In vivo spermatotoxic effect of chromium as reflected in the epididymal epithelial principal cells,...

In vivo spermatotoxic effect of chromium as reflectedin the epididymal epithelial principal cells, basalcells, and intraepithelial macrophages of a nonhumanprimate (Macaca radiata Geoffroy)

Mariajoseph Michael Aruldhas, Ph.D.,a Senthivinayagam Subramanian, Ph.D.,a,b

Pasupathi Sekhar, Ph.D.,a Ganapathi Vengatesh, Ph.D.,a Peranaidu Govindarajulu, Ph.D.,a

and Mohammad Abdulkader Akbarsha, Ph.D.c

a Department of Endocrinology, Dr. ALM Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani

Campus, Chennai, India; b Present address: Texas A & M University System Health Science Center, Temple, Texas; andc Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, India

Objective: To understand, through a simulation experiment in a nonhuman primate model, the potential in vivo

spermatotoxic toxic effect of hexavalent chromium (CrVI) in men who are occupationally or environmentally

exposed to it.

Design: Controlled laboratory study.

Setting: Research laboratory in a department of endocrinology in a university in India.

Animal(s): Male bonnet monkey, Macaca radiata Geoffroy.

Intervention(s): Monkeys were exposed ad libitum to 100, 200, and 400 ppm CrVI, dissolved in drinking water,

for a chronic period of 180 days.

Main Outcome Measure(s): Examination of epididymis with a transmission electron microscope and assessment

of the effect of CrVI in terms of accumulation of sperm-derived lipofuscin (LF) material in the principal cells,

basal cells, and intraepithelial macrophages of the epithelium.

Result(s): The abundance of basal cells and intraepithelial macrophages and the content of LF material in these

cell types increased. The principal cells phagocytosed from the lumen the dead sperm resulting from CrVI

exposure and processed them partially into LF material, which was acquired by the basal cells and intraepithelial

macrophages and processed further. The LF material–laden basal cells and intraepithelial macrophages appeared

to leave the epithelium, accompanied by recruitment of fresh basal cells and intraepithelial macrophages.

Conclusion(s): Occupational or environmental exposure to CrVI, as would occur in the tannery, soap, and other

industries in developing and underdeveloped countries, can be toxic in vivo to spermatozoa. (Fertil Steril� 2006;

86(Suppl 3):1097–105. ©2006 by American Society for Reproductive Medicine.)

Key Words: Basal cell, chromium, epididymis, intraepithelial macrophage, lipofuscin material, principal cell,

spermatotoxicity

Hexavalent chromium (CrVI) is a heavy-metal pollutant that

can cause carcinogenesis and several systemic toxicities

(1–5). A case-controlled study of the male partners of infer-

tile couples who were exposed to CrVI in welding fumes

through the nasal route suggested that low-level exposure to

this heavy metal may not be a major hazard affecting sper-

matogenesis (6). In a recent review, Bonde (7) opined that

evidence to implicate CrVI in male reproductive toxicity is

not adequate. Two recent epidemiological studies, one from

China (8) and another from India (9), also attest poor semen

quality in men who are exposed to Cr in the chrome-plating

and welding industries.

The available information on the male-reproductive toxic

effect of CrVI in human beings is confined to occupational

exposure to CrVI fumes from stainless-steel welding through

the nasal route only. However, occupational exposure to

CrVI varies with the intensity and the route, depending upon

the nature of work. For example, in the case of the tannery,

soap, and a few other industries, the exposure route can be

oral and even through the skin. Tannery effluent in several

developing countries, including India, and in underdevel-

oped countries is let out without much treatment and can

contaminate water bodies, including subsoil water. These

occupational and environmental exposures to CrVI could be

chronic. Therefore, there is pertinent need to investigate the

male-reproductive toxic effect of CrVI in high-level chronic-

exposure groups.

Received December 24, 2005; revised and accepted March 17, 2006.

Supported by the Council of Scientific and Industrial Research, Govern-

ment of India, New Delhi (grant 60(00222)/97/EMR II, to M.M.A.); by the

Department of Science and Technology, Government of India, New

Delhi, under the Fund for Improvement of Science and Technology

program (SR/FST/LSI-206/200 and SR/FST/LSI-112/2002); and by the

University Grants Commission, New Delhi, India, under the Special

Assistance program (F.3-1/99(SAP-III)).

