The effect of Lead Acetate on testicular structure and

Egypt. J. ofHistol Vol. 31, No.2, Dec, 2008: 406 - 418
(ISSN: 1110 - 0559)
Original Article
The Effect of Lead Acetate on Testicular Structure and Protective Effect of
Vitamin E in Adult Albino Rat
Madiha M. M. Makhlouf \ Heba M. S. Eldien1, Dorreia A. M. Zagloul2, Eman E.Abu Dief3 and
Nesreen G. Abd ElHaliem3.
'Departments ofHistology, and2Anatomy, Faculty ofMedicine, Assiut University. 3Department ofHistology, Faculty of
Medicine, Sohag University.
ABSTRACT
Bachground: Lead is considered an important cause of the infertility among occupational workers .The present work
was done to study the histological changes in the testicles of adult albino rats after lead treatment for different periods,
and the role of vitamin E in minimizing these changes.
Material and Methods: Forty five adult rats were used in this study. They divided equally into three groups; Group
I.'-(control). GroupII, was subdivided into subgroups IIa,IIb, and lie that received lead acetate for one, two and three
months respectively. GroupIII, was subdivided into subgroups IIIa& IIIb& IIIc that received prophylactic vitamm
E followed by lead acetate for one, two and three months respectively. The testis was dissected out, processed for
examination by light and electron microscope.
Results: Lead treatment induced shrinkage in some tubules and loss of germ cells while the remaining cells exhibited
pyknotic nuclei with vacuolated cytoplasm. Also proliferation of the interstitial tissue. With increase the duration of lead
acetate treatment there was progression in all previous changes in addition to the appearance of multinucleated giant
cells. Ultrastructurally, the most characteristic features observed were the apoptosis of the germ cells and Sertoli cells
as well as Leydig cells, also degenerative changes specially in mitochondria Combined treatment of vitamin E and lead
exhibited marked improvement in most of the previously mentioned changes.
Conclusion: Oxidative stress is a major cause of lead induced testicular damage. Using an antioxidant as vitamin E
interferes with the reactive oxygen species production and improves lead toxicity.
Key Words: Lead, testis, vitamin E, ultrastructure.
Corresponding Author: Heba M. S. Eldien
E-mail: [email protected]
INTRODUCTION AND AIM OF THE WORK
in observing the histological effect of lead on testis. It
was found that the mechanisms of lead induced cellular
damage are: the generation of reactive oxygen species1617
with subsequent stimulation of lipid peroxidation12. So
oxygen free radicals may therefore play a central role in
lead induced testicular dysfunction18. Free radicals, are
produced in many mammals in both health and disease,
in health, they may arise as regulatory mechanisms, or
as bactericidal agents19. Their production is normally
controlled by the antioxidant defense mechanisms that
include intracellular enzymes for example, glutathione
peroxidase, superoxide dismutase and low molecularweight compounds such as vitamin E or ascorbic acid20.
Oxidative stress arises when there is a marked imbalance
between the production and removal of these free
radicals. This may originate from an overproduction of
these substances or from depletion in the antioxidant
Lead is a heavy metal ad is considered as common
environmental and occupational pollutant. Nowadays, it
has been used in manufacture of electric storage batteries,
glass and ceramic ware. Also it is added as pigment in
paints or plastic formation1. Exposure to lead occurs by
many ways as inhalation and ingestion of contaminated
food and water2. Many studies found that lead has wide
range of toxic biochemical and histological effects where
it deposits in many organs as kidney3, ovary4, liver5,
brain6, blood7and endocrine system8. The toxic effect
of lead on testis was studied mainly physiological9 and
biochemical10. Animal models have been used in a number
of studies to observe histological effect of lead on testis
as rat11, mice12 and monkey13. The effect of lead on testis
is still a matter of controversy where exposure to low
dose of lead was found to arrest spermatogenesis14 or to
have no effect15. Most of these researches were deficient
39(1124-2008)
406
Madiha M. M. Makhloufet al.
defenses. Administration of vitamin E before exposure
to lead could reduce many of its side effects21. The aim
of this work is to reveal :1- The detailed histological and
ultrastructural changes induced by lead on testis. 2- To
know weather the preliminary use of antioxidant such as
vitamin E will improve the lead induced tissue damage
or not.
in cold 1% osmium tetroxide for two hours. Then they
were washed in four changes of cacodylate buffer for 20
minutes each. Dehydration was done by using ascending
grades of alcohol (30, 50, 70, 90 and absolute alcohol)
each for two hours. Clearing in propylene oxide then
they were embedded in Epon 812 using gelatin capsule.
