Egypt. J. Exp. Biol. (Zool.), 2: 81 – 91 (2006)
© The Egyptian Society of Experimental Biology
RESEARCH ARTICLE
Fouad A. Abou-Zaid
Khaled M. Omar1
El Sayed I. Salim
Abeer A. Alam El-Deen
Mohamed M. Bekhite
Can man-made visible light radiation affect the reproductive capacity
of male mice?
ABSTRACT
The present study aimed to evaluate the
effects of isothermal non-ionizing electromagnetic field (EMF), represented by the man made visible light, on the reproductive capacity
of mice. Male mice were exposed to artificial
visible light (intensity of 77 mW /cm 2 on the
area of 72.5 cm 2 and frequency of EM radiation
between 3.9 x10 1 4 Hz and 7.5 x10 1 4 Hz W /cm 2 )
eight hours/day for 5, 7, and 15 days. The
present results indicated that exposure of male
mice to visible light for five or seven days
showed dramatic alterations in the histological
architecture of the testis, moreover, exposure
for 15 days induced a complete degeneration
of germ and Sertoli cells. Gonadotropic
hormones levels; FSH and LH were signifycantly declined by Day 5 and were not
detected absolutely at days 7 and 15.
In general, immunoreactivity of spermatogonia
to proliferating cell nuclear antigen (PCNA)
decreased in exposed mice. Moreover, germ
cell apoptosis assessed by in situ terminal
transferase-mediated end labelling (TUNEL)
increased significantly in 5 days exposed mice
and highly increased in 7 days exposed mice
compared with controls. Bcl-2 and Bax proteins
increased after exposure. Additionally, tumour
necrosis factor-alpha (TNF-α) protein content
decreased significantly in germ cells after
exposure. The present investigation indicated
that there is a strong coincidence between the
effects of man-made EMF on gonadotropic
hormone level expressions and the occurrence
of apoptosis in the testis. These data suggest
that the man-made EMF interferes with stages
of spermato-genesis by disruption of pituitary
gland function. In conclusion, man-made
visible light radiation affects negatively on the
reproductive capacity of male mice.
Key words:
Electromagnetic field, testis, gonadotropic
hormones, apoptosis, mice.
Fouad A. Abou-Zaid, Khaled M. Om ar 1 , El Sayed I.
Salim, Abeer A. Alam El-Deen, Mohamed M. Bekhite
Zoology Department., 1 Physics Department
Faculty of Science,Tanta Univ ersity.Tanta Egypt.
Email: [email protected]
INTRODUCTION
In recent years, the presence of extremely
low frequency electromagnetic field (EMF) in
the environment has become more and more
pervasive. The electromagnetic waves are
produced by transmission power lines and all
equipments using alternating current, including
household appliances.
Generally, while there have been many
undoubted benefits from the widespread use of
electricity, there is a concern that exposure to
electromagnetic fields (EMFs), especially at
the visible light range (400-800 nm) at even
low
levels,
could
have
detrimental
consequences on the human health and the
biological process (NRPB, 1992; Shaw and
Croen, 1993; NRPB, 1994; Tenforde, 1996;
Kheifets and Kelsey, 1997; Stavroulakis,
2003). Some epidemiological studies indicated
that exposure of EMF may be associated with
a variety of effects on reproductive outcome,
including foetal loss, variation in foetal growth,
and congenital malformations (Nordstrom et
al., 1983; W ertheimer and Leeper, 1989;
Juutilainen et al., 1993).
The biological effects of EMF have been
investigated by in vitro and in vivo experiments
with a wide variety of approaches (Stuchly et
al., 1991; Dachà et al., 1993). Male reproductive function was considered (Cerretelli et
al., 1979; Grisset, 1987; Margonato et al.,
1993; Hong et al., 2003), but the results of
these investigations are controversial because
of the variety of methodologies used and the
criteria tested.
Hong et al. (2003) investigated the effects
of extremely low frequency electromagnetic
fields (EMFs) on reproduction of male mice.
