ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 77
OBJECTIVE: To investigate the role of 50-Hz extremely
low frequency pulsed (PEMF) and sinusoidal (SEMF)
electromagnetic fields on the testis using a rat model.
STUDY DESIGN: Twenty-seven male Wistar albino
rats were used. The rats were divided into three groups
(n=9 each): sham group, SEMF group, and PEMF
group. The SEMF and PEMF groups (pulse time: 20
milliseconds, pulse frequence: 50 Hz) were subjected
to 1.5 mT, 50 Hz exposure 6 hours a day, 5 days a
week for 28 days in methacrylate boxes. Formalin-
fixed, paraffin-embedded tissue sections were stained
with H-E and PAS. In addition, E-cadherin and type
IV collagen expressions were examined immunohisto­
chemically.
RESULTS: Seminiferous tubule basement membranes
decreased with EMFs, especially those treated with
SEMF. In addition, expression levels of E-cadherin on
seminiferous epithelium decreased in PEMF and SEMF
groups. The expression level of type IV collagen was
also decreased in perivascular and seminiferous tubule
basement membrane as compared to that in the sham
group.
CONCLUSION: PEMF and SEMF have adverse
effects on the testis at a histopathological level. EMF
leads to a decrease in E-cadherin and type IV collagen
expression levels. (Anal Quant Cytopathol Histpathol
2020;42:77–84)
Keywords: cadherins, cell adhesion, collagen, cyto­
plasm, E-cadherin, EMF exposure, electromagnetic
fields, histopathology, immunohistochemistry, type
IV collagen.
There has been growing research interest on the
mechanisms of interaction between extremely low
frequency electromagnetic fields on biological
systems, since there is remarkable public concern
associated with health effects of exposure to
extremely low-frequency electromagnetic fields
(ELF-EMFs), such as electrical distribution net­
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4203-0077/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Effects of Pulsed and Sinusoidal
Electromagnetic Fields on Rat Testes
Selcuk Tunik, Ph.D., Ozlem Pamukcu Baran, Ph.D., Veysi Akpolat, M.D., Ph.D.,
Ercan Ayaz, M.D., Yusuf Nergiz, Ph.D., M. Salih Celik, Ph.D., and
Ozkan Yumusak, M.D.
From the Department of Histology and Embryology, Faculty of Medicine, University of Dicle, Diyarbakır; the Department of Biophysics,
Faculty of Medicine, University of Dicle, Diyarbakır; and the Department of Histology and Embryology, Faculty of Medicine, University
of Hitit, Çorum, Turkey.
Selcuk Tunik is Associate Professor, Department of Histology and Embryology, Faculty of Medicine, University of Dicle.
Ozlem Pamukcu Baran is Associate Professor, Department of Histology and Embryology, Faculty of Medicine, University of Dicle.
Veysi Akpolat is Professor, Department of Biophysics, Faculty of Medicine, University of Dicle.
Ercan Ayaz is Associate Professor, Department of Histology and Embryology, Faculty of Medicine, University of Hitit.
Yusuf Nergiz is Professor, Department of Histology and Embryology, Faculty of Medicine, University of Dicle.
M. Salih Celik is Professor, Department of Biophysics, Faculty of Medicine, University of Dicle.
Ozkan Yumusak is Research Assistant, Department of Histology and Embryology, Faculty of Medicine, University of Dicle.