These samples were kept in incubator at 35 degree for
one day, then at 45 degree for another day and lastly for
three days at 60 degree23. Semithin sections (0.5-lum)
were prepared by using LKB ultra microtome. The
sections were stained by Toluidine blue, examined by
light microscope and photographed. Ultrathin sections
(500-800A) from selected areas of trimmed blocks were
made and collected on copper grides. The ultrathin sections
were then contrasted in uranyle acetate for 10 minutes,
lead citrate for 5 minutes and examined by electron
microscope "Jeol JEM 1010" in the electron microscopic
unit of Faculty of Medicine, Sohag university.
MATERIAL AND METHODS
Material:
A total number of 45 adult male albino rats was used
in the present study and their weight ranged between 150
- 200 gm. The animals were divided into 3 groups.
Group 1 (15 animals): Were used as control, injected
intrapritoneally with saline.
Group II (15 animals): Were intraperitoneally injected
with lead acetate in a dose of 10 mg/kg body weight 5
days/week for three months 11. This group was subdivided
into 3 subgroups :-
RESULTS
Subgroup Ha: They were sacrificed after one month.
Control animals (Group I):
The parenchyma of the testis was formed of the
seminiferous tubules and the interstitial tissue inbetween.
The seminiferous tubules are oval or rounded according
to the direction of section cutting (Fig. 1). Each tubule is
surrounded by fibrous lamina called the tunica propria.
Each tubule is lined with stratified epithelium which
was seen to be formed of germinal cells and supporting
Sertoli cells. The germinal cells were stacked in the form
of many layers from the basement membrane toward
the lumen of the tubules. These layers are formed of
spermatogenic cells, which are; spermatogonia, primary
spermatocytes, secondary spermatocytes, spermatids and
mature sperms (Fig. 2).
Subgroup lib: They were sacrificed after two months.
Subgroup He: They were sacrificed after three months.
Group III (15 animals): Were injected intraperitoneally
with vitamin E in a dose of 100 mg/kg body weight 12
hours before lead acetate injections in the same previous
dose and duration21. This group was subdivided into 3
subgroups:Subgroup Ilia: They were sacrificed after one month.
Subgroup Illb: They were sacrificed after two months.
The spermatogonia lied next to the basement
membrane. They are of two types; type A spermatogonia
which were the predominant ones and appeared dome
shaped with large oval pale nuclei and prominent nucleoli
(Fig. 2). Type B cells were more rounded in shape with
rounded nucleus containing coarse clumps of marginated
heterochromatin in addition to fine chromatin granules
(Fig. 2). The primary spermatocytes represent the next
row of cells, present in more than one layer and were
considered the largest among the surrounding cells. They
were rounded cells, had acidophilic cytoplasm and large
nuclei with deeply stained chromatin granules of uniform
size which were distributed through the nucleoplasm.
The chromatin appeared in the form of short and thick
filament (pachytene stage) (Fig. 2). The secondary
spermatocytes were smaller and appear for short period
in stage IV. The early or round spermatids were stacked
into several layers (3-6) of rounded cells that had lightly
stained acidophilic cytoplasm and rounded vesicular
nuclei (Figs. 1,2). Sperms may appear in some tubules
with condensed, flattened elongated nuclei and their tails
were directed toward the lumen (Figs. 1,2).
Subgroup IIIc: Ihey were sacrificed after three months.
Preparation of materials:
Lead acetate solution (Merck) was prepared in saline
and replaced daily to minimize lead precipitates. D-Latocopherol (VE) was obtained as a gift from Pharco
Pharmaceuticals.