They exposed adult male mice to 50 Hz
sinusoidal EMFs of 0.2, 3.2 or 6.4 mT for 2 or
4 weeks. The authors reported that the
testicular weight of the exposed mice was
significantly lower than that of the control, no
significant histopathological changes were
observed on the testis of EMFs-exposed mice,
the
sperm
amount
and
motility
were
http://www.egyptseb.org
82
Egypt. J. Exp. Biol. (Zool.), 2: 81 – 91 (2006)
significantly decreased after EMFs exposure,
and the deformity rates of sperm were
significantly higher than those of the control.
Mammalian spermatogenesis consists of
three stages, i.e., (1) proliferation and
differentiation of spermatogonia, (2) meiotic
division of spermatocytes and (3) spermatogenesis (Kierszenbaum, 1994). Moreover,
testis has two important roles; production of
spermatozoa (fertility) and the secretion of
testosterone (T) which is needed for the
expression of secondary sexual characteristics
(virility). Proper functioning of the mammalian
testis is dependent upon an array of hormonal
messengers acting through endocrine, paracrine, and autocrine pathways. Successful and
complete male germ cell development is
dependent on the balanced of endocrine
interplay of the hypothalamus, the pituitary,
and the testis. Follicle stimulating hormone
(FSH) and luteinizing hormone (LH) are
glycoprotein hormones and part of the
transforming growth factor (TGF) β superfamily
that secreted by the anterior pituitary (Pierce
and Parsons, 1981) and are stimulated by
hypothalamic gonadotropins - releasing hormone (GnRH). These hormonal messengers are
critical not only for regulation of male germ cell
development, but also for the proliferation and
function of the somatic cell types required for
proper development of the testis (Sharpe,
1994).
The process of apoptosis is associated with
well-defined morphologically and biochemically
distinct form of the programmed cell death
process in which cells die in a controlled
fashion either spontaneously or in response to
changes in the levels of the specific
physiological stimuli such as exposure to
radiation and chemotherapeutic drugs or
activation by various death factors and their
ligands (McLachlan et al., 2002).
There are two major pathways to activate
proteases from the caspase family for
execution of apoptosis; Fas ligand (FasL) with
its receptor (FasR), and the TNF-α with its
receptors TNFR1 and TNFR2 (Huppertz et al.,
1999). In both pathways the death inducing
signal complex or the procaspases 8 and 9,
recruited by the apoptosomes, are activated
(Salvesen and Dixit, 1999). These caspases
subsequently cleave and activate the execution
caspases, which induce an irreversible degradation of the cell (Mignotte and Vayssiere,
1998).
The present study aimed to evaluate the
effects of isothermal non-ionizing EMF,
represented by the man-made visible light on
the histological structure of the testis and on
the efficiency of spermatogenesis in mice.
MATERIALS AND METHODS
35
Adult (60 day-old) male BALB/c mice (30 –
g) purchased from Touder Bilharzias
http://www.egyptseb.org
Laboratory, Cairo, Egypt, were used in the
study. Animals were housed in a standard
animal facility under controlled temperature 23
± 2° C (50 ± 10 %) humidity and photoperiod
(12 h of light, 12 h of darkness), with free
access to standard chow and water ad libitum.
Linear Source Lamps (LSL) were designed
as square plate with a reflected mirror, fixed
on it eight Neon lamps (60 cm length) at
distance > 0.4 m to irradiate electromagnetic
flux. Photometer was used for measuring the
intensity of electromagnetic flux (El-Bardie,
2003). Mice cages were prepared from a
polymer material, which is not dielectric
material.
Forty mice were divided randomly into 4
groups, 10 animals each. Three experimental
groups were exposed to visible light with
intensity 77 mW /cm 2 on the area of 72.5 cm 2
and frequency of electromagnetic radiation
between 3.9 x 10 1 4 Hz and 7.5 x 10 1 4 Hz W /cm 2
for 8 hours/day for 5, 7, and 15 days. The
fourth shame exposed group served as control.
