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World Applied Sciences Journal 31 (8): 1478-1487, 2014
ISSN 1818-4952
© IDOSI Publications, 2014
DOI: 10.5829/idosi.wasj.2014.31.08.83144
Corresponding Author: Aida I El makawy, Cell Biology Department, National Research Centre,
EL Tahrir Street, 12622 Dokki, Giza, Egypt.
1478
Evaluation of Male-Mediated Reproductive Toxicity Induced by Polyethylene Glycol in Mice
Aida I Elmakawy, Omaima M Abd-Elmoneim, Kawthar AE Diab and Hafiza A Sharaf
11 2 3
Cell Biology Department, National Research Centre, EL Tahrir Street, 12622 Dokki, Giza, Egypt
1
Genetics and Cytology Department, National Research Centre, EL Tahrir Street, 12622 Dokki, Giza, Egypt
2
Pathology Department, National Research Centre, EL Tahrir Street, 12622 Dokki, Giza, Egypt
3
Submitted: Jan 05, 2014; Accepted: Mar 20, 2014; Published: Apr 27, 2014
Abstract: In the present work polyethylene glycol (PEG6000) was given orally by gavage to male mice at three
different doses 50, 100 and 200 mg/kg/day for consecutive 5 days per week up to six successive weeks. 24hr
of treatment, half number of the mice from all experimental groups was sacrificed. Epididymis and testes were
collected to evaluate the reproductive toxicity of PEG6000. The other half of treated male mice was mated with
untreated virgin female mice to evaluate male-mediated genotoxicity of PEG6000 on fetuses. The results
illustrated that PEG6000 decreased sperm motility and count in a dose-dependent manner. However,
PEG6000 resulted in an increased fraction of sperm with abnormal morphology or abnormal chromatin integrity
measured by toluidine blue and acridine orange. Besides, PEG6000 exhibited an increase in the occurrence of
chromosomal aberrations in spermatocytes of treated male and in liver cells of their fetuses. Furthermore,
testicular alternation and reduction of DNA nuclear content in spermatogonia and spermatocytes were
observed in PEG6000 treated mice. In conclusion, chronic exposure of PEG6000 induced male reproductive
impairment which correlated with genetic damage of fetuses generated from mating treated paternal mice with
untreated female mice.
Key words: Polyethylene Glycol Mouse Sperm Morphology Sperm Chromatin Integrity Histopathology
INTRODUCTION Millions of tons of PEGs are involved in industrial process
Polyethylene glycol is a polyether compound with discharged as waste water after industrial utilization [4].
many applications from industrial manufacturing to PEG6000 is permitted as food additives in various foods
medicine. The structure of PEG is also known as according to the European Parliament and Council
polyethylene oxide or polyoxyethylene, depending on its Directive No. 95/2/EC of 20 February1995. Our previous
molecular weight [1]. PEGs of different molecular weights experiments exhibited that PEG6000 has been induced
(200-10000) are widely used in many cosmetic and DNA damage in mouse bone marrow cells using comet
pharmaceutical preparations. In the pharmaceutical assay and micronucleus assay [5]. There is copious
industry, they are used as vehicles for drugs and as evidence of male mediated developmental toxicity in
ointment bases, capsules, tablet and pill binders, experimental models and some evidence showing a
suppositories liquid prescriptions and in veterinary drugs, sensitivity of the male germ line to these transmissible
including topical, ophthalmic, oral and rectal preparations effects. Putative mechanism have been proposed
[2]. PEGs are utilized as a laxative agent for treatment of including direct action such as contamination through the
constipation [3]. Further applications include use as seminal fluid and both genetic and epigenetic pathways
ingredients in soaps and detergents, in the textile and including germ cell mutation, induction of germ–line
leather industry, in plastics and resins, in the paper genomic instability, suppression of germ cell apoptosis or
industry, in printing, in the ceramics and glass industry, interference with genomic imprinting [6] The body of
in the rubber, petroleum, mining and metal industries, for knowledge regarding PEG6000 reproductive toxicity has
wood preservation and as chemical intermediates [1]. been limited and based largely upon knowledge of similar
worldwide every year which of most of them have to be
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1479