Reprint requests: Mariajoseph Michael Aruldhas, Ph.D., Department of

Endocrinology, Dr. ALM Post-Graduate Institute of Basic Medical Sci-

ences, University of Madras, Taramani Campus, Chennai 600113, India

(FAX: 91-44-24926709; E-mail: [email protected]).

10970015-0282/06/$32.00 Fertility and Sterility� Vol. 86, Suppl 3, October 2006doi:10.1016/j.fertnstert.2006.03.025 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

Experiments in laboratory animal models and cell lines

attest impairment of male reproductive function by chro-

mium (10–19), albeit some differences as a result of varia-

tions in the experimental design. It is not possible to carry

out such experiments in human beings, and hence, studies in

an animal model close to human beings, such as nonhuman

primates, that simulate the occupational and/or environmen-

tal exposure to CrVI in the tannery industry for a chronic

period may be pertinent.

Adopting the above rationale, we subjected male bonnet

monkeys (Macaca radiata) to chronic–CrVI toxicity testing.

We reported several adverse changes in the testis, including

cytological changes in different generations of germ cells in

the adluminal compartment of the seminiferous epithelium,

generation of uninucleate and multinucleate giant cells, pre-

mature release of germ cells from the Sertoli cell, occurrence

of macrophages in the lumen of the seminiferous tubules,

and decrease in the activities of enzymatic and nonenzymatic

antioxidants in the testis (20).

Earlier, we reported the presence of undifferentiated

and/or immature uninucleate and multinucleate germ cells in

the lumen of ductus epididymidis, and hypertrophy and

hyperplasia of the cauda epididymal epithelium. These

changes obstructed the distal portions of the male reproduc-

tive tract, resulting in a tendency of sperm to escape through

certain weak patches in the epithelium. To prevent the gen-

eration of auto-antibodies to sperm antigens in sequel to such

an extravasation, microcanaliculi, lined by a hitherto unre-

ported cell type, developed in the epididymal epithelium, in

which these sperm were closeted and secluded from the

circulatory system. It was suggested that these cells were

derived from basal cells (BCs) (21).

In recent years, several reports have ascribed a role to

epididymal epithelial BCs and intraepithelial macrophages

(IEMs) in surveillance of sperm antigens when there is

obstruction to passages of sperm (22–26). Obstruction of

sperm passage leads to increase in the number of BCs and

IEMs and to a dense accumulation of lipofuscin (LF) mate-

rial in their cytoplasm. These are indications of the arrival of

sperm antigens into the epithelium through transcytosis

across the principal cells (PCs) and also through leaky junc-

tions between the tall columnar cells. These changes result in

increased abundance of BCs and IEMs and also in increased

content of LF material in these cell types, which are reflec-

tions of a pathophysiological state of the luminal sperm and

of a defense mechanism to sperm antigens.

Taking a cue from the above background information, we

used a transmission electron microscope to reexamine, using

the same tissue blocks used for our earlier study (21), dif-

ferent segments of the epididymis of monkeys that were

exposed to CrVI. This article reports similar changes in the

BCs and IEMs. The study strongly implicates chronic CrVI

exposure through the oral route in the male-reproductive

toxic effect of bringing about death of epididymal luminal

sperm, but the epididymis responds to the situation with

appropriate changes to process them.

MATERIALS AND METHODS

The animal (Macaca radiata), experimental design, treat-

ment of CrVI, and analysis were the same as reported in our

article published elsewhere (21), except that the recovery

study is not included in the present report. The experimental

design was approved by the institutional animal ethics com-

mittee, which includes a nominee of the Committee for the

Purpose of Control and Supervision of Experiments on An-

imals, Government of India.

Briefly, adult monkeys obtained from the Department of

Forests, Government of Tamil Nadu, India, were subjected

to quarantine and maintained under animal-house conditions.

The monkeys were divided into four groups of three each.

Monkeys in groups 1 to 3 were provided with drinking water

containing 100, 200, and 400 ppm CrVI (potassium dichro-

mate), respectively, for 180 days. Monkeys in group 4

formed the control and received plain water in which CrVI

was not dissolved.