The experiments were repeated three times.
After the end of the exposure, both control
and experimental animals were sacrificed by
cervical dislocation, and the testes were
excised and processed for routine paraffin
embedding for either normal histology or
immuno-histochemistry.
Immediately
after
death, blood samples were collected from the
inferior vena cava of each animal and the
plasma was separated and stored at -20º C for
subsequent hormone assays.
The Terminal deoxynucleotidyltransferase
(TdT)-mediated
dUTP
nick
end-labelling
(TUNEL) technique was performed according
to Gavrieli et al. (1992). Briefly, sections were
hydrated and treated with proteinase-K (Roche
Diagnostics GmbH, 20 µg/mL in 10 mmol/L
Tris-HCl buffer, pH 7.4) for 15-30 min at 37 ºC.
Slides were rinsed twice with PBS. Then, 50 µl
of TUNEL reaction mixture [450 µl nucleotide
mixture
containing
fluoresceinated
deoxyuridine triphosphate (dUTP) in reaction buffer
plus 50 µL enzyme TdT, both from Roche
Diagnostics GmbH] was added for 60 min at 37
ºC. After rinsing, slides were incubated with
antifluorescein antibody, Fab fragment from
sheep, conjugated with horse-radish peroxidase for 30 min at 37º C. Slides were rinsed
twice with PBS. Then, 50-100 µL of diaminobenzidine tetrahydrochloride (DAB) (Sigma),
for 10 min. Sections were washed, counterstained with haematoxylin and mounted.
The number of apoptotic cells was reported
at least in 30 randomly seminiferous tubules in
cross sections of testis. Data were averaged
for each testis and expressed as apoptotic
cells per tubule cross section.
The expression of Bcl-2 and Bax protein
was
determined
immuno-histochemically.
Sections were deparaffinized, hydrated and
then incubated in 2% H 2 O 2 . Sections were
blocked with 5% normal goat serum and
Abou-Zaid F. A. et. al - Can man-made visible light …
RESULTS
The results indicated that the mean value of
the control testis weight was 303.7 ± 11.6 mg.
The mean values of testis weight of the
experimental groups significantly decreased to
205.9 ± 9.2, 151.4 ± 6.7, and 115.3 ± 8.1 mg in
experimental mice exposed to electromagnetic
waves for 5, 7 and 15 days respectively (Fig.
1).
The normal testis is enclosed in a thick
dense connective tissue capsule, the tunica
albuginea. The structure unit of the testis is
the seminiferous tubules. The interstices
between the seminiferous tubules are occupied
by a highly vascular connective tissue contain
Leydig cells (interstitial cells). These cells are
350
300
Testis weight (mgm)
incubated with a 1:400 dilution of Bax or 1:300
dilution
of
Bcl-2
affinitypurified
rabbit
polyclonal antibody (Santa Cruz Biotechnology). Immunoreactivity was detected using
biotinylated goat antirabbit IgG (Chemicon,
1:400) secondary antibody followed by avidinbiotinylated horse radish peroxidase complex
(Santa Cruz Biotech.) visualized with DAB.
Slides were washed, counterstained with
haematoxylin and mounted.
Testis sections were permeabilized for 5
min in citrate buffer (10 mmol/l citrate, pH 6.0).
The sections were blocked with blocking
solution (PBS containing 5% goat normal
serum, 3% BSA, and 0.1% Tween 20) for at
least 30 min at room temperature. The sections
were incubated with goat anti-TNF-α antibod y
(1:150; Santa Cruz Biotechnology) for 60 min
at room temperature, followed by the addition
of biotinylated rabbit anti-goat immunoglobulin
G (Zymed LAB-SA kit) for 30 min. The sections
were then incubated with DAB, counterstained
with haematoxylin and mounted.