Address correspondence to: Selcuk Tunik, Ph.D., Department of Histology and Embryology, Faculty of Medicine, University of Dicle,
21280 Diyarbakır, Turkey (selcuktunik@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. works or from residential and occupational.1 The
effects of ELF-EMFs on human biological systems
were evaluated in several studies.2,3 Some of them
were performed to evaluate the effects of EMFs
on reproductive disorders such as infertility, abor-
tion, prematurity, congenital malformations, and
genetic diseases.4,5 EMF effects on male reproduc­
tion are also being investigated, but most of those
results are still controversial. Adverse effects of
EMF were shown on fertility in male mice.6 Dev­
astating effects of EMF on spermatogenic and
Leydig cells were also reported.7 On the other
hand, several studies reported no alteration in
the male reproductive tract related to exposure to
electromagnetic fields.8-11 It is known that some
members from the superfamilies of adhesion mol-
ecules (i.e., immunoglobulins, integrins, and cad­
herins) were expressed from spermatozoa.12-15
E-cadherin is an epithelial cell adhesion mole-
cule that mediates the establishment and main­
tenance of cell-cell contacts.16 E-cadherin con­
tains an extracellular domain of five tandemly
repeated immunoglobulin-like modules, a single
membrane-spanning region, and a cytoplasmic
tail.17 E-cadherin, CDH1, is a 120-kDa transmem­
brane glycoprotein specifically expressed in epi­
thelial tissues and also present in some types of
neurons and glia cells.18,19 Recently, it has been
reported that E-cadherin can be used as a specific
surface marker for undifferentiated spermatogo­
nia in mice,20 showing that cell-cell interactions via
E-cadherin are essential for the initial cell division
of spermatogonial stem cells.21
Collagens are the fundamental, most abundant
structural proteins of the extracellular matrix in
mammalian tissues. Collagen is also the major
structural element of connective tissues and con­
tributes to the form and stability of tissues and
organs. Basement membranes are extracellular
structures mainly composed of several molecules
such as type IV collagen and laminin.22-24 The goal
of this study was to investigate the role of two
types of extremely low frequency magnetic fields,
pulsed (PEMF) and sinusoidal (SEMF), on the
expression level of E-cadherin and type IV colla-
gen in rat testis histopathologically and immuno­
histochemically.
Materials and Methods
Animals
The experiments were performed on 27 male Wis­
tar albino rats with initial weights of 155–230 g.
The rats were 4 months old at the beginning of
the study and were obtained from the Medical
Science Application and Research Center of Dicle
University. All rats were allowed free access to
water and standard pellet food diet during the
experimental period. The rats were divided into
three groups: sham group (n=9), SEMF group
(n=9), and PEMF group (n=9). The sham group
was not exposed to the electromagnetic fields.
The SEMF and PEMF groups (square pulse width
had 1:1 mark-space ratio that was 50% duty cycle,
the magnetic flux density was 1.5 mT, stable fre-
quency of 50 Hz, signal period of 20 milliseconds,
and duration between square pulses was 10 milli­
seconds) were subjected to 1.5 mT, 50 Hz exposure
for 6 hours a day, 5 days a week for 28 days in
methacrylate boxes (43×42×15 cm). The treated
animals were all exposed to EMF at the same time.
The animals were kept in 14/10 hours light/dark
environment at constant temperature of 22±3°C
and 45±10% humidity.
Exposure to Magnetic Field
The EMF was generated in a device designed
by us, which had two pairs of coils of 70 cm in
diameter in a Faraday cage (130×65×80 cm) that
earthed shielding against the electric component.
This magnet was constructed by winding 125 turns
of insulated soft copper wire with a diameter of
1.5 mm. Coils were placed vertically and facing
one another. The distance between coils was 47
cm. An AC current produced by an AC power
supply (DAYM, Turkey) was passed through the
device. The current in the wires of the energized
exposure solenoid was 40 A for 1.5 mT, which
resulted in 50 Hz magnetic field. The magnetic
field intensities were measured once per week as
1.5 mT in 15 different points of the methacrylate
cage with a Bell 7030 Gauss/Tesla Meter (F.W.
BELL, Inc., SYPRIS, Orlando, Florida, USA) to en-
sure homogeneity of the field during the course
of the experiment by a person who was not in-
volved in the animal experiment. Magnetic field
measurements showed that, at the conditions of
the experiment, the magnetic field exposure sys-
tem produced a stable flux density of 1.5 mT and
stable frequency of 50 Hz with negligible harmon­
ics and no transients. No temperature differences
were observed between exposure and cages dur-
ing the exposure. For the sham, nothing was ap-
plied to the rats in this group, and they completed
their life cycle in the cage during the study period.
78 Analytical and Quantitative Cytopathology and Histopathology®
Tunik et al

3. The rats were free in a methacrylate cage inside
the coils.
Histopathologic Examination
Testicular tissues were obtained from the rats
after high-dose ketamin HCl (Ketalar, Pfizer) anes-
thesia immediately after the last exposure. Tissues
were fixed in 10% buffered formalin for 12 hours
and then washed for 16 hours. They were dehy-
drated with graded alcohol series starting from
30% to absolute ethanol, and then embedded into
paraffin. All sections (histologic and immunohis­
tochemical) were evaluated and photographed by
using a light microscope (Eclipse i80, Nikon, Ja-
pan). Paraffin blocks were cut into 4–5 μm sec-
tions and stained with hematoxylin and eosin
(H-E) and periodic acid–Schiff (PAS). Seminifer­
ous epithelium and interstitial tissues of testes
were examined histopathologically under light
microscope.
