH 7.4) and 2 mM hydrogen peroxide (H2 O2 ),
thereby providing information to determine the pseudo-rst-order The results were analysed using a two-way analysis of variance
reaction constant (k ) of the decrease in H2 O2 absorption [22]. The (ANOVA) test followed by Duncans test. The level of statistical sig-
results were reported as pmol/mg protein. nicance was set at P < 0.05. All analyses were performed using
The GPx activity was measured using a coupled reaction with Statistica Software (Stat Soft, Inc.), version 7.0. The results are pre-
glutathione reductase (GR) in the presence of GSH and NADPH, as sented as the means standard error (SE).
described previously [23]. The oxidised glutathione produced upon
reduction of t-butyl hydroperoxide by GPx is reduced by GR using 3. Results
NADPH as a cofactor. The oxidation of NADPH to NADP+ results
in a decrease in the absorbance at 340 nm, and the GPx activity is 3.1. Determination of thyroid state
dened as the amount of enzyme that catalyses the oxidation of
1 pmol of NADPH per minute. The thyroid state of the different experimental groups was char-
The GST activity was assayed based on the conjugation reaction acterised using the physiological data reported in Table 1. As can
with GSH, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate be observed, H, HR1 and HR10 presented higher T3 and lower TSH
[24]. The assay was performed by using 100 mM sodium phosphate serum concentrations than C, CR1 and CR10 (P < 0.05). Moreover,
buffer (pH 6.5), with GSH and CDNB at a nal concentration of 1 mM hyperthyroid animals H, HR1 and HR10 presented lower gains in
each. The activity was calculated from the changes in absorbance body weight than C, CR1 and CR10 (P < 0.05). The ratio of heart
at 340 nm. One unit of GST activity was dened as the amount of weight to body weight (HW/BW) was calculated to estimate the
enzyme catalysing the conjugation of 1 mol of CDNB with GSH cardiac hypertrophy. This ratio was higher in H, HR1 and HR10 than
per minute at 25 C. in C, CR1 and CR10 (P < 0.05). There were no signicant differences
among H, HR1 and HR10.
2.9. Non-enzymatic antioxidant (tissue sulfhydryl groups)
3.2. Testis, epididymis and prostate weights
To measure GSH, protein-free supernatant was obtained by
adding 0.5 M perchloric acid to the testicular homogenate and The ratio of the weights of the testis, epididymis and prostate
centrifuging the mixture at 7000 g for 5 min. To this supernatant, to the body weight was also calculated in the different groups
0.2 M phosphate buffer (pH 8) and 3.78 mM 5,5 -dithiobis-2- to estimate the hypertrophy of these tissues. The results indicate
nitrobenzoic acid (DTNB) were added and vortexed. The DTNB hypertrophy in the testis of the H and HR10 groups when com-
formed a yellow complex with GSH, and the absorbance was pared with C and CR10 (P < 0.05); however, HR1 did not present an

Table 2
Effects of resveratrol on the weights of the reproductive organs of the rats with induced hyperthyroidism.

Group Variable

TW/BW (mg/g) EW/BW (mg/g) PW/BW (mg/g)

C 5.29 0.14 1.49 0.08 1.19 0.12


CR1 5.31 0.18 1.53 0.05 1.31 0.11
CR10 5.59 0.19 1.59 0.07 1.38 0.22
H 6.27 0.27a 1.75 0.05a 1.76 0.25a
HR1 5.85 0.25 1.73 0.06a,b 1.25 0.21
HR10 6.36 0.17a,b 1.82 0.03a,b 1.36 0.07

The data represent the mean SE of eight experiments.


TW/BW = testis weight/body weight; EW/BW = epididymis weight/body weight; PW/BW = prostate weight/body weight. C = control group; CR1 = control group treated
with 1 mg/kg resveratrol; CR10 = control group treated with 10 mg/kg resveratrol; H = hyperthyroid group; HR1 = hyperthyroid group treated with 1 mg/kg resveratrol;
HR10 = hyperthyroid group treated with 10 mg/kg resveratrol.
a
Denotes that the data are signicantly different from C at P < 0.05.
b
Denotes that the data are signicantly different from the corresponding control group, CR1 or CR10, at P < 0.05.
34 G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139

Fig. 1. Effects of resveratrol on epididymal sperm motility and morphology of rats with induced hyperthyroidism. Each bar represents the mean SE of eight animals.
a
Denotes that the data are signicantly different from C at P < 0.05; b denotes that the data are signicantly different from H at P < 0.05.