Testicular sections were washed with 1%
hydrogen peroxide in 0.1 M PBS containing
0.4% Triton X 100 (PBST) for 10 minutes to
quench the endogenous peroxidase activity,
rinsed 5 times with PBST, and incubated for 48
hours at 4 °C in PBST containing a monoclonal
anti-PCNA antibody (Santa Cruz Biotechnology, 1:500) and 4% normal sheep serum. After
incubation, sections were washed with PBST
and incubated with a biotinylated sheep-antimouse IgG (Chemicon, 1:400), washed, and
incubated with the Vectastain ABC Kit (Vector
Laboratories) for 1 hour. Sections were
washed and the immunoreactivity visualized
using DAB, counterstained with hematoxylin
and mounted.
LH and FSH in frozen plasma of mice were
measured with chemiluminescence assays
(Chiron Diagnostics ACS 180).
Data are given as mean values ± S.D.
GraphPad
InStat-3
software
(GraphPad
Software Inc., San Diego, USA) was applied
for t-test unpaired data. A value of P < 0.05 is
considered significant.
83
250
200
150
100
50
0
C ontrol
5 days
7 days
15 days
Fig. 1: (a) Photograph showing the testes of different
groups. (b) Weight of the testes of different groups
in mg. (scale bar = 5000 µm)
large, polygonal, and eosinophilic with rounded
nuclei. The seminiferous tubules are lined by a
very complex stratified epithelium containing
spermatogenic cells and supporting cells.
Spermatogenic cells include several morphologically distinguishable types: spermatogonia,
primary spermatocytes, secondary spermatocytes, spermatid and spermatozoa. Spermatogonia are dome shaped cells, have ovoid
nuclei, and resting on the basal lamina of the
seminiferous epithelium. The primary spermatocytes are large rounded cells located above
the spermatogonia. The secondary spermatocytes are resulted from the first meiotic
division
of
the
primary
spermatocytes.
Spermatids which are the most mature cells
are attached to the apical portion of Sertoli
cells and border the lumen of the tubules.
Supporting cells are of a single type, the
Sertoli cells. They are separate cells that
extend from the lamina to the free surface of
the epithelium. They are basically columnar
with extensive apical and lateral processes
that surround the adjacent spermatogenic
cells. The mean diameter of the seminiferous
tubules was 267 ± 3.4 µm (Figs 2a & 3).
Five days exposure group showed dramatic
changes in the histology of the testis.
Degeneration and atrophy in the germinal
epithelium of the seminiferous tubules were
observed. The basal lamina of the seminiferous tubules was obviously thin and shrunk.
The seminiferous endothelium showed complex
foldings resulted in irregular shapes of the
tubule outlines. The spermatogonic cells
contained only primary spermatocytes with
shrunk oval nuclei. Moreover, both spermatids
and spermatozoa were absent and the lumen
of the seminiferous tubules contained a lot of
84
Egypt. J. Exp. Biol. (Zool.), 2: 81 – 91 (2006)
large
pleomorphic
vacuoles
filled
with
protienous fluid.
The peritubular interstitial spaces were
filled with markedly numerous interstitial cells
that could be interpreted here as diffuse type
interstitial cell hyperplasia. Also interstitial
oedema (effusion) was obvious elsewhere
within the interstitial spaces. Multinucleated
giant cells were seen elsewhere within the
tubules, as well as in the interstitial areas (Fig.
2b). The diameter of the semini-ferous tubules
was significantly decreased and reached 129 ±
2.2 µm compared with that of the control group
(Fig. 3). Seven days exposure group showed
increasing in the degenerative characteristics
and atrophy in the testis components as
compared with both control and 5 days
exposure groups. The seminiferous endothelium and the myoid cells became more
irregular in shape. The spermatogonic cells
were greatly reduced in number and size. Few
Primary
spermatocytes
and
degenerated
Sertoli cells were observed lining the germinal
wall of the seminiferous tubules with a
complete absence of other types of germ cells
or spermatozoa. The pleomorphic vacuoles
were increased inside the lumens of the
seminiferous tubules. The number of interstitial
cells was decreased and the interstitial
oedemas were still observed (Fig. 2c). The
diameter of the somniferous tubules was
significantly decreased and reached 119.2±1.9
µm compared with both control and 5 days
exposed mice (Fig.3).
