Biol Trace Elem Res
DOI 10.1007/s12011-010-8702-5
Testicular Histomorphometry and Ultrastructure of Rats
Treated with Cadmium and Ginkgo biloba
Fabrícia de Souza Predes & Juliana Castro Monteiro &
Sérgio Luis Pinto Matta & Márcia C. Garcia &
Heidi Dolder
Received: 11 February 2010 / Accepted: 14 April 2010
# Springer Science+Business Media, LLC 2010
Abstract The aim of this study is to investigate the association of a single low dose of Cd
and daily doses of Ginkgo biloba extract (GbE) on the testis and accessory glands of rats.
The animals were treated with a single dose of 3 µmol/kg body weight of cadmium chloride
(CdCl2) and/or 100 mg/kg body weight of GbE. The plasma testosterone levels; corporal,
testicular, and accessory glands weight; gonadosomatic index, volumetric proportion; and
absolute volume of testicular components did not change after the treatments. CdCl2 caused
significant reduction in Leydig cells volume and altered Leydig cell morphology, as well as
vacuolated Sertoli cells cytoplasm, irregular chromatin condensation of late spermatids, and
modified acrosome formation. However, animals that received GbE did not show these
alterations. The reversal of Cd-induced alterations by the extract is a strong indication that
G. biloba is helpful in diminishing the effect of Cd toxicity.
Keywords Wistar rat . Ginkgo biloba . Cadmium . Morphometry and ultrastructure
Introduction
Environmental problems have recently increased exponentially because of industrial
pollution and the rapid growth of the human population. Toxic chemicals such as heavy
metal ions discharged into the air, water, and soil get into the food chain from the
F. de Souza Predes : J. C. Monteiro : M. C. Garcia : H. Dolder
Rua Charles Darwin, s/n, Department of Anatomy, Cellular Biology, Physiology and Biophysics,
State University of Campinas, Campinas CP 6109 São Paulo 13083-863, Brazil
S. L. P. Matta
Department of General Biology, Federal University of Viçosa, Campus Universitário Viçosa,
Minas Gerais, Brazil
F. de Souza Predes (*)
Departamento de Anatomia, Biologia Celular e Fisiologia e Biofísica, Instituto de Biologia,
Universidade Estadual de Campinas-UNICAMP, CP 6109, São Paulo, SP, Brazil 13083-863
e-mail: fpredes@yahoo.com.br
Predes et al.
environment. By entering into the biological system, they disturb biochemical processes,
leading to health abnormalities and, in some cases, to fatal consequences. One of these
heavy metals is cadmium (Cd) [1].
In adult male rats, acute or chronic treatment with Cd induces a well-documented
toxic reaction in the reproductive organs and the testes are particularly susceptible to
Cd [2]. After acute exposure, cadmium-induced damage can be found at interstitial
and tubular levels [3]. Metal-induced testicular dysfunction may arise from disturbances in Sertoli cells, which support spermatogenesis, or Leydig cells, that are responsible for androgen production under control of the hypothalamic–pituitary–testicular
axis [4].
Some studies have suggested that the primary site of cadmium action in the testis is the
Sertoli cell, altering the tight junction (TJ) and disrupting the blood–testis barrier (BTB) [5].
Moreover, this metal causes fragmentation of actin microfilament bundles in the
seminiferous epithelium [5, 6]. The disruption of Sertoli cell TJ at the BTB could lead to
secondary damage in both basal and apical ectoplasmic specializations. This, in turn, leads
to germ cell loss from the epithelium [7]. Hew et al. [8] reported that cadmium began to act
during early stage VIII to induce spermiation failure.
Reports from various experimental studies revealed that damage arising from the
cadmium-induced reactive oxide species (ROS) production may be protected by the
intervention of free radical scavengers and antioxidants. β-Carotene, vitamin C, and
vitamin E [9, 10] are some of them. It was reported that extracts of Hibiscus sabdariffa
[11], Pluchea lanceolata [12], Allium cepa Linn, and Allium sativum Linn [13] extracts and
green tea [14] have also the ability to prevent or attenuate cadmium toxicity in various rat
and mice organs.
Recently, the antioxidant properties of Ginkgo biloba extract (GbE) have been
intensively examined for their potential beneficial action. It has been reported that GbE
scavenges several free radical species in vitro and in vivo [15–17]. GbE protects the testis
from diethylstilbestrol-induced injury [18], uranium-induced genotoxicity and oxidative
stress in albino mice [1], and mercury (II)-induced oxidative tissue damage in rats [19].