For immunohistochemical studies, streptavidin-
biotin peroxidase method was used. Sections ob-
tained from testes were deparaffinized in xylene
for 20 minutes, rehydrated in descending grades
of alcohol, and put into distilled water. Sections
were then washed three times in phosphate-
buffered saline (PBS), pH 7.4 for 5 minutes each.
Endogenous peroxidase was inhibited with 3%
H2O2 for 20 minutes. After washing in PBS (three
times, 5 minutes each), the sections were put into
a microwave oven for antigen retrieval. Antigen
retrieval was achieved for E-cadherin (Santa
Cruz, 1/100, mouse monoclonal), 7+5+3 minutes
in citrate buffer at pH 6.0, for type IV collagen
(Abcam, 1/500, rabbit monoclonal) 7+5 in EDTA
at pH 8.0. Then, the sections were washed three
times in PBS for 5 minutes each. The slides were
covered with protein block serum (Thermo Sci­
entific) for 7 minutes at room temperature. They
were then incubated with the specific primary
antibody (E-cadherin and type IV collagen) for
16 hours at 4ºC. After an overnight incubation
with the primary antibody and washing the sec-
tions with PBS three times (5 minutes each), incu­
bation with a secondary antibody for E-cadherin
and type IV collagen for 15 minutes at room
temperature was performed. Immunohistochemi­
cally positive staining was defined as the pres­
ence of a brown detection by using chromogen
diaminobenzidine (DAB) (Invitrogen). Counter­
staining of the nuclei was done with hematoxylin
for 35 seconds. Sections were dehydrated through
ascending grades of ethanol, cleared with xylene,
and covered with DPX. Stain intensity and the
proportion of immunopositive cells were assessed
also by light microscope. Intensity of staining was
graded on a scale of 0–4, according to the follow­
ing assessment: 0= no detectable staining, 1= weak
staining, 2= moderate staining, 3= strong staining,
and 4= very strong staining. Immunostained slides
were blindly evaluated under light microscope.
Measurement of Seminiferous Tubule Diameters
The diameters of 20 relatively round-shaped sem­
iniferous tubules from each rat were measured
using linear micrometer reticle (Eclipse i80, Nikon,
Japan) and NIS-Elements software program (Ni­
kon) for morphometric analysis.
Measurement of Seminiferous Tubule Basement
Membrane (STMB)
The thicknesses of 20 STBMs randomly chosen
with round or nearly round shape of each rat
stained with PAS were measured. The mean val-
ues of these measurements were calculated, and
mean seminiferous tubule diameter and mean
membrane thickness were determined for each
testicle.
Statistical Analyses
Statistical analysis was performed with the Statis­
tical Package for the Social Sciences for Windows
(version 15.0, SPSS Inc., Chicago, Illinois, USA).
Kruskal-Wallis and the Mann-Whitney U test were
used for the statistics as indicated; tests and results
were expressed as mean±SD. A p value ≤0.05 was
considered significant.
Results
Histopathological Results
There were no pathological alterations in the
sham group sections. All components of the semi­
niferous epithelium and underlying basement
membranes of the seminiferous tubules as well as
interstitial tissue were seen in normal appearance
in the sham group (Figure 1A–B). However, struc­
tural changes were seen in interstitial tissue and
seminiferous tubules of PEMF group sections.
These changes included Leydig cell proliferation,
perivascular edema, and congestion in some blood
vessels. In addition, disorganization and decrease
in spermatogenic germ cell lines and separation
from basement membrane were also observed in
PEMF group sections (Figure 1C–D). Histopathol­
Volume 42, Number 3/June 2020 79
Effects of Electromagnetic Fields on Rat Testes

4. ogical lesions that were observed in SEMF groups
were remarkable as compared to the sham group.
In addition to interstitial edema and Leydig cell
proliferation, absence of spermatogenic germ cells
and existence of only basement membrane were
observed in some seminiferous tubules of testes
sections obtained from the SEMF group. Perforated
and atrophic seminiferous tubules were viewed in
some sections of SEMF, as seen also in Figure 1F–I.
Morphometrical Results
Measurement of tubular diameter showed a signif­
icant decrease in the PEMF group as compared
to the sham group. On the contrary, a decrease in
seminiferous tubule diameter was not statistically
significant in the SEMF group. In addition, the
thickness of seminiferous tubule basement mem­
branes increased in both groups; however, it was
only significant in the SEMF group (Table I).
