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- Original Article -
Protective effects of estrogens and caloric restriction during
aging on various rat testis parameters
Khaled Hamden1, Dorothee
ElFeki1, Serge Carreau2
1Department of Animal Ecophysiology, Faculty of Sciences, University of Sfax, PB 802, Sfax 3018, Tunisia
2USC 2006 INRA- EA 2608, Biochemistry, Caen-14032, France
Aim: To investigate the effects of 17β-estradiol (E2),
Peganum harmala extract (PHE) and caloric restriction (CR)
on various testis parameters during aging.
Methods: Twelvemonth-old male rats were treated for 6 months with
either E2 or PHE, or submitted to CR (40%). Results:
Our results show that estrogens and CR are able to protect the
male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen
receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase
activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate
deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase
and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at
18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level
of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats.
Conclusion: Besides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay
testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the
reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large
quantities in the aged testis. (Asian J Androl 2008 Nov; 10: 837_845)
Keywords: aging; (phyto)estrogens; 17β-estradiol;
Peganum harmala; caloric restriction; rat testis; antioxidant enzymes
Correspondence to: Dr Serge Carreau, Department of Biochemistry, University of Caen, Esplanade de la Paix, Caen-14032, France.
Tel: +33-2-3156-5488 Fax: +33-2-3156-5120
Received 2008-01-24 Accepted 2008-06-16
Aging in men is accompanied by a reproductive senescence associated with a chronic state of oxidative stress
following a functional deficit in Leydig, Sertoli and germ cells [1_3]. The decrease of steroidogenesis associated with
the imbalance between prooxidant and antioxidant activities leads to oxidative damage of various cellular processes
. The risk of oxidative damage and the lipid peroxidation is especially high in steroid synthesizing tissues, because,
in addition to oxidative phosphorylation, they use molecular oxygen for steroid synthesis [5, 6]. Indeed, it has been
shown that free radicals inhibit steroidogenesis by interfering with cholesterol transport to the mitochondria and/or the
catalytic function of P450 enzymes [7, 8], which leads (i) to an increase of lipid peroxidation ; (ii) to an
enhancement of the toxicity indexes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate
dehydrogenase (LDH), gamma-glutamyl transferase (GGT) and alkaline phosphatase (PAL) activities ; and (iii) to the
decline of the antioxidant barrier. To protect against the adverse effects of reactive oxygen species (ROS),
mammalian cells are equipped with various enzymatic and
non-enzymatic antioxidant scavengers. The major enzymatic
antioxidants are superoxide dismutase (SOD), glutathione
peroxidase (GPx) and catalase (CAT) . Among the
non-enzymatic antioxidants are chemicals, such as
estrogens [12, 13], growth factors  or vitamin E ,
and flavonoids (e.g. quercetin and herbacetin), found in
many plants, such as Peganum harmala [16, 17]. These
bio-molecules are known by their capacity to scavenge
free radicals and to modulate the expression of genes
encoding antioxidant enzymes.
Caloric restriction (CR) is also known to slow
aging and to delay the appearance of age-associated
phy-siopathological changes [18, 19] and, therefore, to
induce some benefits on the longevity of rats . The
mechanisms underlying the robust protective effects
of CR remain to be identified; however, it has been
suggested that the most significant effect of CR on aging
is the associated reduction in oxidative stress at the
cellular level. Indeed, CR suppresses the age-related
oxidative damage of lipids, DNA and proteins, and
increases the resistances of cells to oxidative stress 
and/or induces changes in the expression of
stress-response genes in testicular cells of aged rats [22, 23].
We have reported that the rat testicular cells, including
germ cells, are able to synthesize estrogens, and these
cells are also equipped with estrogen receptors,
therefore suggesting a putative role for these female
hormones in spermatogenesis . Because aromatase
and antioxidant strategies are concerned with the
control of male fertility, our working hypothesis was to
check the long-term effect of treatment with either pure
estrogens (or phytoestrogens) or caloric restriction
during aging on aromatase and estrogen receptor gene
expression in rat testis. In addition, some parameters of
oxidative stress, such as lipid peroxydation
(thiobar-bituric acid reactive substance) and antioxidant
enzymatic activities (CAT, SOD and GPx), as well as other
enzyme activities involved in the toxicity index (AST,
ALT, LDH, GGT and PAL), have been measured in testes of rats at the end of the
2 Materials and methods
2.1 Animals and treatments
Male Wistar rats aged 12 months were used. Animals were maintained in the animal house facility (Faculty
of Sciences, University of Sfax, Sfax, Tunisia) at a
constant temperature of 25 ± 3ºC, under 12 h : 12 h
light:dark cycle. The animals (six per group) were fed with
standard chow and were given access to tap water
ad libitum. The 12-month-old rats were divided into four
groups and treated for 6 months: they were either fed
ad libitum (control) or received 17β-estradiol (1
μg/kg body weight, daily)  or Peganum
harmala extract (PHE) (50 mg/kg body weight, daily)  by gastric gavage,
or submitted to caloric restriction (CR) diet. In the CR
group, rats were fed with 60% of the quantity of food
given to the control animals (equivalent to 1 726 KJ/w
daily). The sham-control rats were gavaged with water.