Moreover the GbE 761 (especially at the dose of 50 mg/kg) enhances the copulatory
behavior of male rats [20].
Since GbE is known to exert protective influences against the action of free radicals, we
hypothesized that application of such an extract might attenuate the cadmium-induced
damage in the testis of adult rats. The purpose of this study was to evaluate the cadmium
modifications in testicular morphology with morphometry and ultrastructure and the
capacity of GbE to attenuate this damage.
Material and Methods
Animals
The study was carried out on 90-day-old adult male Wistar rats obtained from the
Multi-research Center for Biological Investigation (State University of Campinas,
Campinas, SP, Brazil). The experiments were carried out according to the Guide for
Care and Use of Laboratory Animals and were approved by the Committee for Ethics
in Animal Experimentation of UNICAMP (registration number: 850-1). Animals were
housed three per cage in cycles of 12-h light, 12-h dark. Food and water were provided
ad libitum.
Testicular Histomorphometry and Ultrastructure of Rats Treated with Cadmium and Ginkgo biloba
G. biloba Extract
GbE (Bioflavin drops) was used as the test herbal product in the present study. It is
produced and marketed by Herbarium Botanical Laboratory LTDA (Brazil). Each milliliter
contained 80 mg of dry extract of G. biloba leaves of which 19.2 mg was ginkgo flavonoids
and 4.8 mg was terpenelactone according to manufacturer's specifications.
Treatment
Twenty-four rats were randomly divided in four groups that received treatment with the
following:
Group
Group
Group
Group
1:
2:
3:
4:
water for 56 days (control)
GbE for 56 days
CdCl2 (single dose) and water for 56 days
CdCl2 (single dose) and GbE for 56 days (Cd + GbE)
GbE was administered daily by gavage in a dose of 100 mg/kg body weight (BW) [20, 21].
The BW was recorded weekly in order to calculate the GbE dose. Cd was injected
intraperitoneally as a single dose of 3 μmol/BW of cadmium chloride (CdCl2) [22]. Groups
1 and 3 received water by gavage to maintain the same conditions. The animals were
euthanatized after 56 days, because this interval represents the duration of spermatogenesis [23].
Hormone Measurement
Rats were intraperitoneally anesthetized with ketamine (80 mg/BW) and xylazine (5 mg/BW).
Blood samples were obtained from the cava vein in a heparinized syringe. The plasma was
separated by centrifugation and stored at -72°C for subsequent hormone assays. Plasma levels
of total testosterone were estimated by Testosterone Total RIA—Catalogue #DSL 4000. The
assay sensitivity was 0.8 ng/ml.
Tissue Preparation
Rats were anesthetized with ketamine (80 mg/BW) and xylazine (5 mg/BW). The animals
were fixed by whole body perfusion. Briefly, after a saline wash to clear the vascular bed of
the testis, they were perfused with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M
sodium phosphate buffer, with pH 7.2 for 25–30 min and then fixed in the same solution for
24 h, at 4°C. Testis, epididymis, prostate, seminal vesicles, and coagulating gland were
removed, postfixed in the same solution overnight, and then weighed. Historesin-embedded
testis fragments were sectioned at 3-µm thickness and stained with toluidine blue/1%
sodium borate.
Morphometry
The testicular albuginea was dissected out and weighed. The weight of testicular parenchyma
was obtained subtracting the mass occupied by the albuginea from the testis total weight, thus
providing the net weight of the organ’s functional portion. The gonadosomatic index (GSI)
was recorded, and the testes weight expressed as a percentage of the total BW, GSI = (testes
weight/total BW) × 100. The volumetric proportions of testicular tissue components were
determined by light microscopy, by projecting a 432-intersection grid in Image Pro Plus
Predes et al.
software associated to an Olympus BX-40 microscope. Ten fields were chosen randomly
(4,320 points) over testicular parenchyma in each animal at 400× magnification. Points were
scored and classified as one of the following testicular components: seminiferous tubule,
Leydig cell, blood vessels, lymphatic space, and connective tissue. The volume of each
component of the testis was determined as the product of the volumetric proportion and
parenchyma volume. For subsequent morphometric calculations, the specific gravity of testis
tissue was considered to be 1.0 [24]. Individual volume of a Leydig cell was obtained from
the nucleus volume and the proportion between nucleus and cytoplasm. For this purpose,
Leydig cell nucleus diameter was obtained from the assessment of 30 cells/animal in Image
Pro Plus software associated to an Olympus BX-40 microscope at 1,000× magnification.