increase in the ratio of the testis weight to body weight when com- treatment with RSV prevented this increase, and the hydroperox-
pared with C and CR1. Moreover, epididymal hypertrophy occurred ide levels in HR1 and HR10 were 32% and 42% lower, respectively,
in H, HR1 and HR10 when compared with C, CR1 and CR10 (P < 0.05). than in H (P < 0.05) (Fig. 2).
In the prostate, hypertrophy was observed only when the H animals The TBARS levels were 39% higher in H than in C (P < 0.05). How-
were compared with the C animals (P < 0.05); this difference did not ever, the RSV treatment reduced the TBARS levels in the HR1 (35%)
occur in the other groups (Table 2). and HR10 (52%) groups compared with the levels in H (P < 0.05),
returning to the C levels. CR1 and CR10 did not present changes in
the lipid hydroperoxide and TBARS levels when compared with C,
3.3. Sperm motility and morphology
showing that RSV alone had no effect (Fig. 2).
The sperm motility was 30% lower in H than in C (P < 0.05).
However, HR1 did not differ signicantly from C. In addition, HR10
3.5. Activities of antioxidant enzymes
presented sperm motility that was 36% higher than H (P < 0.05).
The percentage of total morphological abnormalities in the epi-
The SOD activity did not differ across the various experimental
didymal sperm did not show a signicant difference among the
groups (Fig. 3). The CAT and GPx activities were 31% and 32% lower,
experimental groups (Fig. 1).
respectively, in H than in C (P < 0.05). The RSV treatment prevented
the decrease in the activities of these enzymes because HR1 and
3.4. Oxidative damage HR10 presented an increase of 46% in the CAT activity and of 42% in
the GPx activity when compared with H (P < 0.05) (Fig. 3 and Fig. 4).
Lipid hydroperoxide levels, determined using xylenol orange, In H, the GST activity was 152% higher than in C (P < 0.05). The
were 85% higher in H animals than in C animals (P < 0.05). The treatment with RSV was able to reduce this GST activity increase by
G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139 35

Fig. 2. Effects of resveratrol on the levels of lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS) in the testes of rats with induced hyperthyroidism and
in the controls. Each bar represents the mean SE of eight animals. a Denotes that the data are signicantly different from C at P<0.05; b denotes that the data are signicantly
different from the corresponding control group, CR1 or CR10, at P < 0.05; c denotes that the data are signicantly different from H at P < 0.05.

58% in the in both HR1 and HR10 (P < 0.05). There were no signi- T3 is able to increase mitochondrial respiration via many
cant differences in the antioxidant enzyme activities in the testes of complex changes in the number and activity of mitochondrial
the CR1 and CR10 animals when compared with those in C (Fig. 4). respiratory chain components. Thus, accelerated mitochondrial
electron transport, resulting from a TH-induced hypermetabolic
3.6. Non-enzymatic antioxidant (tissue sulfhydryl groups) state, increases ROS generation, which might easily start the pro-
cess of LPO in many types of cells [25], such as spermatozoa and
The GSH values did not differ across the different experimental testicular cells. These cells are susceptible to oxidative damage
groups (Fig. 4). because of the high content of polyunsaturated fatty acids within
their plasma membranes, and this damage may underlie several
4. Discussion aspects of male infertility [10]. Thus, natural antioxidants might be
helpful in preventing OS-related functional abnormalities in testes.
The results of the present study demonstrated that RSV pre- Some compounds, such vitamin E and curcumin, have been
vented the loss of sperm motility, decreased LPO and prevented demonstrated to protect testes from l-thyroxine(T4)-induced OS,
the OS induced by hyperthyroidism in adult rat testes. mainly by restoring the level of antioxidant enzymes to that in
36 G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139

Fig. 3. Effects of resveratrol on the superoxide dismutase (SOD) and catalase (CAT) activities in the testes of the control and hyperthyroid rats. Each bar represents the
mean SE of eight animals. a Denotes that the data are signicantly different from C at P < 0.05; b denotes that the data are signicantly different from H at P < 0.05.

euthyroid animals [9]. RSV has been reported to have several ben- the dose of 10 mg/kg i.p. has been reported to be effective with
ecial effects on health and has antioxidant properties that are some benecial effects assigned to RSV [16].
considered more effective than those of vitamin E [26]. The results from the present study showed that experimental
Despite the many benecial effects of RSV, low doses of RSV hyperthyroidism induced by T3 administration (100 g/kg) for 45
protect against different diseases, whereas high doses can be days was successfully achieved, as observed from the elevation in
detrimental for health [27]. The mean daily intake of RSV can serum T3 levels associated with a reduction in the serum TSH levels.
be approximated as 0.02 mg/kg in humans given the following This condition was further supported by the decreased body weight
assumptions: red wine is the main dietary source of RSV, the gains presented by the hyperthyroid animals. Cardiac hypertrophy
average concentration of trans-resveratrol in red wine is 5 mg/L, also conrmed the hyperthyroid state. These data are consistent
and moderate daily consumption of wine is 250 ml [28]. Thus, we with the increased catabolic activity described in hyperthyroidism
selected doses that were 50 (1 mg/kg) and 500 (10 mg/kg) times [3,31]. RSV had no effect on these variables, which was acceptable
higher than this estimate to provide a sufciently large safety mar- because we expected RSV to act as an antioxidant and not to alter
gin [29]. Oral RSV shows poor bioavailability because of the high the endocrine status of the animals.
rate of its degradation during metabolism [30]. Therefore, in this Zamoner et al. [3] demonstrated that T3 treatment (80 g/kg)
experiment, RSV was administered intraperitoneally. Additionally, caused testicular hypertrophy in pup rats (8-day-old) and
G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139 37

