Human Reproduction Vol.20, No.10 pp. 2801–2813, 2005
Advance Access publication June 24, 2005.
© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. 2801
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Chronic chromium exposure-induced changes in testicular
histoarchitecture are associated with oxidative stress: study
in a non-human primate (Macaca radiata Geoffroy)
M.Michael Aruldhas1,5, S.Subramanian1,4, P.Sekar1, G.Vengatesh1, Gowri Chandrahasan2,
P.Govindarajulu1 and M.A.Akbarsha3
1Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus,
Chennai 600113, 2Department of Biochemistry, Central Leather Research Institute, Chennai 600025 and 3Department of Animal Science,
Bharathidasan University, Tiruchirappalli 620024, India. 4Present address: Department of Comparative Biomedical Sciences, School of
Veterinary Medicine, Louisiana State University, USA.
5To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
BACKGROUND: Reproductive toxicity of chromium is in dispute despite positive findings in rodents. Recently we
reported epididymal toxicity of hexavalent chromium (CrVI) in bonnet monkeys and in this paper we report its tes-
ticular toxicity. METHODS: Adult monkeys (Macaca radiata) were given drinking water containing CrVI (100, 200,
400 p.p.m.) for 6 months and testes were removed for ultrastructural and biochemical analyses. RESULTS: CrVI
treatment disrupted spermatogenesis, leading to accumulation of prematurely released spermatocytes, spermatids
and uni- and multinucleate giant cells in the lumen of seminiferous tubules. Transmission electron microscopy
revealed granulation of chromatin and vacuolation between acrosomal cap and manchette microtubules of elongated
spermatids and in the Golgi area of round spermatids. Pachytene spermatocytes had fragmented chromatin and
swollen mitochondria with collapsed cristae. Spermatocytes and spermatogonia in the basal compartment were unaf-
fected. Macrophages containing phagocytosed sperm and dense inclusions in Sertoli cells were seen. Specific activities
of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and
glucose-6-phosphate dehydrogenase and concentrations of the non-enzymatic antioxidants glutathione, vitamins A, C
and E decreased, while concentrations of H2O2 and hydroxyl radicals increased in the testis of chromium-treated
monkeys. Withdrawal of chromium treatment for 6 months normalized spermatogenesis and the status of pro- and
antioxidants in the testis. CONCLUSIONS: CrVI disrupts spermatogenesis by inducing free radical toxicity, and
supplementation of antioxidant vitamins may be beneficial to the affected subjects.
Key words: chromium/multinucleate spermatids/oxidative stress/testicular toxicity/testis ultrastructure
Unabated pollution of the environment is considered to be a
major reason for the decline of human semen quality over the
years (Skakkebaek et al., 1991). Occupational, industrial, envi-
ronmental, therapeutic and dietary exposures to a wide range of
chemicals and heavy metals have harmful effects on male fer-
tility (Cheek and McLachlan, 1998; Pant et al., 2003).
Hexavalent chromium (CrVI), used in more than 50 industries,
is an important heavy metal pollutant (Barceloux, 1999). Several
systemic toxicities of CrVI have been demonstrated in experi-
mental animals in vivo and in vitro (Bagchi et al., 2002; Levina
et al., 2003). However, reproductive toxicity of chromium has
been underplayed since the report of Bonde (1993), which
stated that low-level exposure to CrVI might not be a major
hazard affecting spermatogenesis in stainless steel welders.
Even in a recent review, Bonde (2002) emphasized the need
for additional data to recognize the reproductive toxicity of
chromium. Nevertheless, a number of investigations using lab-
oratory animals have pointed out testicular toxicity of CrVI
(Behari et al., 1978; Ernst, 1990; Saxena et al., 1990; Zahid et al.,
1990; Murthy et al., 1991; Ernst and Bonde, 1992; Chowdhury
and Mitra, 1995; Sutherland et al., 2000). Two recent reports
also correlated chronic occupational exposure to CrVI to
abnormal semen quality in men (Li et al., 2001; Danadevi
et al., 2003), though the amount and type of CrVI used by Li
et al. were questioned (Duffus, 2002).
Uptake of CrVI by the testis and its subsequent reduction to
trivalent chromium (CrIII) are well known (Sipowicz et al.,
1997; Sutherland et al., 2000). Recently we reported accumula-
tion of uni- and multinucleate germ cells in the epididymal
lumen of the monkeys treated with CrVI causing ductal
obstruction (Aruldhas et al., 2004). We attributed this to the
disruption of spermatogenesis and testicular histoarchitecture
and this was tested in the present study.
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M.M.Aruldhas et al.
In an attempt to understand the mechanism underlying the
testicular toxicity of chromium, we also studied the status of
antioxidants and pro-oxidants in the testes of these animals. The
involvement of oxidative stress in heavy metal-induced cellular
toxicity is known (Sugiyama, 1994). Maintenance of a critical
balance between pro-oxidants and antioxidants is a physiologi-
cal feature of a cell, to sustain its survival (Aitken, 1989; Yu,
1994). Production of free radicals/reactive oxygen species
(ROS) by sperm (Iwasaki and Gagnon, 1992) and the adverse
effect of excess ROS and peroxidation on sperm motility and
viability were also reported (de Lamirande and Gagnon, 1992;
Kim and Parthasarathy, 1998). The testis, epididymis, sperm
and seminal plasma contain high activities of antioxidant
enzymes, which protect sperm against the deleterious effects of
ROS (Kobayashi et al., 1991; Zini et al., 1993; Potts et al.,
2000). Increased concentrations of ROS and subnormal antioxi-
dant status have been recorded in the seminal plasma of infertile
men (Pasqualotto et al., 2000). In the light of the above back-
ground information, it is hypothesized that chronic exposure to
chromium would disrupt spermatogenesis and testicular histoar-
chitecture by inducing oxidative stress. Bonde and Ernst (1992)
considered only the nasal route of human exposure to CrVI in
the welding industry, whereas workers in tanneries are subjected
to direct exposure to chromium. A number of people who live
around such industries suffer indirectly through contamination
of drinking water due to percolation of untreated or incom-
pletely treated effluents into the ground. There are several such
large-scale leather industries in India and other developing
countries, where pollution control laws may not be implemented
effectively. Therefore, it would be pertinent to revisit the testicu-
lar toxicity of CrVI to man, adopting a protocol simulating oral
exposure and using an animal model closer to the human, in
order to understand and overcome the imminent problem of
chromium-induced infertility/subfertility in such men.