Leydig cell nuclear volume was expressed in μm3 and obtained by the formula (4/3)πR3,
where R = nuclear diameter/2. To calculate the proportion between nucleus and cytoplasm,
a 432-intersection grid was projected in Image Pro Plus software associated to an Olympus
BX-40 microscope at 1,000× magnification. One thousand points over nuclei and
cytoplasm of Leydig cells were counted for each animal. The cytoplasm volume was
obtained by the formula: (% of cytoplasm × nuclear volume)/% nucleus. The number of
Leydig cells per testis and per gram of testis was estimated from the Leydig cell individual
volume and the volume occupied by Leydig cells in the testis parenchyma [25].
Tissue Preparation for Transmission Electron Microscopy
The tissues were postfixed with 1% osmium tetroxide in the same buffer at 4°C, dehydrated
in acetone, and embedded in epoxy resin. Ultrathin sections (20–60 nm) were cut with
diamond knives and stained with 2% uranyl acetate (25 min) and 2% lead citrate (10 min)
prior to observation with a transmission electron microscope (Zeiss, Leo 906).
Statistical Analysis
Comparison of the values of control and treated groups was done by analysis of variance
(ANOVA) followed by Tukey’s test. The results were considered significant for P<0.05.
For all values, the means ± standard error mean (SEM) were calculated.
Results
Hormone Measurement
The plasma testosterone levels are shown in Fig. 1. These measurements were highly
variable; therefore, no clear statistical differences could be found.
Morphometry
No statistically significant differences were observed for the control and treated groups in
BW gain and testis weight, testicular parenchyma, albuginea, and GSI (Table 1).
The weights of epididymis, prostate, and seminal vesicle were similar when compared
with the control group. Statistical evaluation showed the coagulating gland weight of group
4 to be significantly different from the control group (Table 2).
The volumetric proportion of testicular parenchyma components is shown in Table 3. No
significant changes were observed in all groups studied. The absolute volumes of the
Testicular Histomorphometry and Ultrastructure of Rats Treated with Cadmium and Ginkgo biloba
2,50
Plasma testosterone
(ng/mL)
Fig. 1 Plasma testosterone of
adult rats treated with GbE and/or
cadmium (mean ± SEM).
1—Control, 2—GbE, 3—CdCl2,
4—CdCl2 + GbE
2,00
1,50
1,00
0,50
0,00
1
2
3
4
Groups
1- Control; 2- GbE; 3- CdCl2; 4- CdCl2 + GbE
parenchyma components were not significantly different for all the experimental groups
(Table 4).
A statistically significant reduction of nuclear diameter, nuclear volume, cytoplasmatic
volume, and individual Leydig cell volume was observed in the cadmium-treated group
when compared with control groups. The cytoplasmatic volume of Leydig cell reduced
significantly. The number of Leydig cells per testis and per gram of testis was not different
for the groups studied (Table 5).
Transmission Electron Microscopy
In the control and GbE-treated groups, the seminiferous tubule consisted of typical Sertoli
and germ cells. In the interstitium, Leydig cells showed a large nucleus. The cytoplasm
contained Golgi complexes, patches of rough endoplasmic reticulum (RER), well-defined
and abundant SER, and numerous mitochondria (Fig. 2).
In the cadmium-treated group, Sertoli cell cytoplasm showed vacuolation and loss of
cytoplasmic organelles and contained large, lysosome-like vacuoles, with polymorphous
interiors and electron-lucent lipid droplets (Fig. 3a). There was an increase in the
intracellular space at the basal compartment, namely, Sertoli cells, spermatogonia, and
preleptotene spermatocytes. At the adluminal compartment, expanded intercellular spaces
between spermatocytes and spermatids embedded in Sertoli cell prolongations were also
observed (Fig. 3b). The elongated spermatids exhibited heterogeneous and granular
chromatin. Structural alterations in some acrosomes were observed (Fig. 3b, d). Blood
vessels were affected, and the nucleus of endothelial cells was irregular in shape (Fig. 3a).