The other six intact male rats aged 4 months were used
as a reference group. The handling of the animals was
approved by the local Ethical Committee for the care and
use of laboratory animals. At the age of 18 months, the
animals were weighed, then killed by decapitation, and the
trunk blood was collected. The serum was prepared by
centrifugation (1 500 × g, 15 min, 4ºC) and the testes
were removed, cleaned of fat and weighted; all these
samples were stored at _80ºC until used.
2.2 RNA extraction and quantification of transcripts
Total RNAs from testes were extracted using the
guanidium thiocyanate_phenol-chlorofom method of Chomczynski and Sacchi (1987) . Briefly, testes
were homogenized in 600 μL of lysis buffer (1 mol/L
Tris, 4 mol/L guanidium thiocyanate, 0.5% sarcosyl, 1%
β-mercaptoethanol), then 0.1 volume of 2 mol/L sodium
acetate, 1 volume of phenol and 0.2 volume of isoamylic
chloroform-alcohol (v/v : 49/1) were added to the preparation. After 15 min of incubation in a cold bath, the
samples were centrifuged at 10 000 × g at 4ºC for 15
min. RNAs were precipitated at _80ºC by adding 1 volume of
isopropanol. After centrifugation, the pellets were washed
with 75% ethanol, dried and dissolved in 50 μL of diethylpyrocarbonate treated water. They were stored
at _80ºC until used. The quantity of total RNA was
determined by the measurement of the optical density at
260 nm. The purity was evaluated by the ratio 260/280
nm and the integrity was controlled by electrophoresis on a
1.5% agarose gel.
For the semi quantitative reverse
transcription-polymerase chain reaction (RT-PCR), 2 μg of total RNAs were reverse transcribed into cDNA as follows: 1 h at
42ºC with 200 IU Moloney murine leukemia virus
reverse transcriptase (M-MLV-RT), 0.5 mmol/L dNTP,
0.2 μg oligo-dT and 20 IU RNasin in a final volume of
40 μL. Then, cDNAs coding for aromatase, estrogen
receptor alpha (ERα), estrogen receptor beta (ERβ) and
ribosomal protein L19 were amplified by PCR using
specific primers (Table 1) as reprorted elsewhere . We
have chosen L19 transcripts which did not vary among
the samples in order to correct the difference in the
quantities of total RNA used for reverse transcription .
The amplified products were run on a 2%-agarose-gel
stained with ethidium bromide, visualized under UV
transillumination and analyzed with NIH software
2.3 Steroid determinations and measurements of
antioxidant enzymatic activities
After homogenization of testes in a phosphate buffer
(1 g/2 mL), steroids were extracted by diethylether
according to our reported method . The estradiol and
testosterone levels were then measured by RIA using
highly specific antibodies from P.A.R.I.S (Compiègne,
France). The intra- and inter-assay coefficients of
variation were 8% and 5% for estradiol and 4.6% and 7.5%
for testosterone. The sensitivities were 6 pg/tube and
12 pg/tube respectively for estradiol and testosterone.
The lipid peroxidation was determined in the homogenates from control and treated rat testes by
quantification of the thiobarbituric acid reactive substances
(TBARS) using the method of Buege and Aust . The
SOD activity was assayed by the spectrophotometric
method of Marklund & Marklund . The activities of
GPx and CAT were measured by the method of Pagila and Valentine  and Aebi  respectively. The
protein level was determined by the method of Lowry
et al. . The testicular LDH, AST and ALT, GGT, and PAL
activities, were determined using commercial kits from
Sigma (Munich, Germany) and Boehringer (Mannheim, Germany).
For histological studies, pieces of testes were fixed
in a Bouin's solution for 24 h, then embedded in paraffin.