In the earlier experimental studies, rabbits, rats and mice
have been used as animal models to test the testicular toxicity
of CrVI (Behari et al., 1978; Ernst, 1990; Saxena et al., 1990;
Zahid et al., 1990; Murthy et al., 1991; Chowdhury and Mitra,
1995; Sutherland et al., 2000). Fine details of spermatogenesis,
the relative duration of postnatal development and the onset
of sexual maturation differ between rodents and primates
(de Kretser and Kerr, 1994). Taking into consideration all the
above facts, we tested the hypothesis in a non-human primate
model, Macaca radiata subjected to chronic exposure to chro-
mium through drinking water.
Materials and methods
The experimental protocol of the present study on a non-human
primate model (Macaca radiata) was approved by the Institutional
Animal Ethics Committee (IAEC) constituted under the auspices of
the Committee for the Purpose of Control and Supervision of Experi-
ments on Animals (CPCSEA), Ministry of Social Justice and Empow-
erment, Government of India. At the end of the experiments, the
monkeys were handed over to the CPCSEA for rehabilitation.
Adult male monkeys weighing 7–8 kg (6–8 years old as calculated by
the dental formula), trapped by the Department of Forests and Wild
Life, Government of Tamil Nadu State, for creating public nuisance,
and kept in captivity, were procured with the permission of the Chief
Wildlife Warden, Chennai, India. Animals were kept under quaran-
tine, screened for infectious diseases and acclimatized to the animal
house for 2 months before the experiments were initiated. The monkeys
were maintained in cages (60 × 60 × 80 cm) individually, under
natural temperature (28 ± 2°C), and light and dark schedules (12 ± 1 h)
in a well-ventilated animal quarter. All animals were fed ad libitum
with pellet diet (Brooke Bond India, Kolkatta, India), rice cooked with
lentils, vegetables such as potatoes, carrots and beetroot, and season-
ally available fresh fruits such as banana and guava.
The experimental design was the same as reported earlier (Aruldhas
et al., 2004). Briefly, adult male bonnet monkeys (Macaca radiata)
were divided into four groups, each consisting of six animals. Animals
belonging to groups I, II and III were provided with drinking water
containing CrVI (potassium dichromate) at concentrations of 100, 200
or 400 p.p.m. respectively, ad libitum for 180 days. Group IV con-
sisted of control animals, which were provided with drinking water
without chromium. Potassium dichromate was selected as it is highly
soluble in water and is used in many industries. The concentrations of
chromium were selected on the basis of a dose–response study with
reference to reproductive toxicity, as judged by decline in sperm char-
acteristics. Chromium at a concentration of 10 mg/l has been consid-
ered as safe for humans (Barceloux, 1999). Therefore, we tested
increasing concentrations of potassium dichromate, starting a little
above the safe level, i.e. 12.5, 25, 50, 100, 200, 400 and 800 mg/l
(p.p.m.) for reproductive toxicity. The doses (100, 200, 400 mg/l) that
impaired spermatogenesis (sperm count decreased by 50–70% of the
control value of 350 to 375 × 106/ml) by 6 months with the least systemic
toxicity were selected for the study. At the end of the experiments,
testes from three monkeys in each of the experimental groups and the
control group were removed surgically under deep sodium pentathol
anaesthesia (28 mg/kg body weight, i.p. injection). The testes of
remaining three monkeys in each group were removed after withdrawing
CrVI treatment for a period of 180 days. Immediately after removing,
the testes were weighed and used for histological and biochemical
analyses. After removing the testes, the wound was closed using nylon
stitches; antibiotics were applied every day and stitches were removed
on the eighth day.
Tissue processing for light microscopy and transmission electron
Tissues were not subjected to perfusion fixation for TEM, as samples
from the same monkeys were also used for light microscopic and bio-
chemical studies. The entire right testis was immersion-fixed in 2.5%
glutaraldehyde in cacodylate buffer (Hess and Moore, 1993) immedi-
ately after removal. Thin slices of the testis were again fixed in the
same fixative to ensure proper fixation. The tissues were postfixed in
1% osmium tetroxide and embedded in thin viscosity resin (Spurr’s
mix; Sigma, St Louis, MO, USA). Semithin sections (1µm) were
obtained with an Ultratome (Reichert Jung, Vienna, Austria) and
stained in toluidine blue O (TBO) for light microscopic observations.
The diameter of the seminiferous tubules (20 randomly selected
tubules from each animal) was measured using a calibrated ocular
micrometer (Erma, Tokyo, Japan). Ultrathin sections were cut with an
ultramicrotome (Leica Microsystems, Nussloch, GmbH Nussloch,
Germany), stained with uranyl acetate and lead citrate, and observed
in a Phillips 201-C (Amsterdam, Holland) TEM. Image analysis and
processing were done using Axivision image analysis software
(Carl Zeiss, Jena, Germany). Tissues from the recovery group of
monkeys were subjected to light microscopic analysis only.
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Testicular toxicity of chromium
Blood collection and chromium analysis
Blood samples were collected by vein puncture before starting, every
month during and at the end of experimentation, and plasma was
separated and used for estimation of chromium in an atomic absorption
spectrophotometer (Perkin-Elmer Life and Analytical Sciences, Shelton,
CT, USA), following the protocol of the manufacturer. All glassware
used for chromium analysis was washed thoroughly in deionized and
double-distilled water, after overnight soaking in nitric acid.