At the end of the experimental period, the monkeys were

subjected to sodium pentobarbital anesthesia, and the testi-

cles and epididymides were autopsied through an incision in

the scrotum, after which the incision was closed with stitches

of nylon thread. After healing of the wound and removal of

the stitches, the monkeys were released into the reserve

forest as per the guidelines of Committee for the Purpose of

Control and Supervision of Experiments on Animals, Gov-

ernment of India. Under this circumstance, perfusion fixation

was not performed. Thin slices of different segments of the

epididymides were fixed in 2.5% glutaraldehyde prepared in

cacodylate buffer and were postfixed in 1% osmium tetrox-

ide. Semithin sections (1 �m), obtained with a Leica (Gena,

Germany) ultramicrotome, were stained with toluidine blue

O and were used to select areas for ultrathin sections. The

latter, cut with the same ultramicrotome, were stained with

uranyl acetate and lead citrate and observed in a Phillips

201C (Amsterdam, Holland) transmission electron micro-

scope. The images were scanned into a Pentium III com-

puter, and the images were edited and processed using Pho-

toshop version 7.0 software (Adobe, San Jose, CA).

RESULTS

Irrespective of the segments, that is, initial segment (IS),

caput (CA), corpus (CO), and cauda (CU), the epididymal

epithelium of control monkeys possessed very few BCs and

IEMs (Fig. 1A and B). The BCs had the characteristic

arrangement abutting the basal lamina (BL) and possessed

heterochromatic round or irregular nucleus occupying al-

most the entire cell, leaving only little cytoplasm around it.

The cytoplasm was scarce in organelles, which consisted of

mitochondria, endoplasmic reticulum, lysosomes, and endo-

cytic vesicles. The Golgi apparatus was hardly visible (Fig. 1C).

1098 Aruldhas et al. Spermatotoxic effect of chromium in a nonhuman primate Vol. 86, Suppl 3, October 2006

FIGURE 1

Transmission electron micrographs of epididymal epithelium of control (A–D) and CrVI-treated (E, F)

monkeys. (A) Initial segment of control monkey. (B) Cauda of control monkey. (C, D) Portions of caput of

control monkey, showing a basal cell (C) and an intraepithelial macrophage (D). (E, F) Corpus and cauda,

respectively, of 200 ppm CrVI–treated monkeys. In both the segments of CrVI-treated monkeys the

abundance of basal cell has increased, forming an almost continuous row. Note dense lipofuscin material in

the basal cells of corpus and not of cauda. BC � basal cell; BL � basal lamina; ER � endoplasmic

reticulum; IM � intra-epithelial macrophage; LF � lipofucsin material; MI � mitochondria; NU � nucleus;

PC � principal cell; PD � peduncle; and PT � peritubular tissue. Scale bar � 6 �m (A, B), 2 �m (C, D), or

5 �m (E, F).

Aruldhas. Spermatotoxic effect of chromium in a nonhuman primate. Fertil Steril 2006.

1099Fertility and Sterility�

FIGURE 2



Although a few BCs were flat, a few others were elongated

and had a vertical orientation, contacting the BL through a

broad cytoplasmic peduncle (Fig. 1A).

A few IEMs, which were present at different heights along

the epithelium, possessed densely heterochromatic nucleus,

large mitochondria, and scattered endoplasmic reticulum.

The cells were characterized by irregular shape as a result

of the pseudopodia (Fig.1A and B; see also Fig. 3A). Neither

the BCs nor the IEMs possessed any perceptible LF material

(Fig. 1C and D).

In the epididymis of CrVI-treated monkeys, the BCs were

abundant and formed an almost continuous row on top of the

BL in such a way that the epithelium appeared pseudostrati-

fied (Fig. 1E and F). The cytoplasm of every BC of IS, CA,

and CO contained a dense accumulation of LF material,

which was more voluminous than in the control monkeys

(Fig. 1E). Though the BCs were abundant in the CU as in the

proximal segments, the cytoplasm had only sparse LF

material (Fig. 1F).