Immunohistochemical Results
E-cadherin. The immune reaction for E-cadherin
revealed variations between the components of
seminiferous epithelium of testes of each group.
Sham group testes showed strong reaction in
spermatogonia and primary spermatocytes, as
80 Analytical and Quantitative Cytopathology and Histopathology®
Tunik et al
Figure 1 The histopathological view of testes sections stained with hematoxylin and eosin (H-E) and periodic acid–Schiff (PAS) in all
groups. Normal histological features of the testes were observed in the sham group (a, b). Proliferation of Leydig (L) cells and eudema
(E) in the perivascular area, decrease in germ cells and disorganization (d) as well as basement membrane (arrowhead) were observed in
pulsed electromagnetic field (PEMF) group sections (c–e). Interstitial eudema (E), lack of germ cells (asterisk), atrophic (A) and perforated
(P) seminiferous tubules, and thickening of basement membranes were viewed in the sinusoidal electromagnetic field (SEMF) group (f–i).

5. well as a moderate level in Leydig cells. Espe­
cially, both cytoplasm and cell membranes of
spermatogonia of the sham group showed strong
positive immunostaining. Positive immunoreac­
tion was also observed in spermatids and in lu-
men. The immune reaction for E-cadherin de-
creased in both seminiferous tubule epithelium
overall and in Leydig cells of the PEMF group
as compared to the sham group. On the other
hand, there is no variation in the intensity of
the immunoreactivity for E-cadherin between the
SEMF and PEMF groups (Figure 2A–C) (Table II).
Type IV Collagen. The presence of immune reac-
tion for collagen type IV was observed in perivas­
cular connective tissue of interstitial tissue and
basement membranes of the seminiferous tubules.
Sham group testes showed strong reaction in peri­
vascular connective tissue and seminiferous tu-
bule basement membranes. The immune reaction
for collagen type IV decreased in seminiferous tu-
bule epithelium and in perivascular connective
tissue of the PEMF group as compared to the
sham group. In addition, the same findings were
observed for SEMF group sections. There were no
significant differences between the two electro­
magnetic fields for type IV collagen expression
levels (Figure 3A–F) (Table III).
Discussion
The effects of EMF on male reproduction are also
being investigated, but most of these studies still
remain controversial. It was reported that exposure
to consequently divided doses of magnetic field
showed more potential effects for testicular dam­
age than acute exposition.25 In the present study,
we evaluated the effect of extremely low frequency
pulsed and sinusoidal electromagnetic fields on rat
testes. It was suggested that application of EMF
Volume 42, Number 3/June 2020 81
Effects of Electromagnetic Fields on Rat Testes
Table I Comparison of Histopathological Results
Seminiferous
tubule Seminiferous
basement tubule
membrane* diameter*
Group μm μm
Control (n=9)
3.34±0.15
281.73±3.09
PEMF (n=9) 3.45±0.30b 254.34±15.32a,b
SEMF (n=9) 5.62±1.91a 279.99±26.43
Results are presented as mean±standard deviation.
*Kruskal-Wallis test, p<0.05.
ap<0.05 as compared to the control group.
bp>0.05 as compared to the SEMF group.
PEMF = pulsed electromagnetic field, SEMF = sinusoidal electromagnetic
field.
Figure 2 The representative sections of immunohistochemistry for E-cadherin in all groups. Tissue expressing E-cadherin appeared brown
in color. The strong staining for E-cadherin was found in seminiferous epithelium of sham group sections in all layers (a). The moderate
staining of E-cadherin is observed in seminiferous epithelium and Leydig cells of both SEMF and PEMF group sections (b-c). Arrow =
expression in seminiferous epithelium, arrowhead = expression in Leydig cells.
Table II Comparison of E-Cadherin Expression
Group
E-cadherin Control PEMF SEMF
Spermatogonia* 3.1±0.7 2.2±0.4a 2.1±0.3a
Primer spermatocytes* 2.8±0.7 1.1±0.6a,b 2.1±0.3a
Spermatids* 3.1±0.7 1.3±0.7a 1.1±0.6a
Leydig cells* 2.2±0.4 1.2±0.4a 1.0±0.3a
Results are presented as mean±standard deviation.
*Kruskal-Wallis test, p<0.05.
ap<0.05 as compared to the control group.
bp>0.05 as compared to the SEMF group.
PEMF = pulsed electromagnetic field, SEMF = sinusoidal electromagnetic
field.