Tissue preparation. Immediately after removal, the left testis from
each monkey was washed in ice-cold saline and a 10% homogenate
was prepared in 0.1 M Tris–HCl buffer (pH 7.4). To 1.0 ml of 1:10
diluted tissue extract, 0.25 ml absolute ethanol and 0.15 ml chloro-
form were added and the mixture was placed in a mechanical shaker
for 15 min, centrifuged at 3000 g for 10 min at 4°C and the superna-
tant was used for the assay of antioxidant enzymes.
Superoxide dismutase (SOD) (EC 1.15.11). SOD activity was esti-
mated according to the method of Marklund and Marklund (1974).
The percentage inhibition of pyrogallol auto-oxidation at 470 nm was
converted to units of enzyme activity. The amount of enzyme required
to cause 50% inhibition of pyrogallol auto-oxidation was considered
as 1 unit of enzyme activity.
Catalase (EC No. 188.8.131.52). Catalase activity was quantified colori-
metrically (Sinha, 1972) based on the intensity of the green colour
developed due to the conversion of dichromate into chromic acetate.
The amount of enzyme that uses 1 µM of H2O2 per minute is equival-
ent to 1 unit.
Glutathione peroxidase (GPx) (EC 184.108.40.206). GPx activity was
determined colorimetrically by estimating the amount of reduced glu-
tathione (GSH) oxidized per minute, using GSH as the standard
(Rortruck et al., 1973).
Glutathione reductase (GR) (EC 220.127.116.11). GR activity was assayed
colorimetrically by following the rate of reduction of oxidized glutath-
ione (Stall and Vegel, 1969).
Glutathione-S-transferase (GST) (EC 18.104.22.168). GST activity was
determined by estimating the amount of enzyme that catalysed the
conjugation of a known amount of 1-chloro-2,4 dibenzene with GSH,
as described by Habig et al. (1973).
Glucose-6-phosphate dehydrogenase (G-6-PDH) (EC 22.214.171.124).
G-6-PDH activity was assayed spectrophotometrically by following
the rate of oxidation of glucose-6-phosphate to 6-phosphogluconate,
according to Beutler (1983).
γ-Glutamyl transpeptidase (γ-GT) (EC 126.96.36.199). γ-GT activity
was estimated colorimetrically by quantifying the amount of
p-nitroaniline released from γ-glutamyl-p-nitroanilide in a
given time, using the method of Orlowski and Meister (1965)
with p-nitroaniline as the standard.
Reduced glutathione (GSH). GSH concentration was quantified color-
imetrically by measuring the product of the reaction between GSH
and DTNB (Moren et al., 1979).
Vitamin C. Vitamin C was estimated colorimetrically using ascor-
bate as the standard (Omaye et al., 1971).
Vitamin E. Vitamin E content was quantified by the colorimetric
method of Desai (1984), using α-tocopherol as the standard.
Vitamin A. Vitamin A content was quantified by the colorimetric
method of Bayfield and Cole (1974), using β-carotene as the
Reactive oxygen species
Hydrogen peroxide (H2O2). H2O2 production was assessed spectropho-
tometrically (Holland and Storey, 1981) by estimating the oxidation
product of ferrocytochrome.
Hydroxyl radical. The production of hydroxyl radical was quantified
by the colorimetric method described by Puntarulo and Cederbaum
Data were subjected to one-way analysis of variance, and whenever the
F value was significant, the data were analysed by Duncan’s multiple
comparison test to find the within-group significance at the P < 0.05 level.
The plasma concentration of chromium increased up to ten-fold
in monkeys that were provided with drinking water containing
chromium and it returned to the normal level in the recovery
group (Table I).
The absolute weights of the testis did not show any appreciable
change due to CrVI treatment. However, there was a statistically
significant decrease in the relative weight of the testes of monkeys
that were exposed to CrVI The weight of the testes in monkeys
belonging to the withdrawal group showed a trend of recovery
to control level (Table I). (Author please re-check this.
Light microscopic observations
Control testes. The seminiferous tubules in the testes of control
monkeys had the typical organization (Figure 1a), with different
generations of germ cells associated with Sertoli cells (Figure 1b).
The Leydig cells also had their characteristic organization
Testes of chromium-treated monkeys. The seminiferous tubules
of the chromium-treated monkeys were disorganized, with
decreased diameter (0.61 ± 0.08, 0.52 ± 0.07 and 0.97 ± 0.11 mm
for the 100, 200 and 400 p.p.m. groups, respectively, versus the
control value of 1.32 ± 0.13 mm). Depletion of germ cells
and hyperplasia of Leydig cells (Figure 1c–e) were the
Table I. Plasma chromium concentration (µg/l) and testicular weight
(g/kg body weight) in monkeys treated with different doses of CrVI
Each value is mean ± SEM of three values.
A, CrVI-treated (24 h after the 180th day of treatment).
B, withdrawal group (180 days after the last day of chromium treatment).
TreatmentPlasma CrTesticular weight
46.5 ± 2.103.95 ± 0.25
150.0 ± 6.3*
48.81 ± 2.5
3.05 ± 0.20*
3.68 ± 0.13
230.0 ± 10.5*
47.52 ± 2.8
2.55 ± 0.10*
3.50 ± 0.27
410.3 ± 20.2*
48.67 ± 3.1
2.60 ± 0.17*
3.75 ± 0.23
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M.M.Aruldhas et al.
common features in all the experimental groups. Spermatids
were the most affected germ cells in most of the tubules and
were totally absent in a few tubules. Most of the germ cells in
the adluminal compartment were prematurely released into the
lumen, resulting in Sertoli cell fibrosis. Even those spermatids
that adhered to the epithelium had vacuoles around them, indi-
cating that they were detaching from the Sertoli cell (Figure 2a).