Table 1 Basic Data (g) and GSI (%) of Adult Rats Treated with GbE and/or Cadmium (Mean ± SEM)
Parameters
Control
GbE
CdCl2
CdCl2 + GbE
Body weight gain
93.83±16.84
44.40±8.78
70.50±1.65
Final body weight
415.33±23.85
387.00±16.68
442.00±8.40
415.67±8.74
80.00±17.23
1.59±0.08
Testis weight
1.61±0.03
1.5±0.12
1.63±0.04
Testicular parenchyma
1.53±0.03
1.47±0.12
1.56±0.03
1.51±0.08
71.50±3.64
71.00±2.39
74.50±4.62
76.33±4.34
0.79±0.05
0.79±0.05
0.74±0.02
0.77±0.04
Albuginea (mg)
GSI
n=6 for each group
Predes et al.
Table 2 Organ Weights (mg) of Adult Rats Treated with GbE and/or Cadmium (Mean ± SEM)
Control
Epididymis
535.83±19.29
543.80±33.96
544.00±12.73
538.00±23.24
443±40.31
430.40±37.67
532.67±33.17
444.33±28.94
Ventral prostate
GbE
CdCl2
CdCl2 + GbE
Parameters
Dorsolateral prostate
349.83±31.26
373.80±15.13
414.83±31.64
Coagulating gland
Seminal vesicle (g)
207.67±22.09
1.16±0.14
235.40±13.14
0.95±0.06
214.33±6
0.99±0.04
365±24.70
254.83±9.73*
1.08±0.07
n=6 for each group
*Indicates significant differences (P<0.05) between control and treated groups
The Leydig cells showed a nuclear envelope with many deep indentations, dense
cytoplasm, and poorly defined organelles such as SER and mitochondria (Fig. 3c).
The administration of GbE is effective in maintaining normal ultrastructure of rat
seminiferous tubules and the interstitium (Fig. 4).
Discussion
A low Cd dose was considered more appropriate for this study, since this dose is the
smallest that causes testicular damage in Wistar rat according to Cabral [26] and also
because environmental contaminations usually occur in low doses. The dose of GbE was
calculated to be proportional to that routinely used by patients. If a high dose of Cd had
been used, this dose of GbE would probably have had little or no effect.
In this study, the BW gain was not statistically significant. Beek et al. [27] affirm that in
long-term toxicity studies in rats (27 weeks), GbE did not induce any significant toxic effect
up to the dose of 500 mg/kg daily. According to this report [28], animals that received
0.45 mg/kg BW of CdCl2 injected subcutaneously did not change their BW. The present
investigation showed no change in the testis weight, which compares favorably with
previously published results using cadmium [29] and GbE [27]. On the other hand, in
several studies of cadmium treatment, decreased testicular weight was found [28, 30, 31].
However, these studies used different doses, exposure routes, and rat strains, making them
difficult to compare.
The treatments used in this study did not alter the weight of epididymis, prostate, and
seminal vesicle. The coagulating gland weight was higher in the group that received GbE
Table 3 Volumetric Proportion (%) of Testicular Parenchyma Components of Adult Rats Treated with GbE
and/or Cadmium (Mean ± SEM)
CdCl2 + GbE
Parameters
Control
GbE
CdCl2
Seminiferous tubule
75.77±4.98
79.58±1.44
76.42±2.86
Interstitium
24.23±4.98
20.42±1.44
23.58±2.86
22.40±1.79
Lymphatic space
14.67±3.84
11.96±1.31
14.25±1.96
14.35±1.65
77.60±1.79
Blood vessels
3.38±0.5
2.81±0.30
3.37±0.69
2.39±0.35
Connective tissue
0.93±0.41
1.07±0.13
1,00±0.23
0.63±0.12
Leydig cells
5.25±0.76
4.55±0.35
4.95±0.65
5.03±0.30
n=6 for each group
Testicular Histomorphometry and Ultrastructure of Rats Treated with Cadmium and Ginkgo biloba
Table 4 Absolute Volume (mL) of Testicular Parenchyma Components of Adult Rats Treated with GbE and/
or Cadmium (Mean ± SEM)
Parameters
Control
GbE
CdCl2
CdCl2 + GbE
Seminiferous tubule
1.166±0.087
1.161±0.095
1.189±0.051
1.174±0.068
Interstitium
0.368±0.070
0.305±0.030
0.366±0.043
0.358±0.031
Lymphatic space
0.222±0.054
0.180±0.023
0.222±0.03
0.217±0.028
Blood vessels
0.052±0.008
0.041±0.005
0.052±0.01
0.036±0.005
Connective tissue
0.015±0.006
0.016±0.002
0.016±0.004
0.009±0.002
Leydig cells
0.080±0.010
0.067±0.007
0.077±0.01
0.075±0.005
n=6 for each group
and cadmium. Except for the coagulating gland, these results are similar to those reported
by some GbE studies [21, 32]. However, according to some studies [28, 30, 33], cadmium
has been reported to have a pronounced effect on sex organ weight, which is the primary
indicator of possible alteration in androgen status. The unaltered weight of the accessory
sex glands thus supports the results of unchanged testosterone plasma level after 56 days of
the treatment with a single dose of cadmium. However, histopathologic changes were found
at lower Cd concentrations than those necessary to obtain significant effects on other
parameters and organs [34].