Methods:
1-Light Microscopy: The specimens were taken
from the testis of the control and treated animals and
were fixed in 10% Formalin for Haematoxyline and Eosin
stain,Toluidine blue-stained semithin sections (0.5-lum)
for testis were examined22.
2-Transmission Electron microscopy: Immediately
after sacrificing the animals, small pieces were fixed
in 5% gluteraldehyde for 24 hours. The specimens
were then washed in 3-4 changes of cacodylate buffer
(PH 7.2) for 20 minutes in every change and post fixed
407
The Effect of Lead Acetate on Testicular Structure and Protective Effect of Vitamin E in Adult Albino Rat
The Sertoli cells rest on the basement membrane of
the tubule and extends to the lumen. They are elongated
or pyramidal in shape and partially enveloped the
spermatogenic cells. Each cell exhibits ill defined outlines,
lightly stained cytoplasm, irregular pale vesicular nucleus
and prominent large nucleolus (Fig. 2).
highly vacuolated with deeply stained pyknotic nuclei.
Proliferation in the interstitial cells was observed
(Fig. 9).
Thickening and irregularities of the basement
membrane were observed in some tubules (Fig. 10).
Some of the Spermatogonia, the Sertoli cells as well
as the interstitial cells of Leydig had vacuolated deeply
stained cytoplasm (Fig. 10).
The interstitial tissue appeared as a granular
acidophilic ground substance containing Leydig cells
and blood vessels (Fig. 1).
Ultrastructurally, the degenerative changes were
observed in almost all germ cells types as well as the
Leydig cell, as regard type A spermatogonia showed
apoptotic changes in the form of nuclear chromatin
condensation and electron dense cytoplasm that contained
swollen mitochondria with destructed cristae (Fig. 11).
Ultrastructurally, the type A spermatogonium had
oval large nucleus with fine euchromatin and scanty
electron lucent cytoplasm containing numerous free
ribosomes and few mitochondria (Fig. 3). The type B
spermatogonium exhibited more or less rounded nucleus
with coarse clumps of marginated heterochromatin.
The cytoplasm contained numerous free ribosomes,
mitochondria and few strands of rough endoplasmic
reticulum (RER) (Fig. 4).
Early signs of degeneration in some spermatids were
observed in the form of destructed nuclear envelope.
Multiple residual bodies with large electron dense bodies,
lipid droplets and occasional vacuoles in their cytoplasm
were observed (Fig. 12).
The primary spermatocyte contained large rounded
nucleus with coarse chromatin threads evenly distributed
within the granular chromatin. The cytoplasm contained
numerous small mitochondria, strands of RER and free
ribosomes (Fig. 5).
The Sertoli cells revealed irregular electron dense
nuclei and the cytoplasm contained many dilated SER,
numerous mitochondria with destructed cristae which
sometimes appeared dilated and ballooned, also large
lipid droplets and secondary lysosomes were clearly
seen. (Fig. 13).
The rounded spermatid appeared as rounded cell
smaller than the primary spermatocyte. The nucleus was
large rounded with evenly distributed euchromatin and
the cytoplasm contained small spherical mitochondria
with electron lucent matrix. They were arranged in row
beneath the cell membrane. There were numerous free
ribosomes, vesicular profiles of smooth endoplasmic
reticulum (SER) and the acrosomal granule (Fig. 6).
Lead treated rats for two months (Subgroup lib):
After two months of lead treatment more degeneration
in the spermatogenic cells and the basement membrane
were observed. Arrested spermatogenesis at the level
of round spermatids or primary spermatocytes was
commonly observed while mature sperms were rarely
seen. Exfoliated cells or cell remnants were observed in
the lumen of some tubules. Multinucleated giant cells
were demonstrated within the lumina of the seminiferous
tubules. They exhibited acidophilic cytoplasm and two or
more rounded nuclei (2-6) (Fig. 14).