Of the round and elongating spermatids, the former appeared
to be the earlier target for chromium as the round spermatids
were missing or being lost in many of those tubules which
should possess two generations of spermatids, whereas the
elongating spermatids were present (Figure 2a).
In several tubules, which were in the process of losing
round spermatids, the adluminal compartment contained
multinucleate giant cells (Figure 1e). The organization of the
nuclei of the multinucleate giant cells suggested they were
spermatids (Figure 2b), and in a few such giant cells the
nuclei were abnormally shaped, with marginalized chromatin,
creating a hollow in the centre (Figure 2c). In rare cases, the
cytoplasm of the multinucleate spermatids appeared vacu-
olated and the nuclei were pycnotic (Figure 2d).The lumen
was invariably filled with prematurely released germ cells
and cell debris (Figures 1c, e and 2b, d).
In several postzygotene spermatocytes, the chromatin
appeared fragmented and discontinuous (Figure 3a), whereas
in those with chromosomes in the metaphase plate the spindle
fibres were not discernible (Figure 3b). Cells resembling
macrophages were noticed in the adluminal compartment
(Figure 3a). In most of the prematurely released germ cells,
the chromatin was either intact or marginalized; in a few
instances hydropic swelling and necrosis were also noticed
(Figure 3c). Depending upon the extent of the loss of germ
cells, the seminiferous epithelium reflected prominence of
Sertoli cells and a few such tubules reflected the Sertoli cell-
only syndrome (Figure 3d), but closer examination revealed
the presence of spermatogonia in the basal compartment
Accumulation of electron-dense granules in the basal and
perinuclear cytoplasm of Sertoli cells was a common mani-
festation of chromium toxicity (Figure 3c, d).
In the recovery group of monkeys (i.e. 6 months after the
withdrawal of chromium exposure) the diameter (100 p.p.m.,
1.18 ± 0.12 mm; 200 p.p.m., 1.22 ± 0.08 mm; 400 p.p.m., 1.25 ±
0.14 mm) and the histological organization of the seminiferous
tubules were comparable to those of control monkeys. Sperma-
togenesis recovered fully, though a few prematurely released
germ cells were still noticed in the lumen (Figure 4a, b).
Control testes. Low-power electron micrographs of the testes
of control monkeys revealed closely packed Sertoli cells
Figure 1. (a) Semithin section of testis of a control monkey showing
seminiferous tubules (ST) and Leydig cells (LC). Scale bar, 20 µm.
(b) Seminiferous epithelium of a control monkey showing Sertoli cells
(SC) and the different generations of germ cells (PS, pachytene sper-
matocyte; RS, round spermatid; ES, elongating spermatid). Scale bar,
6 µm. (c, d) Seminiferous tubules (ST) and the Leydig cells (LC) of
CrVI-treated monkeys (c, 100 p.p.m.; d, 200 p.p.m.) showing regression
of seminiferous tubules and degeneration of seminiferous epithelium.
Scale bar, 20 µm. (e) A seminiferous tubule of a CrVI-treated
(400-p.p.m.) monkey showing mild effect in the seminiferous epithe-
lium (SE) with uninucleated germ cells (UN) and multinucleate giant
cells (GC) in the lumen. Scale bar, 20 µm.
Figure 2. (a) Seminiferous epithelium of a CrVI-treated monkey
(200 p.p.m.) showing premature release of round spermatids from the
Sertoli cells (arrowheads). Scale bar, 6 µm. (b) Seminiferous tubular
lumen of a CrVI-treated monkey (200 p.p.m.) showing occurrence of
a multinucleate giant cell (MN). Scale bar, 6 µm. (c) A multinucleate
giant cell (MN) produced in the seminiferous tubule of a CrVI- treated
monkey (100 p.p.m.) with some of the nuclei indicating apoptotic
morphology (AP). Scale bar, 3 µm. (d) A multinucleate giant cell
(MN) with vacuolated cytoplasm and pycnotic nuclei present in the
seminiferous epithelium of a CrVI-treated monkey (100 p.p.m.). Scale
bar, 10 µm.
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Testicular toxicity of chromium
contacting the basement membrane. The spermatogonia and
spermatocytes up to preleptotene stage were present in the
basal compartment, whereas spermatocytes beyond prelepto-
tene stage and spermatids were present in the adluminal com-
partment of Sertoli cells. The cell types reflected their
characteristic ultrastructural organization (Figure 5a).
Testes of chromium-treated monkeys: elongating spermatids.
Sertoli cells in which sperm heads were embedded
developed vacuoles around the latter and had electron-dense
granules around the posterior aspect of the sperm nucleus. The
cytoplasm of the differentiating midpiece was also vacuolated
(Figure 5b). Other important manifestations in spermatids of
the monkeys exposed to chromium included: (i) chromatin
becoming heterogeneous and granular; (ii) the appearance of a
dense, plaque-like acrosomal cap; (iii) association of the nuclear
cap with microtubule-like structures; (iv) the presence of a
large vacuole at the junction between the acrosomal cap and
the manchette microtubules; (v) vacuoles in the developing
midpiece (Figure 5c); (vi) formation of the manchette microtu-
bules into a continuous patch (Figure 5d); and (vii) the mid-
piece lagging in pace behind that of the head (Figure 5e).
Round spermatids. In the 400 p.p.m. chromium-treated
group the round spermatids were prematurely released from
the Sertoli cells, with little pathological change (Figure 6a),
whereas in the 100 and 200 p.p.m. groups the round spermatids
developed vacuoles in the cytoplasm, particularly in the Golgi
area (Figure 6b). The mitochondria were swollen, with col-
lapsed cristae (Figure 6c).