15 days exposure group showed complete
atrophy of the testes and severe degeneration
of the seminiferous tubules and germinal
epithelia.
Germinal
epithelium
of
the
seminiferous tubules was completely devoid of
both germ and Sertoli cells. Interstitial spaces
were highly vacuolated and contained very few
interstitial cells (Fig. 3d). The diameter of
seminiferous
tubules
was
significantly
decreased and reached 97.6 ± 7.8 µm
compared with control, 5, and 7days exposure
groups (Fig. 3).
280
Seminiferous tubule diameter (µ)
260
Fig. 2: Photomicrographs of testes sections of
different experimental groups. (a) Control
(b) 5 days (c) 7 days (d) 15 days exposure
groups. H. Haemorrhage; h. Hyperplasia: IC.
Int erstitial cell; my. myoid cells SC. Sertoli cell;
SG. Spermatogonia; SP. Spermatids; SZ.
Spermatozoa; V. v acuoles. (scale bar = 50 µm).
240
220
200
180
160
140
120
100
80
Control
5 days
7 days
15 days
Fig. 3: The effect of exposure on the diameter of the
seminiferous tubules (Mean ± SE).
Abou-Zaid F. A. et. al - Can man-made visible light …
85
trend towards a significant increase of
apoptosis began at 5days exposure group and
reached its maximum significance in 7 days
exposure group (Figs 4b-d & 5). However, in
15 days exposure group, which is completely
devoid of both germ and Sertoli cells, no
apoptotic cells were observed (Figs 4d & 5).
Mean number of apoptotic germ cells
16
14
12
10
8
6
4
2
0
-2
Control
5 days
7 days
15 days
Fig.
5:
Number of apoptotic cells in different
experimental groups.
The immunoreactivity for Bcl-2 for control
testis was positive in the cytoplasm of some
germ cells (Fig. 6a). The immunoreactivity for
Bcl-2 was decreased in 5 and 7 days exposure
groups (Figs 6b&c). However, in 15 days
exposure group, Bcl-2 staining was negative
due to the absence of germ cells (Fig. 6d).
Control
testis
showed
a
reasonable
immunoreactivity for Bax in the cytoplasm of
some germ cell nuclei (Fig. 7a). In 5 and 7
days exposure groups, a higher percentage of
Bax staining was observed in the germ cell
nuclei (Fig. 7b&c).
Due to the absence of germ cells in 15 days
exposure group, Bax staining was observed to
be negative (Fig. 7d).
The TNF-α protein detected by immunohistochemistry in the primary spermatocytes of
different
groups
revealed
a
significant
decrease in 5 and 7 days exposure groups
compared with that of control (Fig. 8a-c). This
down-expression of TNF-α was negative in 15
days exposure group (Fig. 8d).
Efficiency of spermatogenesis
Fig. 4: Sections of the testes of various experimental
groups showing germ cell apoptosis. Presence of
apoptotic germinal cells (arrowhea ds) detected by
TUNEL technique. (a) Control, (b) 5 days (c) 7
days, (d) 15 days exposure groups. (Scale bar = 50
µm)
Evaluation of germ cell apoptosis
The TUNEL technique was used to
document the induction of germ cell-specific
apoptosis following exposure to EMF.
TUNEL stain shows spontaneous apoptosis
of germ cells in control mice; primarily in the
spermatogonia and spermatocytes (Fig. 4a). A
PCNA protein was conducted to evaluate
the effect of man-made visible light on the
number of proliferating germ cells and to allow
more consistent identification of testicular
function. In control testis, an intense PCNA
immune-reactivity was observed in both
primary and secondary spermatocytes (Fig. 9
a). Since PCNA was shown to be expressed
during the pre-meiotic S phase. Exposure to
visible light alter the appearance of cell
positivity to PCNA In 5 and 7 days exposure
groups, reduction in the number of PCNApositive germ cells was observed (Fig. 9 b&c).