Sections of 5-μm thickness were stained with
hematoxylin-eosin and examined under Olympus CX41 light
microscope (Olympus Industrial America Inc., Orangeburg,
Data are presented as means ± SEM. The
determinations were performed from six animals per group and
the differences were examined by the one-way analysis
of variance (ANOVA) followed by the Fisher test, and
the significance was accepted at P < 0.05 (StatView;
SAS Institute, Cary, NC, USA).
3.1 Aromatase, ERα and ERβ gene expression in testis
of control and treated animals
We performed a semi-quantitative RT-PCR to
determine whether the amounts of aromatase and estrogen
receptors (ERs) transcripts in testes were affected by
aging and the various treatments. In the 18-month
untreated rats, the amount of aromatase transcripts was
significantly lower compared to that of 4-month-old
animals. Conversely, in the E2, PHE and CR groups the
level of aromatase mRNA was increased by 62%, 44% and 65%, respectively, compared to 18-month-old
control rats and was significantly higher than that in
12-month-old rats (Figure 1A).
The amount of ERα mRNA was decreased of 56% between 4 and 18 months in control rats (Figure 2A).
Conversely, in the three other groups, the levels of
transcripts were significantly higher (66%, 60% and 58%,
respectively, in E2, PHE and CR-treated rats) compared
to control rats aged 18 months. The levels of ERβ
transcripts were significantly decreased by 85% in control
rats between 4 and 18 months (Figure 3A). However, in
18-month-old rats the treatments induced a significant
(P < 0.01) increase of the level of ERβ transcripts: 87%,
37% and 77%, respectively, in E2, PHE and CR animals
compared to controls of the same age.
3.2 Effects of various treatments on testosterone and
estradiol concentrations in testis
The levels of testosterone were significantly
decreased (P < 0.001) in 18-month-old rats compared to
animals aged of 4 months (Figure 4C). After treatment
with either E2 or PHE and CR, an increase of the
endo-genous testosterone concentrations (54%, 49% and 42%
respectively, compared to control rats) was recorded and
the levels were identical to that of 12-month-old rats.
The testicular level of estradiol was diminished by
33% between the age of 4 and 18 months (Figure 4B).
However, in E2, PHE and CR-treated rats the
estradiol levels were enhanced by 103%, 87% and 18%,
respectively, compared to the control rats (Figure 5B).
As far as the blood estradiol levels were concerned,
significant (P > 0.05) variations were observed in control
rats as well as in CR animals; conversely, and as
expected in E2 and PHE-treated rats, the levels of estradiol
were increased by 127% and 60% (Figure 4C).
3.3 SOD, CAT, GPx activities and TBARs levels in
testes of control and treated rats (Table 2)
After either estradiol, plant extract or caloric
restriction treatment, a significant (P < 0.05) increase in SOD
activities (49%, 66% and 33%, respectively) was recorded compared to the control rats. The CAT activity
was enhanced by 19%, 21% and 27%, respectively, in
E2, PHE and CR animals compared to untreated 18-month-old rats. It is of note that these enzyme activities
were higher than those measured in 1-year-old rats. The
GPx activity was increased in the three groups of treated
rats (23%, 39% and 18%, respectively), but not
significantly (P > 0.05) compared to the control animals. The
testicular TBARs levels were significantly increased in
the control rats aged 18 months compared to 1-year-old
animals. When rats were treated either with E2 or PHE,
or submitted to CR, a significant (P < 0.05) decrease in
TBARs levels by 20%, 16% and 26% was observed compared to the control animals of the same age.
3.4 LDH, AST, ALT, GGT and PAL activities in rat
testes (Table 2)
In control rats aged 12 and 18 months a significant
increase in the activity of testicular LDH, GGT, PAL, AST
and ALT was observed when compared to 4-month-old animals. In 18-month-old E2, PHE and
CR-treated rats, a significant decrease in all these enzyme activities
compared to the untreated rats of the same age was recorded.
3.5 Histological changes in testes of control and treated
Because a positive effect of E2, PHE and CR was
observed on the relative testicular weight, we performed
histological analyses of spermatogenesis (Figure 5).
Although not evaluated quantitatively, a qualitative
evaluation of spermatogenesis was performed: a depletion of
germ cells at various stages of development was observed in old-untreated rat (Figure 5C), when compared
to 4-month-old rats (Figure 5A). In E2 (Figure 5D),
PHE (Figure 5E) and CR (Figure 5F)-treated rats,
spermatogenesis proceeded normally and was similar to that
of 12-month-old rats (Figure 5B).
Our results indicate that in male rats aging is
accompanied by an increase in the production of free radicals.