Spermatocytes and spermatogonia. In spermatocytes at the
advanced pachytene stage, the chromatin appeared fragmented
and the mitochondria underwent hydropic swelling accompa-
nied by collapse of cristae (Figure 6d). A few spermatocytes
showed necrotic changes with thoroughly damaged chromatin
(Figure 7a). However, the pale and dark spermatogonia and
preleptotene spermatocytes manifested little pathological
change (Figure 7b,c), though mitochondria were slightly dam-
aged in the pale spermatogonia (Figure 7d).
Sertoli cells. Sertoli cells of CrVI-treated monkeys accumu-
lated large electron-dense bodies in the basal and perinuclear
cytoplasm, and at a higher magnification these bodies appeared
to be pale, transitional and dark lysosomes (Figure 8a). In
certain rare cases, deformed sperm heads were seen in a vacu-
ole in the Sertoli cell cytoplasm (Figure 8a). The structure of
inter-Sertoli cell junctions (Figure 8b) and the Sertoli cell–germ
cell junctions appeared to be affected (Figures 6a and 8b).
Multinucleate giant cells and their origin. Ultrastructural
analysis revealed the multinucleate giant cells to be spermatids
possessing two or more nuclei in a common cytoplasm. The
presence of an acrosome in the nuclear cap in some of these
cells suggested the initiation of spermiogenesis, though in respect
of the different constituent cells this was not uniform (Figure 9a).
Figure 3. (a) Seminiferous epithelium of a CrVI-treated monkey
(100 p.p.m.) showing fragmented chromatin in pachytene spermatocytes
(PS). Picture also shows occurrence of a macrophage (MA) associat-
ing with a cell in metaphase of meiotic division (arrowhead). Scale
bar, 3 µm. (b) Seminiferous epithelium of a CrVI-treated monkey
(200 p.p.m.) showing disruption of spindle fibres in meiotic metaphase
cells (MS). Pachytene spermatocytes (PS) are also seen. Scale bar, 3 µm.
(c) Seminiferous epithelium of a CrVI-treated monkey (100 p.p.m.)
showing death of round spermatids through necrosis (NE) following
hydropic swelling (HY). Scale bar, 3 µm. (d) Seminiferous epithelium
of a CrVI-treated monkey (200 p.p.m.) showing a continuous row of
Sertoli cells (SC), with dense granules in the cytoplasm, without any
trace of germ cells. Scale bar, 3 µm.
Figure 4. (a) Seminiferous tubules (ST) of a monkey in the recovery
group showing almost complete recovery of spermatogenesis; the
Leydig cells (LC) also appear normal. Scale bar, 20 µm. (b) Epithe-
lium in Figure 4a magnified, showing normal Sertoli cells (SC),
pachytene spermatocytes (PS), round spermatids (RS) and elongating
spermatids (ES). Scale bar, 3 µm.
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M.M.Aruldhas et al.
Occasionally, the nuclear envelope in the binucleate spermatid
was indented at varying degrees, and the mitochondria that
were destined to develop into the mitochondrial sheath accu-
mulated at this pole of the nucleus (Figure 9b). The formation
of bi- and multinucleate spermatids appeared to be due to
incomplete cytokinesis (Figure 9c), as a result of which two
nuclei shared a common nuclear cap with a common Golgi
apparatus lying on top of it (Figure 9d).
Macrophages in the seminiferous epithelium. The occur-
rence of macrophages in the seminiferous epithelium was con-
firmed in the TEM study, and such macrophages were seen to
align close to sperm heads that detached from the Sertoli cell
(Figure 10a). In some cases, damaged macrophages containing
germ cell fragments were found within the Sertoli cell cyto-
plasm (Figure 10b).
Chromium treatment for 6 months led to a dose-dependent
decrease in the specific activities of testicular SOD, catalase,
GPx, GR and G-6-PDH, with the maximum decrease in monkeys
exposed to 400 p.p.m. chromium (Figures 11 and 12). The spe-
cific activity of γ-GT (Figure 11) decreased in the testes of
monkeys exposed to 400 p.p.m. chromium alone. Unlike the
other enzymes, GST activity (Figure 12) increased in the testes
of chromium-treated monkeys in all groups.
The concentration of reduced glutathione (Figure 12) increased
in the testes of monkeys exposed to chromium, whereas the
concentration of antioxidant vitamins A, C and E (Figure 13)
The concentrations of H2O2 and OH– increased significantly in
the testes of monkeys exposed to chromium, in a dose-dependent
manner; the maximum concentration was recorded in monkeys
exposed to 400 p.p.m. chromium (Figure 14).
Figure 5. (a) A low-power transmission electron micrograph (TEM)
of the seminiferous epithelium of a control monkey showing Sertoli
cells (SC) and different generations of germ cells, viz., pachytene
spermatocytes (PS), round spermatids (RS) and elongating spermatids
(ES). Scale bar, 3 µm. (b) TEM showing presence of vacuoles in and
around elongating spermatids (ES) in a CrVI-treated monkey (100 p.p.m.).
H, head; M, midpiece with cytoplasmic vacuolation. Arrowhead
points to a large vacuole containing electron-dense granules. Scale
bar, 1µm. (c) TEM of an elongating spermatid of a CrVI-treated mon-
key (200 p.p.m.) showing head (H) with dispersed chromatin, and
microtubule-like structures (MT) connected to the head. Arrowheads
point to vacuolated areas at the junction of manchette microtubules
with the head cap. Scale bar, 1 µm. (d) TEM of an elongating spermatid
showing manchette microtubules (MA) occurring as a continuous
patch, with a large vacuole at the point of origin (arrowhead). Scale
bar, 1 µm. (e) TEM showing an unusual organization of the elongat-
ing spermatid, with much advanced development of the head (H),
leaving behind the development of midpiece (M), containing vacuoles (V)
and lysosomes (LY). Scale bar, 1 µm.