The Sertoli cell was identified by irregular shape and
illdefined outlines. The nucleus was large, located at the
base of the cell and showed indentation in the nuclear
membrane with homogenous nucleoplasm and prominent
large nucleolus. The cytoplasm contained mitochondria,
SER cisternae, few strands of RER, free ribosomes,
some lipid droplets and lysosomes (Fig. 7). Tight
junction was observed between the cell membrane of the
adjacent Sertoli cells above the level of spermatogonia
(Figs. 11,16).
Ultrastructurally, more degenerative changes were
encountered in germ cells in addition to the basement
membranes that appeared highly enfolded with thickened
lamina densa. The myoid cells appeared dense with
vacuolated cytoplasm (Fig. 15).
The interstitial cell of Leydig was rounded or oval in
shape with rounded euchromatic nucleus and prominent
nucleolus. The cytoplasm contained well developed
Golgi bodies, vesicular and tubular profiles of SER, few
lipid droplets, and numerous mitochondria with tubular
cristae (Fig. 8).
Variable degrees of degeneration were observed in the
primary spermatocytes. Their nuclei showed numerous
clumps of heterochromatin and dilation in the perinuclear
spaces. The cytoplasm contained mitochondria with
destructed cristae, dilated SER and free ribosomes
(Fig. 16).
Treated animals:
Multinucleated cells were observed and appeared to
be formed of fusion of two or more spermatids contained
two or more nuclei. The cytoplasm contained, free
ribosomes, mitochondria (Fig. 17).
Lead treated rats for one month (Subgroup Ha):
Treatment with lead for one month led to slight
degenerative changes with reduced diameter in some of
the seminiferous tubules, most of these cells appeared
408
Madiha M. M. Makhloufet al.
The Leydig cells showed euchromatic nuclei with
prominent nucleoli and dilated perinuclear spaces.
Some cells appeared binucleated. The cytoplasm
contained numerous dilated cisternae of SER and some
mitochondria with electron dense granules inside their
matrix (Fig. 18).
Lead and vitamin E treated rats for three months
(Group IIIc):
Most of the seminiferous tubules were more or less
similar to those of the control group, Some tubules
showed mild degenerative changes in the form of multiple
vacuoles in germ cells. (Fig. 26).
Lead treated rats for three months (Subgroup lie):
Treatment with lead for three months led to marked
degenerative changes in most of the seminiferous
tubules. The seminiferous tubules appeared shrunken
with irregularity in their basement membranes and lined
with one or two layers of small acidophilic cells with dark
nuclei and several layers of completely degenerated cells.
Different germ cells especially mature sperms were rarely
seen in most of the seminiferous tubules. Multinucleated
giant cells were observed in the lumina of these tubules
The proliferating interstitial cells appeared as numerous
cellular masses of highly acidophilic cells with deeply
stained nuclei (Fig. 19).
Ultrastructurally, similar observations in the
spermatids as those previously reported in the previous
Subgroup (lib).
Fig. 1: A section in the testis of control animal (GI) showing, group of
seminiferous tubules (T) with interstitial tissue (I) in between them .
II and E X 200.
The Sertoli cells had indented nuclei and prominent
nucleoli. Their cytoplasm showed aggregation of multiple
apoptotic bodies and large irregular secondary lysosomes
(Fig. 20). Some of Leydig cells showed apoptotic nuclei
and their cytoplasm contained multiple lipid droplets, few
dilated cisternae of SER, mitochondria and lysosomes
(Fig. 21).
Combined treated animals:
Lead and vitamin E treated rats for one month
(Subgroup Ilia):
Obvious improvement in the affected tubules
compared to that treated with lead only. Most of the
seminiferous tubules were more or less similar to those
of the control group while other tubules showed small
vacuoles in some germ cells (Fig. 22).
Fig. 2: A scmithin section in the testis of control animal (GI) showing,
type B spennatogonia (B), primary spermatocyte at pachytene stage
(P), early or rounded spermatids (Es), late spermatids(arrow) and
Sertoli cells (S) with oval nuclei and prominent nucleoli.
toluidine blue X 1000.
Ultrastructurally, most of the germ cells were more
or less similar to those of the control .However, some
spermatids appeared with destructed nuclear envelopes
and juxtanuclear chromatoid body (Fig. 23).