These changes are likely the consequence of a lower
number of Leydig, Sertoli and germ cells in old rats
compared to younger animals, as reported by Chen
et al.. These decreases in the numbers of testicular cells
might lead to lower levels of aromatase and ERα/β
transcripts and, therefore, to a diminution of testosterone
and estrogen syntheses. The low steroids output
synthesized mainly by the Leydig cells could be also related
to the production of free radicals, which induce
damages to the cell macromolecules content, as shown by
the increase of the toxicity indexes, such as AST, ALT,
PAL, GGT and LDH activities and the lipid peroxidation
in testes [9, 10]; in parallel, the testicular SOD, CAT and
GPx contents are diminished . All these changes
can alter the testicular cells, including spermatozoa and,
therefore, the sperm production leading to an alteration
of the male fertility. Although we did not perform
quantitative analyses of the germ cells, it was clear that the
spermatogenesis was altered in the untreated
18-month-old rat testes, which likely accounts for the decrease in
testicular weight reported by Henkel et al.  and in
the agreement with our previous study in which a
diminution of the number of spermatozoa has been observed
in aged rats .
However, in rats treated with either estrogens or plant
extract over 6 months, the activities of the above
enzymes were back to their normal control levels,
suggesting an antioxidant protective role of estrogens .
Moreover, caloric restriction could slow aging and
induce some benefits on the longevity of rats .
Because we have demonstrated that most of the rat
testicular cells express aromatase and are equipped with
estrogens receptors  we have herein analyzed the effects
of either estrogens or CR over a period of 6 months
(12_18 months), corresponding to aging in the male rat. The
various treatments induce a positive and a significant
effect on aromatase and ERs genes expression in aged rat
testes; moreover, the classical parameter of oxidative
stress (TBARs) and antioxidant enzymatic activities
(catalase, superoxide dismutase and glutathione peroxydase) have been affected, particularly by the
estrogen treatment. Therefore, we could speculate that an
increase in free radicals formation consecutive to a
diminution of the aromatase and ERs genes expression
associated with a lower level of testosterone in testis of
18-month-old rats (compared to younger animals), likely
accounts for the observed alterations of spermatogenesis
in aged animals, as reported by Chen et al. . The
mechanisms concerned by the protective effect of
estrogens treatment are not clearly understood up to now.
However, estrogens enhance the antioxidant enzyme activities, such as SOD, CAT and GPx  and, therefore,
could protect the testicular cells against damage and death
produced by free radicals. Moreover, this suggests that
the beneficial effect of a caloric restriction diet could be
in part mediated by estrogens via a parallel increase of
ERs expression, as reported elsewhere . Indeed,
lower estrogen levels are responsible for enhanced-free
radical generation [40, 41], leading to an increase in lipid
peroxidation, and a concommittant reduction of the
antioxidant barrier activity, especially in the testis .
Estrogens can exert an antioxidant role by scavenging free
radicals  and, therefore, they may prevent any
damage induced by these free radicals on cell protein and
DNA contents . Consequently, E2, PHE and CR treatments appear to be an adaptive strategy to preserve
testicular integrity and function during aging in male rats.
The diminution of the mitochondrial ROS production
could also be evoked as a putative target for estrogens
and caloric restriction . As reported by Gancarczynk
et al.  in the bank vole, additionnal studies are
necessary to elucidate the mechanisms of actions of estrogens
in the testicular cells and, in particular, at the
In conclusion, according to the localization of
aromatase in Leydig cells as well as in germ cells of
rodents , and taking into account the widespread
distribution of ERs in the testicular cells , an
antioxidant role of estrogens is possible during aging.
According to our observations in aged rats, the testicular
expression of aromatase and ERs in estrogen and
PHE-treated rats, as well as in animals submitted to CR, are
higher than in control animals. That suggests a protective, physiologically relevant effect of estrogens,
especially in lowering oxidative stress via the increase
of the cellular antioxidant defense system, as suggested
by Borras et al.  and Nam et
al. . In our preliminary study , a decrease in testicular estradiol
levels associated with diminutions in the number and motility
of epididymal spermatozoa were recorded during aging.
In addition, caloric restriction was beneficial to support
full spermatogenesis in old rats. Therefore, a low
caloric diet might improve the protection of the cells against
ROS via an improvement of the cellular antioxidant
defense system in which estrogens are probably concerned,
as demonstrated by the effectiveness of the estradiol
Parts of this work were supported by grants from
the French Ministry of Research and Education and from
the National Institute of Research in Agronomy (Paris,
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