Figure 6. (a) TEM showing detachment (arrowhead) of round sper-
matids (RS) from the Sertoli cell (SC). Scale bar, 1.5 µm. (b) TEM
showing appearance of vacuoles in the Golgi area and mitochondria of
spermatocytes (asterisks). Scale bar, 2.5 µm. (c) TEM showing swelling
of mitochondria and collapse of their cristae (arrowhead) of a round
spermatid (RS). The developing acrosome (AC) is also affected.
Scale bar, 0.5 µm. (6) TEM showing chromatin fragmentation and
mitochondrial swelling (arrowhead) in a pachytene spermatocyte
(PS) remaining attached to a Sertoli cell (SC), which has abundant
lysosomes (LY). Scale bar, 0.2 µm.
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Testicular toxicity of chromium
Data on plasma chromium ascertain the supraphysiological levels
of the metal in circulation in experimental monkeys. The
histopathological changes in the testis presented here and those
of the epididymis reported earlier (Aruldhas et al., 2004) in mon-
keys exposed to CrVI ascertain the male reproductive toxicity of
this heavy metal. The important observation of the present study
is that chromium intoxication affects germ cells in the adluminal
compartment to a great extent, leaving those in the basal com-
partment and the basal portion of the Sertoli cells unaffected,
providing scope for recovery of spermatogenesis. The reversibility
of chromium-induced histopathological changes in the sem-
iniferous tubules is clearly distinct from the normal pattern of
testicular histology observed in monkeys of the withdrawal
group. The normozoospermia observed in these monkeys by the
sixth month of withdrawal period (data not shown here) ascer-
tains the recovery of normal spermatogenesis in these animals.
CrVI as a genotoxic agent
The histopathological changes observed in the testis of
chromium-treated monkeys suggest the cytotoxic effect of the
metal. The impact of chromium on meiotic germ cells (sperma-
tocytes) is interesting because the chromosomes appeared to
undergo breakage in two ways, subsequent to pairing in the
zygotene stage. In most instances, chromosomes of the pach-
ytene spermatocytes underwent fragmentation and clumping,
leading to karyolysis and necrosis. In a few cells, which had
developed the metaphase plate, the chromosomes failed to pro-
ceed towards the poles and underwent fragmentation to form
dot-like chromatoid bodies.
Chromium is known to induce chromosomal aberrations such
as single-strand breaks and 8-oxo-guanosine substitutions
(Sugiyama et al., 1993; Wise et al., 1993; Qi et al., 2000). Direct
interaction of reactive chromium intermediates with DNA, lead-
ing to DNA–chromium binding, DNA–DNA crosslinks and
DNA–protein crosslinks are known (Misra et al., 1994). Interac-
tion of chromium with GSH results predominantly in chromium–
DNA binding and strand breaks (Aiyar et al., 1991). Therefore,
the increased concentration of H2O2, OH– and GSH observed in
the present study may favour the operation of such a mechanism
in the testes of monkeys exposed to CrVI, resulting in damage to
the chromosomes/chromatin of meiotic/postmeiotic germ cells.
The data on antioxidants and free radicals in the present
study indicate free radical toxicity in the testis of chromium-
treated monkeys, as there was a significant increase in the
concentration of H2O2 and OH– and subnormal activity of most
of the antioxidant enzymes tested, accompanied by decreased
concentrations of the antioxidant vitamins A, C and E. Thus,
chronic exposure to chromium appears to result in oxidative
stress in the testis due to poor scavenging of free radicals The
histopathological changes in the testis suggest that spermatocytes
Figure 7. (a) TEM showing necrosis of pachytene spermatocytes
(PS) remaining attached to Sertoli cell (SC) in CrVI- treated monkey
(200 p.p.m.). Scale bar, 3 µm. (b) TEM of an intact dark spermatogo-
nium (DSG) in the seminiferous epithelium of a CrVI- treated monkey
(200 p.p.m.). Scale bar, 1.5 µm. (7) TEM of an intact light spermato-
gonium (LSG) in the seminiferous epithelium of a CrVI-treated monkey
(200 p.p.m.). Scale bar, 1.5 µm. (7) TEM of a light spermatogonium
(LSG) in a CrVI-treated monkey (200 p.p.m.), showing slight damage
to mitochondria (arrowheads). Scale bar, 2 µm.
Figure 8. (a) TEM of basal cytoplasm of a Sertoli cell, showing the
electron-dense inclusions to be polymorphic forms of lysosomes
(arrowheads). Picture also shows sperm heads inside an endocytic
vesicle (arrow). Scale bar, 0.4 µm. (b) TEM showing disruption of
inter-Sertoli cell (SC) junctions (arrows). Arrowhead points to a large
gap between the Sertoli cells. Scale bar, 0.4 µm.
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M.M.Aruldhas et al.
and spermatids are susceptible to chromium toxicity, whereas
spermatogonia are resistant to the hostile environment of
increased free radicals in the tissue. GSH is a major cellular
reductant, and plays a pivotal role against the nefarious actions
of hydroxyl radicals (Pal et al., 1993). The increased concen-
tration of GSH observed in the testis of monkeys exposed to
chromium probably acted as a protector of spermatogonia. The
mechanism underlying such a cell-specific effect of GSH may
be another area of future research for a better understanding of
the mechanism of the testicular toxicity of chromium.
γGT couples γ-glutamyl moiety to a suitable amino acid
acceptor for transport into the cell and makes it suitable for the
intracellular synthesis of GSH (Markey et al., 1998). γGT also
mediates the cleaving of the dipeptidyl cysteinyl glycine,
which provides cells with cysteine, a rate-limiting factor for the
synthesis of GSH (Pal et al., 1993). GR converts oxidized glutath-
ione into GSH (Sen, 1997). Therefore, the reduced activity of γGT
(specifically in 400 p.p.m. chromium-treated monkeys) and GR
(in all groups) observed in the present study suggests decreased
degradation as a possible mechanism underlying increased con-
centration of GSH in chromium-treated monkeys. Presumably,
this is a defence mechanism against chromium-induced free
radical toxicity. Increased activity of GST in all groups and
γGT in monkeys exposed to 50 p.p.m. CrVI (data not shown)
also indicates an attempt by the testis to overcome the toxic
effect of chromium. GST catalyses the conjugation of the
electrophilic xenobiotics to the -SH group of glutathione, and
thus increases their water solubility to facilitate excretion (Sen,
1997). Therefore, the enhanced activity of testicular GST
observed in chromium-treated monkeys may be a pointer to
augmented detoxification of xenobiotic compounds.