In several BCs of IS, CA, and CU, the LF material

occupied almost the entire cytoplasm (Figs. 1E and 2A), and

the nucleus was indented at one pole to accommodate the

large mass of LF material (Fig. 2A). The LF material of BCs

occurred as small to large membrane-bound vesicles, each

containing a material of varying electron densities. In some

vesicles, the content at the periphery was more electron

dense than that at the core (Fig. 2B). Cells matching BCs in

organization and loaded with LF material were present in the

peritubular tissue (Fig. 2C). Leukocytic cells matching BCs

but not containing LF material were observed to make access

into the epithelium from the peritubular tissue through the

BL (Fig. 2D).

The IEMs also increased in abundance in the IS, CA, and

CO. In a few cases, the IEMs were engaged in phagocytotic

uptake of cell debris including sperm, which in all probabil-

ity were dead (Fig. 3A). Most of the IEMs, however, con-

tained LF material, some of which were more electron dense

than the BCs (Figs. 2B and 3B). The IEMs acquired the LF

material from outside through phagocytosis (Fig. 3C).

The PCs of IS, CA, and CO also contained LF material in

the supranuclear as well as perinuclear cytoplasm (Fig. 3D

and E). The cytoarchitecture of the PCs was invariably

normal, containing clathrin-coated vesicles in the apical

plasma membrane indicating endocytotic activity (Fig. 3D),

prolific Golgi stacks in the supranuclear cytoplasm, and

vacuolated perinuclear cytoplasm (Fig. 3E). Several PCs

contained spermatozoa in the cytoplasm at various locations

from apical to the base (Fig. 4A–C). The spermatozoa were

in the process of disintegration inside large membrane-

bound vesicles, and LF material was found in the vicinity of

such vacuoles (Fig. 4D). These were spermatozoa that were

phagocytosed at the apical plasma membrane of the PCs

(Fig. 4E). Once inside the cytoplasm, the spermatozoa were

closeted in membrane-bound vesicles (Fig. 4F).

The responses increased with the dose of CrVI treatment;

that is, from 100 to 400 ppm (data not presented).

DISCUSSION

The present study indicates that CrVI exposure leads to

increase in abundance of BCs and IEMs in the monkey

epididymal epithelium and that the cytoplasm of both the

cell types in the IS, CA, and CO is laden with LF material.

Both the cell types appeared to acquire the LF material from

the PCs, produced as a result of partial digestion of sperma-

tozoa, which were phagocytosed through the apical plasma

membrane. The spermatozoa thus phagocytosed are to be

considered dead because live sperm are not phagocytosed,

excepting in the case of autoimmune response to sperm

antigens (27). The results support our earlier findings (20,

21) that exposure of monkey to CrVI through the oral route

for a chronic period of 180 days can bring about toxic effects

on the testis and epididymis, including spermatotoxicity.

Basal cells are the second largest population of epithelial

cell type that is common to all segments of the epididymis,

next only to PCs (22). Consistently found in the epididymis

of the different mammalian species, the BCs are established

only after the other cell types (23). For example, in the

mouse epididymis, the BCs are not present before days 12,

14, and 16 postpartum in the CU, CA, and CO epididymides,

respectively. The BC number increases in all segments be-

tween days 20 and 27 postpartum (23). The temporal pattern

in the first appearance of BCs in the epididymal epithelium

FIGURE 2 Continued

Transmission electron micrographs showing basal cells in the epididymal epithelium (A, B) and a basal cell–

like cell (C) and leukocytes (D) in the peritubular tissue of CrVI-treated monkeys. (A) Basal cell of corpus

showing dense accumulation of lipofuscin material and, also, indentation of nucleus at the apical pole to

accommodate it (arrowhead). (B) Portion of a basal cell of initial segment showing the heterogeneous nature

of lipofuscin material. (C) A basal cell–like cell, loaded with lipofuscin material (arrowhead), in the peritubular

tissue. (D) Entry of leukocytes from the peritubular tissue into the epithelium (arrowheads). BC � basal cell;

BL � basal lamina; CA � capillary; EN � endothelial cell around capillary; LE � peritubular tissue

leukocytes; LF � lipofuscin material; NU � nucleus; PC � principal cell; and PT � peritubular tissue. Scale

bar � 5 �m (A), 2 �m (B, D), or 3 �m (C).