Lead and vitamin E treated rats for two months
(Subgroup Illb):
Most of the seminiferous tubules were more or less
similar to previous group. Ultrastructurally, most of the
germ cells appeared more or less similar to the control
group. Some spermatids still showed signs of nuclear
degeneration in the form of condensation of the peripheral
chromatin and decrease in the size of nuclei (Fig. 24).
The cytoplasm of Sertoli cells contained phagocytosed
apoptotic bodies and numerous primary lysosomes. The
late spermatids were frequently seen compared lo that
treated with lead only (Fig. 25).
Fig. 3: An electron micrograph of type A spcrmatogonium of
control animal (GI) showing, large oval euchromatic nucleus (N)
and the cytoplasm contains numerous free ribosomcs (R) and few
mitochondria (M).
Note: the tight junction between the spcrmatogonium and adjacent cells
(T) (upper right).
X 8000.
The Effect of Lead Acetate on Testicular Structure and Protective Effect of Vitamin E in Adult Albino Rat
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Fig. 4: An electron micrograph of type B spermatogoniuni of control
animal (Gl) showing, oval dichromatic nucleus (N) with marginated
hoterochromatin. The cytoplasm contains free ribosornes, numerous
mitochondria (MI and few strands of RER.
X 8000.
Fig. 5: An electron micrograph of primary spermalocyte of control aninia
(Gl) showing, a large rounded to oval nucleus (N) with multiple chromalin
granules, flic cytoplasm contains numerous mitochondria (M). free
rihosomes and strands of RER.
X 6000.
leSi
* V.'
Fig. 7: An electron micrograph of Sertoli cell of control animal ((il)
showing, enfolded dichromatic nucleus (N) with prominent nucleolus
(Nu). The cytoplasm contains mitochondria (M). SF.R cislernae, some
lipid droplets (L) and lysosotncs (Ly).
Note: the inter Sertoli cell junction (arrow head),
X 5000.
Fig. 8: An electron micrograph of Lcydig cell of control tcstis (Gl)
showing, rounded dichromatic nucleus (N) with prominent two nucleoli
(Nu). The cytoplasm contains numerous mitochondria (M), well
developed Golgi body (G). SF.R cislernae and lipid droplets (L),
X 8000.
iff*
i&2fcfaL.
a
y«a
I
V"
■
Fij». 6: An electron micrograph of rounded spennatid of control animal
(Gl) showing, rounded euehromatic nucleus (N) with acrosomal vesicle (f)
and acrosomal granule (Ag). The cytoplasm contains free ribosornes anil
mitochondria (M) with electron lucent matrix arranged in raw just beneath
the cell membrane.
X 8000.
Fig.'): A section in the leslis ol treated aninia Subgrouplla) showing.
multiple affected tubules contain highly vacuolaied cells (V) with
deeply stained pyknolic nuclei.
Note: -proliferation in the interstitial cells (I).
Hand EX 200.
410
MaUiha M, M. Makhloufel til.
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10
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Fig. 10: A semithin section in (he tcstis of treated animal (Subgroup Ila)
showing, type (A) and (B) spermatogonia have dark nuclei and vacuolalcd
cytoplasm. Sertoli cell (S) has dark irregular homogenous nucleus (N)
with prominent nucleolus and vacuolalcd (V) cytoplasm. Leydig cells
(arrow head) exhibit similar changes.
Note: the irregularity in the basement membrane of the seminiferous
lubule(t).
toluidine blue X 1000.
Fig. 13: An electron micrograph of Sertoli cell of treated animal
(SubgroupIIa) showing, enfolded electron dense nucleus (N), numerous
dilated SER cistemac (S). mitochondria (M) with partially destructed
cristae. secondary lysosomes (Ly) and large lipid droplets (L).
X 5000.
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• _**
•» V? .
Fig. II: An electron micrograph of type A spermatogonium of treated
animal (SubgroupIIa) showing, heterocbromatic nucleus (N) and the
cytoplasm contains swollen mitochondria (M) with destructed cristae, few
strands of RER and few lipid droplets. Note: intact junction between the
Sertoli cells (f).