According to Cabral [26], three days after acute intoxication of rats with 2.5 µmol of
CdCl2, some damage in the interstitium can be observed, such as, edema, hyalinization, and
blood vessel congestion, although, normal organization was observed for seminiferous
epithelium, using light microscopy. Yano [22] observed after 5 and 10 days the same
alterations described by the author above. But 15 days after cadmium administration, these
alterations were partially recovered. In the present study, the morphometric parameters,
such as the volumetric proportion and absolute volume of testicular compartments and
interstitial components, did not vary significantly between the groups studied. These results
corroborate the studies above. This may have occurred due to the time allowed to complete
a spermatogenic cycle, which also permitted the partial recovery of the testicular
parenchyma. Long-term exposure (6 and 12 months) to low oral doses of cadmium
decreases tubular volumetric densities, epithelial percentages, and the volume fraction of
the interstitium, while they increase seminiferous tubule and lumen diameters in mice testis
Table 5 Nuclear Diameter (μm), Individual Leydig Cell Volume (μm3), and Number of Leydig Cells in
Testis of Adult Rats Treated with GbE and/or Cadmium (Mean ± SEM)
Parameters
Nuclear diameter
Nuclear volume
Control
GbE
CdCl2
CdCl2 + GbE
6.32±0.07
6.11±0.13
5.30±0.07*
6.15±0.08
132.37±4.27
120.11±7.39
78.27±3.28*
122.03±4.67
Cytoplasmic volume
325.08±28.77
270.70±17.41*
183.88±8.38*
318.27±17.61
Leydig cell volume
457.45±32.94
390.81±23.04
262.15±11.07*
440.30±19.63
No. per testis (106)
186.7±41.2
179.1±30.4
294±36.3
174.3±15.6
No. per gram of testis (106)
128.5±29.8
119.4±11.9
189.7±24.5
n=6 for each group
*Indicates significant differences (P<0.05) between control and treated groups
115.6±8.8
Predes et al.
Fig. 2 Testis of control rats (a, c, and e) and rats treated with GbE (b, d, and f). a, b Seminiferous
epithelium showing Sertoli cell (SC) and spermatogonia (G) in the basal compartment and round spermatids
(RS) in the adluminal compartment. Interstitium showing lymphatic space (LY) and blood vessels (B). c, d
Adluminal compartment showing round spermatids (RS) in different stages of differentiation and residual
bodies (RB). e, f Leydig cells (LC) showing large nucleus and cytoplasm rich in mitochondria (m),
endoplasmatic reticulum (r), and Golgi complex (gc). Scale bars = 5 μm
Testicular Histomorphometry and Ultrastructure of Rats Treated with Cadmium and Ginkgo biloba
Fig. 3 Testis of rats treated with cadmium. a Seminiferous epithelium showing Sertoli cell (SC) with large
vacuoles (V), a lipid droplet (L), and normal spermatogonia (G) in the basal compartment. S spermatocyte, I
interstitium showing a damaged blood vessel (B) and the lymphatic space (LY). Expanded intercellular space
(*). b Presence of deformed elongated spermatid (ES) without condensed chromatin and altered acrosome
(A) enclosed in fragmented Sertoli cell (SC) cytoplasm. Expanded intercellular space (*). c Leydig cell (LC)
showing large nucleus with prominent nucleolus. Presence of dense cytoplasm with endoplasmic reticulum
(r) and few other cytoplasmic organelles. d Adluminal compartment with elongated spermatids (ES),
condensed chromatin, and altered acrosomes (A) and round spermatids (RS) near a vacuolated Sertoli cell
(SC). Scale bars = 5 μm
[3]. These authors also affirm that cadmium withdrawal leads to initial recovery of
morphology; however, cadmium could have caused irreversible testis damage.