FIGURE 3

Transmission electron micrographs of intraepithelial macrophages (A–C) and principal cells (D, E) in

the epididymal epithelium of CrVI-treated monkeys. (A) Two intraepithelial macrophages, lying closer to

the lumen, in corpus, in the process of phagocytosing sperm and cell debris from intercellular space.

(B) The lipofuscin material in the intraepithelial macrophage in initial segment is darker and denser than in

the principal cells. (C) An intraepithelial macrophage in corpus in the process of acquiring lipofuscin material

(arrowheads) from the intercellular space. (D) Principal cells of caput showing lipofuscin material in the

cytoplasm. (E) Principal cells of corpus showing lipofuscin material in the cytoplasm. BC � basal cell;

BL � basal lamina; CD � cell debris; CP � coated pits; CV � coated vesicles; ER � endoplasmic reticulum;

EV � endocytic vesicles; GA � Golgi apparatus; IM � intraepithelial macrophages; LF � lipofuscin material;

LY � lysosomes; MI � mitochondria; MV � microvilli; NU � nucleus; PS � pseudopodial processes; and

SM � sperm. Scale bar � 2 �m (A–C) or 0.5 �m (D, E).



FIGURE 4

Transmission electron micrographs of principal cells of proximal epididymal segments of CrVI-treated

monkeys showing spermatozoa in the cytoplasm, their acquisition and processing. (A) Sperm heads and

tails in the cytoplasm. (B) The spermatozoa are found closer to the basal lamina. (C) A spermatozoon,

in longitudinal section, in the cytoplasm. (D) Spermatozoa partially processed by lysosomal activity.

(E) Acquisition of spermatozoa through deep indentations between microvilli (arrowheads). (F) The

spermatozoa are closeted in a membrane-bound vesicle in the apical cytoplasm. BC � basal cells;

BL � basal lamina; ER � endoplasmic reticulum; IM � intraepithelial macrophages; LU � lumen;

LF � lipofuscin material; MI � mitochondria; MV � microvilli; NU � nucleus; PC � principal cells;

SM � sperm; and VA � membrane-bound vesicle. Scale bar � 4 �m (A, B), 0.5 �m (C, D, F), or 1 �m (E).



has been shown in the rat, ram, rabbit, goat, cat, and human

(23, 28). The hypotheses that the BCs are progenitors of PCs

(29) and that the columnar cells give rise to the BCs (30)

have been set aside (26). The BCs have been shown to

express macrophage antigens in human (22) and in monkey

and mouse (23–26), suggesting that the BCs may be derived

from monocytes, which arrive at the epididymal epithelium

for transformation into macrophages (26). The ultrastructural

similarities between BCs and interstitial macrophages also

attest this suggestion (22).

Several lines of evidence suggest that the BCs are a part of

the immune defense system of epididymis (22–26). A link

between macrophage antigen expression in the BCs and

presence of damaged spermatozoa in the epididymis has

been suggested. In the normal male, the proportion of dam-

aged spermatozoa and the expression of macrophage anti-

gens in the BCs are low in the CU (23). Experimentally

induced sperm cell damage in situ demonstrated that the BCs

respond to the presence of sperm antigens in the lumen with

increased number and expression of macrophage antigens.

This would prevent interaction of sperm antigens with the

immune system and abrogate the risk of immunological

infertility (25).

The relative abundance of LF material in the BCs is a

reflection of the extent of scavenging of sperm antigens. The

LF material in BCs increases with age (22), and it depends

on the arrival of sperm antigens in the epithelium under

experimental or pathophysiological conditions (25). The in-

crease of LF material in the PCs and its prolific occurrence

in the BCs of CrVI-treated monkeys indicates the arrival of

sperm antigens, which could have been processed already by

the PCs. Because the release of antigens from the sperma-

tozoa occurs in pathophysiological states (25, 26), the results

of the present study indicate CrVI-induced pathophysiolog-

ical state of the spermatozoa. This is substantiated by the

increase in the abundance of IEMs with LF material. Active

uptake of LF material by the IEMs from the intercellular

spaces has been documented (22). It also has been shown

that the LF material arrives at the intercellular spaces from

the PCs and thus becomes available to the BCs and IEMs

(22). The PCs acquire sperm antigens from the damaged

spermatozoa in lumen through adsorptive and/or fluid-phase

endocytosis (27).