Fig. 12: An electron micrograph in the leslis of treated animal (SubgroupIIa)
showing, multiple abnormal residual bodies (Rb) containing large electron
dense bodies!arrow head), lipid dioplels(L) and vacuoles.
Note: rounded spermalid with destructed nuclear envelop (]).
X 4000.
411
r„ .
Fig. 14: A section in the testis of treated animal (Glib) showing, some
germ cells with deeply stained nuclei (N) and vacuolalcd cytoplasm (V).
Multinucleated giant cell (G) and exfoliated germ cells (E) appear in the
lumina of the seminiferous tubules.
Note; pro) i feral ion in the interstitial cells (J).
I and E X 200.
Fig. 15: An electron micrograph of seminiferous tubule of treated animal
(Subgroupllb) showing marked enfolding in the basement membrane
(BM) with thickened lamina densa (LD) containing dense niyoid cells
(Y) with vacuolalcd cytoplasm.
Note: (ibroblasl cell (F) encircles the tubule from outside.
X 4000.
The Effect of Lead Acetate on Tcsticular Structure and Protective Effect of Vitamin E in Adult Albino Rat
Fig, 16: An electron micrograph of primary spcrmalocytes of treated
animal (SubgroupIIb) showing, rounded nucleus (N) with numerous
clumps of heteroehromatin and dilated perinuclear spaces (f). The
cytoplasm contains mitochondria (M) with destructed cristae. dilated SER
cisternae (C) and free ribosomes.
X 6000.
Fig. 19: A section in the testis of treated animal (Subgroupllc) showing.
variable degree of degenerative changes in the seminiferous tubules. Some
tubules (*) are lined with one or two layers of small acidophilie cells with
dark nuclei (N) and vacuolated cytoplasm (V). Multinueleated giant cells
(G) arc seen. Some tubules (T) have completely degenerated cells.
Note: proliferation in the interstitial cells (I).
11 and E X 200.
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ft
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Fig. 17: An electron micrograph of multinueleated giant cell of treated
animal (SubgroupIIb) showing, two rounded nuclei (N) of round
spcrmatids sharing the same acrosomal vesicle (Av). The cytoplasm
contains mitochondria (M) and numerous free ribosomes.
X 5000.
Fig. 20: An electron micrograph of Sertoli cell of treated animal
(Subgroupllc) showing, indented nucleus (N) with prominent nucleolus
(Nu). multiple apoptotic bodies (Ab) and large irregular secondary
ysosomes(Ly).
X 5000.
Fig. 18: An electron micrograph of Lcydig cell of treated animal
(SubgroupIIb) showing, two dichromatic nuclei (N) with prominent
nucleoli (Nu) and dilated perinuclear spaces ( | ) . The cytoplasm contains
numerous dilated cisternae of SER (C) and mitochondria.
Note: electron dense granule in some mitochondria (M).
X 8000.
412
*
i
A£J
v
•*
An electron micrograph of lcydig cell of treated animal
(Subgroupllc) showing, apoptotic nucleus (N). multiple lipid
droplets (I.), few dilated cisternae of SER (('). mitochondria (M) and
lysosomes(l.y).
X 8000,
Madiha M. M. Makhloufet al.
Fig. 22: A section in the tcslis of treated animal (SubgrouplNa) showing,
number of seminiferous tubules (T) more or less similar to those of control.
Other tubules have small vacuoles (V) among the lining epithelium.
Hand EX 100.
Fig. 25: An electron micrograph of the Sertoli cell cytoplasm of treated
animal (SubgroupTITb) showing, phagocylosed apoplotic bodies (Ab)
and numerous primary lysosomes (Ly).
Note: the late spermatic! (Ls) al the cytoplasmic process of the Sertoli
cell.
X 5000.
Fig. 23: An electron micrograph of rounded spermalid of treated animal
(SubgroupFIIa) showing, rounded nucleus (N) with acrosomal vesicles
(Av) and some areas show deslrucled nuclear envelope at certain parts
(t). The cytoplasm contains Golgi body(G) and numerous mitochondria
and juxtanuclear chromatoid body (C).
X 8000.