Despite the reduction of Leydig cell cytoplasm in the GbE group compared with the
control, these data are comparable with previous studies with rats [24, 35, 36]. The
significant reduction of Leydig nuclear diameter that occurred in the cadmium treated group
in accordance with the Leydig cell nuclear shrinkage and hyperchromatic images was
reported by Blanco et al. [37], who also found less circular Leydig cell nuclei.
Ultrastructural observations, such as a diminished amount of endoplasmatic reticulum,
mitochondria, and dense cytoplasmatic matrix, were found for Leydig cells. Taken together,
these findings suggest that cadmium is directly toxic to Leydig cells, supporting the results
of earlier studies [38].
The increased number of Leydig cells in the cadmium group of this study may be due to
the differentiation of immature precursor cells that have the capacity to repopulate the testis,
after destruction of the mature cell population due to cadmium treatment [39, 40]. Also,
Predes et al.
Fig. 4 Testis of rats treated with CdCl2 and GbE on the first day and only GbE on the following days. a
Seminiferous tubule showing basal compartment with Sertoli cell (SC) and spermatogonia (G) and adluminal
compartment with round spermatids (RS). b Adluminal compartment with normal round spermatids (RS) and
elongated spermatids (ES) without condensed chromatin enclosed in Sertoli cell (SC) cytoplasm. c Leydig
cell (LC) showing large nucleus and cytoplasm rich in mitochondria (m) and endoplasmatic reticulum (r). d
Normal blood vessel with an elongated endothelial cell nucleus. Scale bars = 5 μm
Blanco et al. [3] affirms that the cadmium withdrawal led to a hyperplasia of this cell
population, which could be an answer to offset the morphologic damage incurred.
The Sertoli cells have been indicated as the most common target cells for Cd toxicity in
the seminiferous epithelium [2, 41], and the main morphologic responses are the
vacuolization [33], alteration of intercellular junctions [4, 42], and the accumulation of
lysosome-like structures with polymorphous interiors and residual bodies [33, 43]. In the
present study, elongated spermatids without condensed chromatin and/or altered acrosomes
were also observed. The results observed in this study demonstrate the toxicity of cadmium
even at lower doses. Various studies affirm that the Sertoli cell damage was caused due to
the disruption of TJs in BTB; however, these changes were observed in studies using a high
dose of cadmium (3 mg/kg) [5, 7]. However, Chung and Cheng [44] reported that 5 µM of
CdCl2 in primary Sertoli cells cultured in vitro can disrupt the inter-Sertoli TJs. These
Testicular Histomorphometry and Ultrastructure of Rats Treated with Cadmium and Ginkgo biloba
authors believed that this dose was noncytotoxic to Sertoli cells because the DNA content
of CdCl2-treated cultures was not different from the untreated ones.
Although many studies have elucidated the effects of cadmium in the testis, few studies
have quantified the morphologic consequences of short-term low doses of cadmium. This
study emphasizes the importance of morphometry in conjunction with morphologic
analysis, since, without the morphometry, the fine alterations caused by acute low dose of
cadmium were not detected.
Taken together, the results show that the association of GbE and cadmium in a low dose
is effective to maintain Leydig and Sertoli cell morphology and ultrastructure. The reversal
of the effect of Cd by the extract is a strong indication that G. biloba is helpful in protecting
testis from the toxic effects of cadmium. However, further studies are required to elucidate
whether the protection afforded can clearly be linked to the G. biloba antioxidant property.