Thus, it is clear that testicular tissue tends to overcome the
adverse effects of the free radical toxicity induced by chronic
exposure to chromium. Spermatogonia and preleptotene sper-
matocytes appear to be the preferred cell types for such protec-
tive measures in the testis, which help the revival of
spermatogenesis after the withdrawal of chromium treatment.
Reversibility of the toxic effect of chromium on the testis was
vividly reflected in the normal organ weight and histoarchitecture
of the testis and normal sperm counts (data not shown here) in
monkeys that were kept free of chromium exposure for 6 months.
It is well established that it takes about 10 weeks for a spermato-
zoon to complete testicular and post-testicular maturation and to
be ready for ejaculation in monkeys (Amann et al., 1976; Shrape,
1994). In fact, normozoospermia was achieved in experimental
monkeys in the present study by the end of 3 months after the
withdrawal of chromium treatment (Aruldhas et al., 2000).
In Wistar rats exposed to chromium water for 30 days,
concurrent supplementation with vitamin C or vitamins C and E
protected the testis from the adverse effects of chromium, as evi-
dent from the normal histoarchitecture and antioxidant enzymes
(Subramanian, 2001). Vitamin A and E are also essential for the
maintenance of normal spermatogenesis (Huang and Hembree,
1979). Vitamin E deficiency was reported to induce sperma-
togenic arrest (Bensoussan et al., 1998). Therefore, decreased
concentrations of vitamins A, E and C in the testes of chromium-
treated monkeys might also have contributed to the premature
release of germ cells into the lumen. This proposal was ascer-
tained by the finding of the maintenance of normozoospermia in
monkeys supplemented with any one of these vitamins while
concurrently treated with chromium (data not shown).
Multinucleate giant cells
The multinucleate spermatids appearing in the seminiferous
epithelium of chromium-treated monkeys, irrespective of the
Figure 9. (a) TEM of a multinucleate giant spermatid (MN). The
constituent cells are at different steps in spermiogenesis. AC, acro-
somal vesicle; GA, Golgi apparatus. Scale bar, 1.5 µm. (b) TEM of a
multinucleate giant spermatid (MN) showing indented nuclear enve-
lope (arrowheads) and the mitochondria crowding around that area.
Scale bar, 1.5 µm. (c) TEM showing incomplete cytokinesis between
spermatids (arrow). Scale bar, 1.5 µm. (d) TEM of a binucleate sper-
matid, without any trace of cytokinesis (arrow). There is a single
Golgi apparatus (GA) on the top of the two. Scale bar, 1 µm.
Figure 10. TEM showing the occurrence of macrophage in the
seminiferous epithelium of CrVI-treated monkeys. (a) The macrophage
(MA) is in close proximity to a damaged sperm (SM), as seen in the
vacuoles surrounding it. Scale bar, 1 µm. (b) The macrophage (MA)
has engulfed spermatozoa (SM). Scale bar, 0.7 µm.
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Testicular toxicity of chromium
dose, may explain another mechanism of chromium toxicity,
independent of its genotoxic effect. It is also important to note
that the formation of multinucleated giant cells and the degen-
erative features described in the present study are common in
animals exposed to a wide range of toxic compounds (Russel
et al., 1990). This may be due to disruption of the cytoplasmic
bridges connecting germ cell clones or failure of cytokinesis
(Stanley and Akbarsha, 1992). The binucleate spermatids with
the cap of the two nuclei remaining fused and the incomplete
cytokinesis in a binucleate spermatid, observed in the present
study, are clear indications of failure of cytokinesis as the
probable mechanism underlying the generation of multinucle-
ate spermatids in the context of chromium toxicity.
Premature loss of germ cells and Sertoli cell toxicity
The results of the present study clearly point to the germ cells
present in the adluminal compartment of the seminiferous epi-
thelium as the principal target of chromium toxicity in the testis.
The manifestations are clearly pathological and the affected
cells have become non-viable or dead and hence do not go
through the process of spermatogenesis and/or spermiogenesis.
Such cells are lost from any epithelium through either necrosis
or apoptosis (Levin et al., 1999). The seminiferous epithelium
responds to such a situation with premature release of the
affected cells, which reach the epididymal duct for further
processing and removal (Agnes and Akbarsha, 2001; Aruldhas
et al., 2004). The TEM observations in the present study suggest
occasional necrosis in the testis of chromium-treated monkeys.
Though DNA labelling and in situ staining by the TUNEL
method are the most reliable techniques for the confirmation of
apoptosis, marginalization of nuclear chromatin is a morpho-
logical indication of apoptosis (Flickinger et al., 1999), the
mode of cell death caused by chromium (Blankenship et al.,
ROS produced during the reduction of CrVI was shown to
be responsible for initiation of p53-induced apoptosis (Ye
et al., 1999). Data on enzymatic and non-enzymatic antioxidants
Figure 11. Effect of chronic chromium exposure on the specific activities of testicular superoxide dismutase (a), catalase (b), glutathione perox-
idase (c) and γ-glutamyl-transpeptidase (d). Each bar represents the mean and the vertical line above denotes the SEM. n = 3. Statistical signifi-
cance of differences among groups at P < 0.05: aControl versus experimental; b100 p.p.m. vs 200/400 p.p.m.; c200 p.p.m. versus 400 p.p.m.
by guest on June 9, 2013
M.M.Aruldhas et al.
and free radicals clearly establish the development of oxidative
stress in the testis of CrVI-treated monkeys. Therefore, it may
be logical to propose that ROS-mediated apoptosis may occur
in the testis of CrVI-treated monkeys. This could be another
mechanism in the system towards maintenance of a viable epi-
thelium to provide scope for revival of spermatogenesis, apart
from the preservation of spermatogonia.