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I'ig. 24: An electron micrograph ol number ol rounded spennatids ol
treated animal (Subgroup lllb) showing, small hetcrochromatic nuclei (|)
in some spennatids and others contain rounded dichromatic nuclei (N).
X 3000.
413
Fig. 26: A section in the testis of treated animal (Subgrouplllc)
showing, multiple seminiferous tubules . Note: multiple vacuoles(*)
in the germ cells.
H and E X 200.
DISCUSSION
The degenerative ehanges observed in the present
study with lead toxicity appeared more obvious after three
months. This might result from increasing the duration of
lead intake and caused excess production of free radicals.
Similar observations were delected in reproductive system
of rat24. As well as in rat brain whereas the effect of lead
is a lime dependent one 25 . However, it hits been found
that no further increase in the disruption of reproductive
axis after 60 days of exposure to lead that explained by
the lead saturation of the soft tissue at this time, resulting
in mobilization of lead into the circulation2''.
In the present work, there was gradual increase in
the thickness of the basement membrane with increasing
duration of lead toxicity. This thickening might be result
from increase in the amount of collagcnous fibers that
could result from either over production of collage
The Effect of Lead Acetate on Testicular Structure and Protective Effect of Vitamin E in Adult Albino Rat
In the present study lead induced apoptotic changes
in most of the germ cells. These cells might be the most
affected cells owing to their proliferating character so
they might be the target for toxic effect of lead42. In
accordance to these observation ,it has been found that
lead affect mitotic spindle in lead treated rats43. These
changes might be due to the induction of the oxidative
mechanisms by lead which induced both apoptotic and
degenerative effects in the germ cells or secondary to
Sertoli cell injury. Similar observations were reported in
the germ cell of lead treated rat testis by using TUNEL
technique44. However , reactive oxygen species (ROS)
induced cell death might occur through apoptosis or
necrosis45. Also, they might be implicated in the defect
in speraiiogenesis, and residual body-like changes,
including multiple lipid droplets in lead treated mice12.
fibers by fibroblasts or decrease the rate of collagen
phagocytosis27,28. So lead toxicity might cause disruption
of collagen binding to phagocytic fibroblasts29. This
tendency towards fibrosis may be one of the possible
explanations for the shrinkage of seminiferous tubules
especially in late period.
On the other hand, the irregularities in the basal
lamina in the present work could be secondary to tubular
shrinkage in degenerated seminiferous tubules or as a
result of contraction of myoid cells. Similar observations
were reported by some investigators in lead treated rat
whereas they noticed the movement of myoid cells closer
to each other30. These changes might have an impact on
the process of differentiation and growth of spermatogenic
and Sertoli cells in lead treated monkey31.
The observed multinucleated giant cells might result
from widening of the intercellular bridge between adjacent
spermatids resulting in subsequent fusion of two or more
cells.This explanation was confirmedbyourultrastructural
finding. Same explanation was reported by some authors
in rat treated with nitrofurazone, they reported that it
might be a sign of germ cell degeneration46,47. However
it might result from karyokinesis without cytokinesis of
spermatid48.
In the present study, prolonged treatment with lead
showed that some seminiferous tubules were virtually
depleted of germ cells. This finding might be due to
loss of those populations via apoptosis or differentiation
failure. Similar explanation was reported in mutant
mice32.These changes might be explained by the
reduced expression of Sertoli cell growth factor (Glial
cell line-derived neurotrophic factor (GDNF) as well as
retraction of the Sertoli cells cytoplasmic processes that
are normaly supporting germ cells, that might depress
the spermatogonial differentiations33-34. Lead induced
mutation in the stem cell population of the embryonic
gonad might be a cause of this germ cell depletion35.
Another explanation was reported by some authors based
on the lead induced reduction of testosterone that might
be implicated in disruption of sertoli junctions10'36.
In the present work, proliferation of the Leydig
cells, dilated perinuclear space and appearance of
some binucleated cells. These findings might reflect
degeneration of these cells and decrease in testosterone
level which simultaneously arrested spermatogenesis.