Acknowledgements The authors thank Dra. Karina Carvalho Mancini (Federal University of Espírito
Santo, University Center of North of Espírito Santo, Department of Health, Biological and Agricultural
Sciences) for her help in preparing the manuscript, Dra. Regina Célia Spadari Bratfisch (Department of
Physiology—UNICAMP) for the testosterone analysis and critical analysis of the article, Júnior de Souza
Predes for elaborating a cell counting program for the morphometric measurements, and Dr. Ronaldo Wada
for his help with the statistical analysis. This study was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP, Grants 2006/00040-9, Brazil), Fundo de Apoio ao Ensino, à Pesquisa e à
Extensão (FAEPEX), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
References
1. Çavuşoğlu K, Yapar K, Yalçin E (2009) Royal Jelly (Honey Bee) Is a potential antioxidant against
cadmium-induced genotoxicity and oxidative stress in albino mice. J Med Food 12:1286–1292
2. Ren X, Zhou Y, Zhang J (2003) Metallothionein gene expression under different time in testicular Sertoli
and spermatogenic cells of rats treated with cadmium. Reprod Toxicol 17:219–227
3. Blanco A, Moyano R, Vivo J et al (2007) Quantitative changes in testicular structure in mice exposed to
low doses of cadmium. Environ Toxicol Pharmacol 23:96–101
4. Bizarro P, Acevedo S, Niño-Cabrera G et al (2003) Ultrastructural modifications in the mitochondrion of
mouse Sertoli cells after inhalation of lead, cadmium or lead-cadmium mixture. Reprod Toxicol 17:561–566
5. Wong C, Mruk DD, Lui W, Cheng CY (2004) Regulation of blood-testis barrier dynamics: an in vivo
study. J Cell Science 117:783–798
6. Hew KW, Heath GL, Jiwa AH, Welsh MJ (1993) Cadmium in vivo causes disruption of tight junctionassociated microfilaments in rat Sertoli cells. Biol Reprod 49(4):840–849
7. Wong C, Mruk DD, Siu MKY, Cheng CY (2005) Blood–testis barrier dynamics are regulated by α2macroglobulin via the c-Jun N-terminal protein kinase pathway. Endocrinol 146:1893–1908
8. Hew KW, Ericson WA, Welsh MJ (1993) A single low cadmium dose causes failure of spermiation in
the rat. Toxicol Appl Pharmacol 122:15–21
9. El-Demerdash FM, Yousef MI, Kedwany FS et al (2004) Cadmium-induced changes in lipid
peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective
role of vitamin E and β-carotene. Food Chem Toxicol 42:1563–1571
10. Gupta RS, Kim J, Gomes C et al (2004) Effect of ascorbic acid supplementation on testicular
steroidogenesis and germ cell death in cadmium-treated male rats. Mol Cell Endocrinol 221:57–66
11. Asagba SO, Adaikpoh MA, Kadiri H et al (2007) Influence of aqueous extract of Hibiscus sabdariffa L.
petal on cadmium toxicity in rats. Biol Trace Elem Res 115:47–57
12. Jahangir T, Khan TH, Prasad L et al (2005) Pluchea lanceolata attenuates cadmium chloride induced
oxidative stress and genotoxicity in Swiss albino mice. J Pharm Pharmacol 57:1199–1204
13. Ola-Mudathir KF, Suru SM, Fafunso MA et al (2008) Protective roles of onion and garlic extracts on
cadmium-induced changes in sperm characteristics and testicular oxidative damage in rats. Food Chem
Toxicol 46:3604–3611
14. El-Shahat AE, Gabr A, Meki A et al (2009) Altered testicular morphology and oxidative stress induced
by cadmium in experimental rats and protective effect of simultaneous green tea extract. Int J Morphol
27:757–764
Predes et al.