The participation of Sertoli cells in the process of premature
loss of germ cells is indispensable. The germ cells in the adlu-
minal compartment are held in position by the characteristic
inter-Sertoli cell junctions and the Sertoli cell–germ cell junctions
(Bardin et al. 1994). These junctional complexes are main-
tained by cytoskeletal elements and the ectoplasmic specializa-
tion of the Sertoli cell cytoplasm (Bardin et al., 1994). Once a
germ cell has arrived at the adluminal compartment, it is
released only at the time of spermiation in the normal course
(de Kretser and Kerr, 1994). During toxic manifestations, as in
the present case, the Sertoli cells may release the germ cells
prematurely, and this would necessarily involve alterations in
the junctional complexes and the cytoskeletal framework of the
Sertoli cells (de Kretser and Kerr, 1994). When such a
response to chromium toxicity is imminent, the Sertoli cell
itself becoming a target of the toxicant cannot be ruled out. The
changes occurring in the mitochondria of the Sertoli cell and
the accumulation of pale, transitional and dark lysosomes in
the basal aspect of the Sertoli cell cytoplasm, as observed in
the present study, may be reflections of such consequences of
One of the important histopathological observations in the
present study is that the germ cells of the adluminal compart-
ment alone are irrevocably affected, whereas those in the basal
compartment remained unaffected. It is an established fact that
Figure 12. Effect of chronic chromium exposure on the specific activities of testicular glutathione reductase (a), glutathione-S-transferase (b),
glucose-6-phosphate dehydrogenase (c) and concentrations of reduced glutathione (d). Each bar represents the mean and the vertical line above
denotes the SEM. n = 3. Letters a, b and c denote statistical significance of differences among groups at P < 0.05: acontrol versus experimental;
b100 p.p.m. versus 200/400 p.p.m.; c200 p.p.m. versus 400 p.p.m.
by guest on June 9, 2013
Testicular toxicity of chromium
nutritive and/or regulatory substances reach germ cells of the
adluminal compartment through the Sertoli cell (Bardin et al.,
1994), and chromium may also take the same route. Germ cells
at different stages of development require specific nutrients,
such as lactate, pyruvate, fatty acids and regulatory peptides
(de Kretser and Kerr, 1994). Chromium toxicity probably
affects the supply of such specific nutrients and/or regulatory
substances required by meiotic/postmeiotic germ cells, resulting
in their premature death.
Macrophages in the seminiferous epithelium
Because of the thick tunic around the seminiferous tubules and
the consequent absence of a direct blood supply (Setchell et al.,
1994), macrophages cannot enter the seminiferous tubules
under normal circumstances (Russell et al., 1990). Thus, the
present study is unique in reporting macrophages in the sem-
iniferous epithelium of CrVI-treated monkeys. Chromium
treatment has probably damaged the blood–testis barrier
(Pereira et al., 2002), paving the way for the entry of macro-
phages into the seminiferous tubules. It is an established fact
that Sertoli cells phagocytose the residual bodies and the other
cell debris under physiological conditions as well as in certain
pathological states (Russell et al., 1990). The presence of degen-
erating spermatozoa in vacuoles in the Sertoli cell cytoplasm
of chromium-treated monkeys indicates the operation of such
a mechanism of cleaning cell debris in the testes of these
animals. The presence of macrophages inside the seminiferous
tubules may enhance cleansing the damaged cellular materials
from the tubules along with the apical portions of the Sertoli
cells, as the latter could not assimilate such large volumes of
degenerating cells, a situation which can attract macrophages
(Russell et al., 1991).
Thus, from the present study on the testis and our earlier
findings on the epididymis of monkeys subjected to chronic
chromium exposure (Aruldhas et al., 2004), and in the light of
recent reports of poor reproductive health in men experiencing
occupational exposure to chromium (Li et al., 2001; Danadevi
et al., 2003), it may be concluded that occupational and/or
environmental exposure to CrVI can affect the male reproduc-
tive health of primates. Our results support the hypothesis that
chromium-induced changes in the histoarchitecture of the testis
Figure 13. Effect of chronic chromium exposure on testicular concentrations of vitamins C (a), A (b) and E (c). Each bar represents the mean and
the vertical line above denotes the SEM. n = 3. Letters a, b and c denote statistical significance of differences among groups at P < 0.05: acontrol
versus experimental; b100 p.p.m. versus 200/400 p.p.m.; c200 p.p.m. versus 400 p.p.m.
by guest on June 9, 2013
M.M.Aruldhas et al.
are due to increased ROS, leading to oxidative stress in the
organ, and that these changes can be prevented by supplemen-
tation with antioxidants and are reversible. Since antioxidant
supplementation has been advocated for infertile men (Irvine,
1996), it becomes likely that the management of reproductive
health of men who have occupational exposure to chromium
may benefit from supplementation of antioxidants.
The financial assistance from Council of Scientific and Industrial
Research (CSIR), Government of India, New Delhi (Grant No.
60(00222)/97/EMR II), University Grants Commission (UGC),
New Delhi (Grant No. F.3–1/99(SAP-II) and the Department of Sci-
ence and Technology (DST), Govt. of India, New Delhi (DST-FIST
No. SR/FST/lSI-206/2000) is gratefully acknowledged. The assist-
ance from the Welcome Trust Research Centre, Christian Medical
College and Hospital, Vellore, India for ultra cut and TEM is also
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Submitted on December 6, 2004; resubmitted on May 5, 2005; accepted on
May 20, 2005
by guest on June 9, 2013