Fig. 4. Effects of resveratrol on the glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities and on the sulfhydryl group (GSH) concentrations in the testes
of the control and hyperthyroid rats. Each bar represents the mean SE of eight animals. a Denotes that the data are signicantly different from C at P < 0.05; b denotes that
the data are signicantly different from H at P < 0.05.
38 G.M. Ourique et al. / Reproductive Toxicology 37 (2013) 3139

attributed this nding to the accelerated testes maturation induced in the testes of T3-induced hyperthyroid rats, supporting a possible
by TH. Our data showed hypertrophy in the testis, epididymis and role of this compound in ROS inactivation and in the modulation
prostate of adult hyperthyroid animals, which might be caused by of antioxidant defences. These effects may result from its ability to
the accelerated metabolic rate present during hyperthyroidism. directly scavenge some ROS, including OH and O2 .
The hyperthyroid animals treated with RSV at 1 mg/g did not To evaluate non-enzymatic antioxidants, the GSH content was
present hypertrophy in the testes. The same result was observed measured. The present study demonstrated that the GSH levels
in the prostate of hyperthyroid rats treated with both RSV doses (1 did not differ in the testes of rats among all experimental groups.
and 10 mg/kg), showing a possible effect of RSV in preventing the There is no consistent change of this non-enzymatic antioxidant
hypertrophic effect of T3 in these organs. in the testes of hyperthyroid rats because several authors reported
In this study, we showed that hyperthyroidism reduced sperm a reduction [8,9], whereas other showed an increase in the GSH
motility, while no signicant differences were observed in the content in this tissue during hyperthyroidism [3,11]. These con-
morphology of the sperm. These data agree with clinical studies tradictory ndings may be attributed to the duration of drug
indicating that hyperthyroidism is associated with poor semen administration.
quality in humans, particularly impaired motility [32]. The RSV Another question to be discussed is that the treatments with 1
treatment prevented this loss of sperm motility induced by T3. and 10 mg/kg of RSV did not modify the sperm quality or the testi-
One of the most important causes of defective sperm function is cular oxidative status in the control groups. This result shows that
OS [7]. At the level of isolated spermatozoa, ROS can induce LPO and RSV protects rat sperm and testes from changes induced by hyper-
DNA fragmentation, decreasing sperm motility; while at the level thyroidism but did not alter these variables in healthy animals.
of the testis, OS is capable of reducing the steroidogenic capac- In conclusion, the results demonstrated that RSV prevented the
ity of Leydig cells and the capacity of the germinal epithelium to loss of sperm motility that may occur in hyperthyroidism. Further-
differentiate normal spermatozoa, affecting male fertility [33]. more, the data indicate that the treatment with RSV was able to
To evaluate whether this protection of sperm motility was prevent OS in the testes of hyperthyroid rats by preventing lipid
related to the antioxidant capacity of RSV, indicators of antioxidant peroxidation and avoiding changes in the activity of the antioxi-
status were measured. Previous studies reported that hyperthy- dant enzymes. It is difcult to predict which cell population of the
roidism was associated with increased LPO levels in testes [3,9,11]. testis is more susceptible to oxidative damage in a hyperthyroid
The early stage of LPO was detected using measurements of lipid state because the whole testis was used in this study. However,
hydroperoxides, whereas the nal products of LPO were measured the decrease in sperm motility in the hyperthyroid animals con-
using TBARS [34]. Our data showed that the T3 treatment increased rms that hyperthyroidism interferes with reproductive function
both the lipid hydroperoxide production and the TBARS levels in and that RSV may contribute to improving this situation by preven-
the hyperthyroid rat testes, supporting a disturbance of the nor- ting OS in the testis. Therefore, RSV has potential as a therapeutic
mal cell balance between the production of ROS and the capacity intervention for preserving sperm motility and testicular function
to neutralise their action, leading to OS. Moreover, our results during hyperthyroidism. Future research might be performed to
demonstrated, for the rst time, that RSV was able to minimise the determine the molecular mechanisms underlying the protective
oxidative damage in the testes of T3-induced hyperthyroid rats by effects of RSV on rat sperm and testes during hyperthyroidism.
decreasing lipid hydroperoxide production and TBARS levels. The
antioxidant mechanism of RSV may be related to its ability to detox- Conict of interest statement
ify O2 and nitrite anions, trapping the peroxyl radical and/or OH
and donating the hydrogen electron from its hydroxyl groups [15]. The authors declare that there are no conicts of interest.
This protective effect of RSV against LPO may also be explained by
the modulation of the enzymatic antioxidant defence by RSV.
In addition to the increase in LPO levels, changes in antioxidant Acknowledgements
defences have also been described in the testes of hyperthyroid rats
[3,8,9]. To evaluate the effect of RSV on antioxidant enzymes, we The authors are grateful to CAPES and CNPq for providing nan-
rst analysed the SOD activity. The data from this study showed cial assistance during the research.
that the SOD activity did not change across the different experi-
mental groups. Similarly, Lee et al. [4] showed that SOD activity References
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