Consistent with present findings, degenerative changes
were observed after diazepam treatment in rat testis49.
and human50. The presence of binucleated cells means
that mitotic division might occur as a compensatory
mechanism of Leydig cells affection which occurred
secondary to positive feed back on pituitary gland.
However, the number of Leydig cells was not necessarily
correlated with the hormone production. This finding
was confirmed by some investigators in some cases of
an increased number of Leydig cell that was associated
with testosterone production in few Leydig cells
only39. Reduced testosterone level was also detected in
workmen5'and in rat52 after lead and phthalate treatment
respectively. However, the observed dilatation in SER
cisternae in the present work might reflect the entrapped
testosterone hormone inside these cisternae and so
reduced its level in blood. Similar observations were
reported after withdrawal of testosterone in rat testis53 and
also in diabetic rat54. Also, tumor necrosis factor might be
implicated in the lead induced inhibitory effect on Leydig
cells55,56. On the other hand ROS might disrupt Leydig cell
mitochondria through the inhibition of steroidogenic acute
regulatory protein (StAR) expression57. In contradiction
to the present results, other workers observed diminution
of the surface area of SER in the Leydig cells in some
lead treated rats58.
Several large irregular secondary lysosomes, and
multiple apoptotic bodies that were observed in sertoli
cells in the present work might be as a result from
phagocytosis of the degenerating cells, consistent with
present fmding,a changes in the integrity of lysosome
and increase their size was observed in the fibroblast of
lead treated human37.
The inter-Sertoli junctional complex was observed
to be intact. This might be explained by the resistance
of these junctions to lead and or the paracrine action of
the neighboring Leydig cells in spite of its low level in
the blood, similar finding was reported in lead treated
animals3839. However, others, found that these junctions
were not hormonal dependent and not affected by changes
in gonadotropin release so they remained intact40. The
negative correlation between these morphological
findings and the previous results of desquamation of the
cells might reflect molecular affection of these junctions.
This suggestion was confirmed by the detection of certain
cell adhesion molecules, particularly the cadherins and Ca
(2+)-independent cell adhesion molecules, which might
be important early targets on which toxic metals such as
a cadmium and lead act to produce their toxic effects41.
From all of the above the state of these junctions will
need further investigations on the molecular basis.
The observed mitochondrial changes might be
414
Madiha M. M. Makhloufet al.
considered as early manifestation of apoptosis and an
adaptative process to unfavorable environments as
excess exposure of the cell to free radicals at the level
of intracellular organelles. These suggestions might be
confirmed by the successful suppression of these changes
by free radical scavengers59.
testis from toxic effect of lead. On the other hand certain
mechanisms might be implicated in the lead inducing
testicular damage other than production of ROS. Lead
could accumulate in cell nuclei associated with nuclear
proteins and chromatin and change their structure70'71. It
has been found that lead induced DNA alterations might
be irreversible12. In agree with these finding, it was
proved that vitamin E did not produce any appreciable
effect on reduced glutathione (GSH) status and other
related enzymes in animals after prolonged exposure to
lead21.
In the present work the dense granules that were
observed in the mitochondrial matrix of some germ cells
might be deposited lead. The same deposits found in
Sertoli cells of lead treated animals11. It has been found
that competitive inhibition occurs between lead and
calcium in the mitochondria that lead to increase in the
intracellular calcium60.
In conclusion, the results of the present study
suggested that oxidative stress is a major cause of lead
induced testicular damage. Using an antioxidant as
vitamin E interferes with the reactive oxygen species
production and improves lead toxicity.
In the present study combined treatment with lead and
vitamin E led to improvement in most of lead induced
apoptotic changes. However, some vacuoles and empty
spaces were observed among the lining epithelium. This
improvement might be secondary to antioxidant ability
of vitamin E which attacks ROS and so antagonizes their
harmful effects on the tissues. Consistent with these
suggestions, it was proved that vitamin E was capable of
dose-dependent regulation of mitochondrial generation
of superoxide and hydrogen peroxide61. Also it has been
found that lead toxicity is associated with decreased level
of alpha tocopherol that explains the importance of the
prophylactic use of the vitamin E in these conditions62.
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