6. for 2 hours each day for 10 days did not change
seminiferous tubule diameter and volume of Ley­
dig cells.26 In addition, it was observed that long-
term cell phone use27 and 2.45 GHz use of EMF28
did not alter the seminiferous tubule diameter.
On the contrary, in the present study we observed
that seminiferous tubule diameter decreased in
both EMF groups, but it was significant in the
sinusoidal EMF group, and we also observed
that the number of Leydig cells increased in the
EMF groups. The decrease in seminiferous tubule
diameter and increase in Leydig cells can be
attributed to the effects of sinusoidal EMF.
Epithelia are characterized by junctional com­
plexes between adjacent cells, which consist of an
apical-basal membrane sequence of a tight junc-
tion, adherens junction, and desmosome.29 Adher­
ens junctions, typically having a role in cell adhe­
sion, are also thought to be involved in signaling
events.30 The important adhesion molecules asso-
ciated with adherens junctions are cadherins and
nectins. Cadherins are calcium-dependent trans-
membrane proteins that are linked to the actin cy-
toskeleton via the intracellular proteins α-catenin
and β-catenin.31 The best characterized cadherins
are E-cadherin, which is expressed in all epithe­
lia.32 Expression of E-cadherin has been shown
in human and rat gametes13,33,34 and gingival epi­
thelium.35 E-cadherin has been reported to be a
specific surface marker for undifferentiated sper­
matogonia in mice20 and rats.36 In the present
study we have shown E-cadherin expression on
spermatogonia, spermatocytes, and spermatid as
well as Leydig cells in interstitial tissues. The
82 Analytical and Quantitative Cytopathology and Histopathology®
Tunik et al
Figure 3 The representative sections of immunohistochemistry for type IV collagen in all groups. Tissue expressing type IV collagen
appears brown in color. The strong staining for type IV collagen was found in perivascular connective tissue and basement membrane
of seminiferous epithelium of sham group sections (a, b). The moderate staining of type IV collagen is observed in basement membrane
and perivascular connective tissue of both SEMF (c, d) and PEMF group sections (e, f). Arrow = expression in seminiferous epithelium,
arrowhead = expression in perivascular connective tissue.
Table III Comparison of Type IV Collagen Expression
Group
Type IV collagen Control PEMF SEMF
Perivascular connective tissue* 2.3±0.5 1.1±0.3a 1.1±0.6a
Seminiferous tubule basement
membrane* 2.1±0.6 1.0±0.5a 0.8±0.6a
Results are presented as mean±standard deviation.
*Kruskal-Wallis test, p<0.05.
ap<0.05 as compared to the control group.

7. expression level of E-cadherin diminished in both
sinusoidal and pulsed electromagnetic field–
exposed rats immunohistochemically. Breakdown
of E-cadherin facilitates the initial invasive behav-
ior of some epithelial derived cancers.36,37
Collagens are the major structural component
of the extracellular matrix. Type IV collagen is a
crucial component of the basal lamina in organ­
isms.38 In addition, type IV collagen has a role
in the initial formation of basement membranes
during embryonic development.39 It was shown
that seminiferous tubule basement membranes
express type IV collagen. The basal lamina of the
seminiferous tubules composed of thin extracel­
lular structures included type IV collagen, hepa-
ran sulfate proteoglycan40 and some other extra­
cellular matrix protein, a thin collagen layer, and
a layer of peritubular myoid cells.41 The effect of
pulsed and sinusoidal electromagnetic field on
type IV collagen expression was nearly the same
as in our study. It was shown that electromag-
netic fields lead to a decrease in type X collagen
expression level in cultured mesenchymal stem
cells.42 On the other hand, the densitometric re-
verse transcription–polymerase chain reaction re-
sults revealed that pulsed electromagnetic fields
did not change the expression of collagen types I
and II on cultured intervertebral disc cells in hu-
mans.43 In the present study, we observed that
type IV collagen expression decreased under the
effect of extremely low frequency electromagnetic
fields, both pulsed and sinusoidal, immunohisto­
chemically.
In conclusion, in the present study it was ob-
served that rats exposed to pulsed and sinusoidal
electromagnetic field had histopathological altera­
tions and a decrease in E-cadherin and type IV
collagen expression in the testes, indicating that
electromagnetic fields adversely affect testes.
Acknowledgments
We are grateful to Professor Erhan Unlu for the
use of his photomicroscope and to English Lecturer
Ibrahim Tunik for kindly checking the grammar
and spelling of the manuscript.
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