PCNA
immunoreactivity
was
completely
negative in 15 days exposure group (Fig. 9 d).
86
Fig. 6: Immunohistochemistry of Bcl-2 (arrowheads)
in the testes of different experimental groups.
(a) Control, (b) 5 days, (c) 7 days, (d) 15 days
exposure groups. (Scale bar = 50 µm)
Egypt. J. Exp. Biol. (Zool.), 2: 81 – 91 (2006)
Fig. 7: Immunohistochemistry of Bax (arrowheads)
in the testes of different experimental groups.
(a) Control, (b) 5 days, (c) 7 days, (d) 15 days
exposure groups. (Scale bar = 50 µm).
Abou-Zaid F. A. et. al - Can man-made visible light …
Fig. 8: Immunohistochemistry of TNF-α (arrowheads) in the testes of different experimental
groups. (a) Control, (b) 5 days, (c) 7 days, (d)
15 days exposure groups. (Scale bar = 50 µm).
87
Fig. 9: Immunohistochemistry of PCNA (arrowheads) in the testes of different experimental
groups. (a) Control, (b) 5 days, (c) 7 days, (d)
15 days exposure groups. (Scale bar = 50 µm)
88
Egypt. J. Exp. Biol. (Zool.), 2: 81 – 91 (2006)
Hormon concentration ng/ml
0.08
0.06
FSH
LH
0.04
0.02
0.00
Control
5 days
7 days
15 days
Fig.
10: Plasma FSH and LH lev els in different
experimental groups.
Gonadotropic hormone levels:
Plasma FSH and LH levels of control mice
were 0.0533 ± 0.00047 and 0.0667 ± 0.00047
ng/ml respectively. These levels were declined
significantly to 0.00097 ± 0.00047 and 0.0150
± 0.00041 ng/ml in 5 days exposure group.
However both hormones were suppressed in 7
and 15 days exposure groups (Fig. 10).
DISCUSSION
Exposure to magnetic fields (MFs) has
become an active area of biophysical research.
Discussion over the possible biological effects
of electromagnetic fields (EMF) first began in
the late sixties (Adey, 1997).
In the present study, testis showed marked
histological changes after exposure to manmade visible light radiation. Moreover, a
significant decrease in the weight and the
diameter of seminiferous tubules was detected.
These results agree with many previous
studies (kim et al., 1997, W ilson et al., 1999;
Lee et al., 2004; Ozguner et al., 2005; Kim et
al., 2006). In 5 and 7 days exposure groups,
marked degeneration and atrophy of the germ
cells were observed. Moreover, in 15 days
exposure group, seminiferous tubules were
completely devoid of both germ and Sertoli
cells.
TUNEL staining indicated a significant
increase in the number of apoptotic cells of
both 5 and 7 days exposure groups. The
stainability was completely negative in 15 days
exposure group. The dramatic changes which
observed in the testis components after
exposure to visible light flux might be
associated with an increased incidence of
germ cell apoptosis.
The process of apoptosis is associated with
well-defined morphological and biochemical
changes including a reduction in cell volume,
blebbing of cell membrane, chromatin condensation and migration and formation of apoptotic
bodies (Jacobson et al., 1997). Batistatou and
Greene (1993) reported that a unique biochemical event in apoptosis is the activation of
calcium-magnesium-dependent endonuclease
http://www.egyptseb.org
activity, which specifically cleaves cellular
DNA between regularly spaced nucleosomal
units. Such DNA fragments are considered the
hallmark of apoptosis.