15. Bridi R, Crossetti VM, Henriques AT (2001) The antioxidant activity of standardized extract of Ginkgo
biloba (Egb 761) in rats. Phyto Res 15:449–451
16. He S, Luo J, Wang YP et al (2006) Effects of extract from Ginkgo biloba on tetrachloride-induced liver
injury in rats. World J Gastroenterol 12:3924–3928
17. Huang S, Luo Y, Wang L et al (2005) Effect of Ginkgo biloba extract on livers in aged rats. World J
Gastroenterol 11:132–135
18. Wang W, Zhong X, Ma A et al (2008) Effects of Ginkgo biloba on testicle injury induced by
diethylstilbestrol in mice. Am J Chin Med 36:1135–1144
19. Şener G, Sehirlia Ö, Tozanb A et al (2007) Ginkgo biloba extract protects against mercury (II)-induced
oxidative tissue damage in rats. Food Chem Toxicol 45:543–550
20. Yeh K, Pub H, Kaphle K et al (2008) Ginkgo biloba extract enhances male copulatory behavior and
reduces serum prolactin levels in rats. Horm Behav 53:225–231
21. Al-yahya AA, Al-majed A, Al-bekairi AM et al (2006) Studies on the reproductive, cytological and
biochemical toxicity of Ginkgo biloba in Swiss albino mice. J Ethnopharmacol 107:222–228
22. Yano C L (2001) Aspectos estruturais e ultra-estruturais de testículos de ratos submetidos ao tratamento
com cádmio e paracetamol. Campinas, SP: State University of Campinas. Thesis
23. Russell LD, Ettlin RA, Hikim APS et al (1990) Histological and histopathological evaluation of the
testis, 1st edn. Cache River Press, Clearwater
24. Mori H, Christensen K (1980) Morphometric analysis of Leydig cells in the normal rat testis. J Cell Biol
84:340–354
25. França LR, Godinho CL (2003) Testis morphometry, seminiferous epithelium cycle length, and daily
sperm production in domestic cats (Felis catus). Biol Reprod 68:1556–1561
26. Cabral FHC (1996) Alterações morfológicas testiculares provocadas pelo cádmio, paracetamol e cádmio
associado ao paracetamol em ratos. (Thesis)
27. Beek TAV, Morazzoni P, Bombardelii E, Peterlongo F (1998) Ginkgo biloba L. Fitoterapia 69:195–244
28. Biswas NM, Gupta RS, Chattopadhyay A et al (2001) Effect of atenolol on cadmium-induced testicular
toxicity in male rats. Reprod Toxicol 15:699–704
29. Laskey JW, Rehnberg GL, Laws SC et al (1984) Reproductive effects of low acute doses of cadmium
chloride in adult male rats. Toxicol Appl Pharmacol 154:256–263
30. Gupta RS, Sharma R, Chaudhary R et al (2003) Effect of textile waste water on the spermatogenesis of
male albino rats. J Appl Toxicol 23:171–175
31. Zeng X, Jin T, Zhou Y (2003) Changes of serum sex hormone levels and MT mRNA expression in rats
orally exposed to cadmium. Toxicol 186:109–118
32. Castro AP, Mello FB, Mello JB (2005) Avaliação toxicológica do Ginkgo biloba sobre a fertilidade e
reprodução de ratos Wistar. Acta Scient Veterin 33:265–269
33. Creasy DM (2001) Pathogenesis of male reproductive toxicity. Toxicol Pathol 29:64–76
34. Blottner S, Frölich K, Roelants H et al (1999) Influence of environmental cadmium on testicular
proliferation in roe deer. Reprod Toxicol 13:261–267
35. Russell LD, França LR (1995) Building a testis. Tissue Cell 2:129–147
36. Kim I, Yang H (1999) Morphometric study of the testicular interstitium of the rat during postnatal
development. Korean J Anat 32:849–858
37. Blanco A, Moyano MR, Molina AM et al (2009) Quantitative study of Leydig cell populations in mice
exposed to low doses of cadmium. Bull Environ Contam Toxicol 82:756–760
38. Yang J, Arnush M, Chenc Q (2003) Cadmium-induced damage to primary cultures of rat Leydig cells.
Reprod Toxicol 17:553–560
39. Keeney DS, Ewing LL (1990) Effects of hypophysectomy and alterations in spermatogenic function on
Leydig cell volume, number, and proliferation in adult rats. J Androl 11:367–378
40. Ichihara I, Kawamura H, Nakano T et al (2001) Ultrastructural, morphometric, and hormonal analysis of
the effects of testosterone treatment on Leydig cells and others interstitial cells in young adults rats. Ann
Anat 183:413–426
41. Queiroz EK, Waissmann W (2006) Occupational exposure and effects on the male reproductive system.
Cad Saúde Públ 22:485–493
42. Fiorini C, Tilloy-Ellul A, Chevalier S et al (2004) Sertoli cell junctional proteins as early targets for
different classes of reproductive toxicants. Reprod Toxicol 18:413–421
43. Morales E, Horn R, Pastor LM et al (2004) Involution of seminiferous tubules in aged hamsters: an
ultrastructural, immunohistochemical and quantitative morphologic study. Histol Histopathol 19:445–
455
44. Chung NPY, Cheng CY (2001) Is cadmium chloride-induced inter-Sertoli tight junction permeability
barrier disruption a suitable in vitro model to study the events of junction disassembly during
spermatogenesis in the rat testis? 142:1878–1888