The present observation in CrVI-treated monkeys shows

that the spermatozoa may be in a pathophysiological state

and are acquired by the PCs and processed in their cytoplasm

by lysosomal activity, resulting in LF material. According to

Gadhially (31), the LF material is the residual body that is

left behind in the cells after lysosomal activity. The abun-

dance of this material increases in the cells of older individ-

uals, and therefore it has been referred to as age pigments,

wear-and-tear pigments, and womb-to-tomb pigments. The

LF material is looked upon as a conglomerate of undigested

residues from lysosomal hydrolysis and modification of se-

questrated material before and after digestion. The power of

lysosomal hydrolases in degrading lipids is limited, and

hence, the lipids are left behind and undergo stepwise oxi-

dation, resulting in overloading of LF material. The LF accu-

mulation is engendered by various noxious influences (31).

We have shown elsewhere that CrVI exposure leads to

impairment of spermatogenesis as a result of the develop-

ment of oxidative stress in the testis of monkeys (20). In

another study published elsewhere, we showed that CrVI

treatment causes obstruction of the distal CU epididymidis

and vas deferens, leading to stress-induced sperm death (21).

Such stress-induced sperm death in the epididymis was at-

tributed to excess free radicals generated and poor scaveng-

ing of the same. Our observation of increased abundance of

LF material in the PCs, BCs, and IEMs attests the above

conclusions.

It is to be inferred that the sperm death caused by CrVI

intoxication occurs in such a large number that the luminal

macrophages do not adequately cleanse them. Therefore, the

PCs take over the role of phagocytosing dead spermatozoa

(32). The digestion of the spermatozoa acquired into the PC

cytoplasm is incomplete and results in LF material. Because

the PCs do not possess the mechanism to process the huge

quantity of LF material, the latter is discharged into the inter-

cellular spaces to be acquired by the BCs and IEMs. The LF

material–laden BCs and IEMs may leave the epithelium and

arrive at the peritubular tissue (22–26).

The poor content of LF material in the BCs and IEMs of

CU makes an interesting observation. Paracrine factors in-

duce macrophage antigen expression once the BCs have

taken up residence in the epithelium and the spermatozoa

and testicular fluid arrive at the epididymis (22). Thus, there

is greater expression of Mac-1 in the BCs of adult CA, and

its expression in the CU occurs only much later, suggesting

a link between macrophage antigen expression and the pres-

ence of spermatozoa and/or testicular fluid in the epididymis.

In the normal male, the proportion of damaged spermatozoa

in the CU was found to be low, whereas in the CrVI-treated

monkeys, the entire epithelium at the CU underwent hyper-

trophy and hyperplasia, contributing to obstruction to the

passage of spermatozoa (21). The consequent limited occur-

rence of spermatozoa in the CU may be responsible for the

fewer LF inclusions in the BCs and IEMs.

Thus, this article provides further strong evidence for the

male-reproductive toxicity of chronic exposure of a nonhu-

man primate to CrVI. The results indicate damage to the

epididymal luminal spermatozoa, as a consequence of which

the PCs take up the role of phagocytosis of the damaged

spermatozoa, which is followed by an increase in the number

of BCs and IEMs with dense accumulation of LF inclusions.

This may be another mechanism by which the epididymis

overcomes the adverse effects of chronic CrVI exposure on

the sperm apart from microcanalization to closet the viable

sperm that might get access into the epithelium (21). This

and our earlier articles on the testis and epididymis of

CrVI-treated monkeys (20, 21), supported by the two recent


reports of increased infertility in men with occupational

exposure to Cr (8, 9), strongly suggest Cr as a vulnerable

disrupter of male fertility.

Acknowledgments: The authors thank Anna Pulimood, M.D., for permis-

sion to use the transmission electron microscope facility of the Welcome

Trust Research Laboratory, Christian Medical College and Hospital, Vel-

lore, India, and also thank B. Kadalmani, M.D. for helping in image

processing.

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In vivo spermatotoxic effect of chromium as reflected in the epididymal epithelial principal cells,...

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