O’Donnell et al. (1996) demonstrated that
spermatid might lose either from cell death
mechanisms that do not require DNA
fragmentation or by other means, such as
physical sloughing from Sertoli cells. The latter
might result from down-regulation of cell
adhesion protein (e.g. cadherins) expression in
Sertoli cells (Perryman et al., 1996). However,
these data together with the previous
speculation suggested that the lost of germ
cells that observed in the 5 and 7 days
exposure groups might be a result of apoptosis
involving DNA fragmentation.
Another important distinguishing feature of
apoptosis is the rapid clearance of dead cells
by ‘professional’ phagocytes (such as macrophages or neighbouring cells) before they can
lyse, spill their noxious contents and cause an
inflammatory reaction (Vaux and Strasser,
1996). This may explain the absence of all
stages of germ cells in 15 days exposure
group.
In the present work, immunohisotological
staining for Bcl-2 and Bax indicated the
decreasing of Bcl-2 and the increasing of Bax
in the exposed testes. This pattern is generally
consistent with the increasing TUNEL-positive
apoptotic labelling. Oltvai, et al. (1993) found
that ratio of Bcl-2 to Bax dictates a cell's
susceptibility to an apoptotic stimulus, with an
excess of Bcl-2 resulting in cell survival but an
excess of Bax resulting in cell death. In
addition, Sinha Hikim and Swerdloff (1999)
explained that rapid increasing of Bax was
capable of causing cytochrome C release,
thereby activating the caspases cascade. This
activation of caspases complex is apparently
followed by significant increase in apoptosis of
germ cells. The present results suggest that
exposure to visible light flux stimulates
apoptosis by elevating Bax synthesis.
Bcl-2 is considered one of the best studied
survival genes that regulate cell death
(Nagata, 1997). The same author added that it
contains both pro-apoptotic such as (Bax) and
anti-apoptotic (Bcl-2) proteins, constitutes a
critical, intracellular checkpoint within a
common cell-death pathway that determines
the
susceptibility of
a
cell
apoptosis.
Generally, the ratio of pro-apoptotic and antiapoptotic Bcl-2 family proteins is the critical
determinant of cell fate, with an excess of Bcl2 resulting in cell survival but an excess of Bax
resulting in cell death (Sinha Hikin and
Swerdloff,
1999).
On the
other
hand,
Korsmeyer (1995 & 1999) found that Bcl-2
family members are located in the outer
membrane of the mitochondria and function, at
least in part, by blocking the release of
cytochrome C from the mitochondria. The
author hypothesized that Bax induces the
Abou-Zaid F. A. et. al - Can man-made visible light …
release of cytochrome C by inhibiting Bcl-2
function. Lee et al. (1999) reported that some
members of the Bcl-2 family are involved in
apoptosis after withdrawal of androgen support
of the testis after treatment with ethane
dimethanesulfonate, a Leydig cell cytotoxin.
The present data showed that immunoreactivity for TNF-α protein was evident at the
primary spermatocytes of control sections.
This immunoreactivity was declined at 5 and 7
days and completely absent at 15 days
exposure groups. TNF-α is a pleiotropic
cytokine that exerts a wide range of cellular
effects (Beutler, 1995; Mauduit et al., 1996;
Baker and Reddy 1998). In the testis, TNF-α is
produced by male germ cells and is held to be
one of the testicular paracrine factors that,
together
with
the
hormones
of
the
hypothalamic-pituitary- testicular axis, regulate
spermatogenesis (De et al., 1993). Pentikainen
et al. (2001) demonstrated that, in mice testis,
TNF-α inhibits germ cell apoptosis and
downregulates Fas ligand, another member of
the TNF-α superfamily of death ligands. The
Fas system is also a widely recognized
apoptosis signal transduction pathway in which
a ligand-receptor interaction triggers the cell
death pathway. This system has been
implicated in the activation of germ cell
apoptosis in response to a variety of
proapoptotic stimuli, including T withdrawal.
There is an increasing evidence for the
involvement of LH and FSH in the regulation of
TNF-α synthesis (Mauduit et al., 1996).
Although high levels of TNF-α have been
demonstrated to exert antiapoptotic effects and
to promote germ cells survival, the low levels
of TNF-α have been demonstrated to be
involved in signal transduction cascades that
regulate Bcl-2 and Bax (Cory and Adams
2002). Thus, the change of spermatogonial
immunoreactive pattern obtained with both
antibodies reflects the differences associated
with spermatogenic impairment.
In the present work, the immunoreactivity
for PCNA protein was observed in both
spermatogonia and primary spermatocytes of
control testis. This immunoreactivity was
decreased in 5 or 7 days exposure groups and
was completely absent in 15 days exposure
group. Immunoreactivity of PCNA that detects
specific S-shape related protein was used to
investigate the mitotic activity of the different
types of spermatogonia and the possible
involvement in the spermatogenic efficiency.
Johnson et al. (1990) found that efficiency of
spermatogenesis depends on the proliferative
activity of spermatogonia and the loss of germ
cells during meiosis and spermiogenesis.
Thus, the change of spermatogonial immunereactive pattern obtained with PCNA antibody
in the exposed mice reflects the differences
associated with spermatogenic impairment.
Mammalian spermatogenesis is tightly
regulated
by the
pituitary gonadotropic
89
hormones; LH and FSH through their direct
effect on the testis. The effects of LH are
mediated via stimulation of Leydig cell T
synthesis, whereas FSH action is mediated via
specific receptors located exclusively on
Sertoli cells. It is generally accepted that in the
mice LH stimulates T formation in Leydig cells.
LH and T stimulate growth, differentiation, and
spermatogenic activity of the testis (Follett,
1984).
In the present investigation, plasma FSH
and LH levels were declined significantly in 5
days exposure group but not detected in 7 and
15 days exposure groups, a result which is
parallel with apoptotic germ cells observation.
This result was in accordance with Kula (1991)
who found that low efficiency of spermatogenesis including reduced numbers of spermatogonia was found to be correlated with low
levels of FSH after exposure of male mice to
EMF. Also, Shetty et al. (1996) found that
specific immunoneutralization of FSH accelerates germ cell apoptosis in the adult rats. The
inhibition of synthesis and release of
gonadotrophin hormones lead to inhibition of T
hormone. Kretser et al. (1998) suggested that
FSH may play a role in stimulating mitotic and
meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes as well
as in preventing apoptosis of pachytene
spermatocytes and round spermatids. In
addition, Sinha Hikim et al. (1997) and Sinha
Hikim and Swerdloff (1999) found a similar
relationship between germ cell apoptosis and T
deprivation in adult rat treated with GnRHantagonist
that
was
also
deficient
in
gonadotropic hormones. In the light of the
obtained data together with the previous works
(Ozguner et al., 2005), it may suggest that
initiation of testicular degeneration, mediated
by apoptotic processes, appears to be
depending on FSH and LH concentrations. The
indiscernible of these hormones at the 7 and
15 days exposure groups is correlated with the
complete absence of germ cells.
Previous studies showed that the reduction
in the number of rounded spermatids and the
complete failure of their maturation to
elongated spermatids were the key features of
T inhibition after EMF exposure (Zirkin et al.,
1989). This loss is suggested to be due to
failure of Sertoli cells to produce the adhesion
molecule N-cadherin, the production of which
appears to require both FSH and T. The
requirement of T for meiosis may be even more
crucial than its requirement for spermatogenesis (Tesarik et al., 1998).
In conclusion, the present investigation
indicated that exposure of male mice to manmade visible light caused degeneration of
testis in the form of germ cell apoptosis. This
degeneration is dependent on the decline of
the pituitary gonadotropins concentration.
The present study delt with some serious
biological effects, caused by one kind of manhttp://www.egyptseb.org
90
made electromagnetic fields. Further researches are necessary for deeper investigation of
the recorded effects, at biochemical and
biophysical levels. As it becomes clear from
the present results, man-made EMFs, can
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