compounds. Therefore, the objective of this study is to Reproductive Toxicity Evaluation Epididymal Sperm
provide information regarding reproductive toxicity of Motility, Count and Morphology: The cauda epididymides
PEG6000 in mice. were cut in a pre-warmed saline solution at 37°C and
MATERIALS AND METHODS 5 min. To determine sperm motility, one drop of sperm
Chemicals: PEG6000 was purchased from Al-Gomhoria covered with cover-slip and observed under a standard
pharmaceutical Industry, Cairo, Egypt. Cyclophosphamide optical microscope at 400X magnification. A minimum of
monohydrate (CP) was manufactured by Baxter oncology five microscopic fields were assessed to determine the
GmbH, Frankfurt-Germany. PEG6000 and CP were percentage of motile sperm on at least 200 sperm for each
dissolved in distilled water. animal. To count sperm, 5 µl of sperm suspension was
Animals: Adult BALB/c mice of both sexes (Mus was transferred into hemocytometer and sperm were
Musculus), 6–8 weeks old and weighing approximately counted under light microscope and data were expressed
25–30 g, were obtained from Animal House of National as the number of sperm per ml. For analysis of sperm
Research Center, Dokki, Egypt. The animals were morphology, a drop of sperm suspension was smeared
acclimated for a period of two weeks before the beginning onto cleaned slides, allowed to air dry and stained with
of the experiments. Mice were housed in stainless-steel 1% aqueous solution of eosin Y. At least 1000 sperm per
cages, maintained in a room designed for control of each mouse were examined under light microscope at
temperature, humidity and light cycle. Mice were fed 1000X magnification. Sperm abnormalities were recorded
standard granulated diet with tap water supplied with ad according to Wyrobek and Bruce [7].
libitum. The study protocol was approved by the Ethics
Committee for Animal Care of National Research Center, Sperm Chromatin Integrity Assay
Dokki, Egypt. Toluidine Blue Assay: The assay was performed
Treatment Schedule: Male mice were randomly assigned et al. [8]. Briefly, air-dried sperm smears were fixed in 96%
to five experimental groups of ten mice per group. Control ethanol-acetone (1:1) at 4°C for 30 min. The slides were
group: mice were given distilled water. Positive control staining with 0.05% TB in 50% Mcilvaine citrate
group: mice were given intraperitoneally CP at single dose phosphate buffer for 10 min. Following staining, the slides
of 20 mg/kg. PEG6000 groups: Three groups of mice were were gently rinsed in a stream of distilled water and air
administrated orally PEG6000 at three doses 50, 100 or 200 dried again. A total 1000 spermatozoa per animal was
mg/kg/day for consecutive 5 days per weeks up to six examined under light microscope at 1000X magnification.
successive weeks. Chromatin quality of sperm was assessed in following
Experimental Design: At the end of treatment schedule (abnormal chromatin).
half number of the mice from each experimental group was
sacrificed by dislocation of neck vertebra. Testes and Acridine Orange Assay: The assay was performed
epididymis were collected for different assays. Fifty according to the standard protocol described by
untreated virgin female mice (10 female/group) were Martins et al. [9]. Briefly, air-dried smears were fixed for
cohabited with other half of male mice from all groups for 1 hr in Carnoy’s solution at 4°C and allowed to dry. The
two weeks. Each treated male mouse was housed in a smears were incubated in 1 N HCl at 75°C for 15 min then
separate cage with two virgin untreated females of the stained with AO dye (0.2 mg/ml) for 10 min. Slides were
same strain. The day of sperm positive vaginal smear was washed with distilled water and air-dried. At least 1000
considered as day‘0’ of gestation. Pregnant female was spermatozoa per mouse were analyzed under fluorescence
removed from the male’s cage and placed in her own microscope (490/530 nm excitation/barrier filter) at 1000X
individual cage. The dams were killed by cervical magnification. Two types of staining patterns were
dislocation on day 15-17 of gestation and fetuses' livers considered in sperm head; green spermatozoa (double-
were collected for mitotic chromosome aberrations stranded DNA), yellow or orange spermatozoa (single-
analysis. stranded DNA).
minced with scissors and placed in 37°C incubator for
suspension was placed on warmed microscope slide,
diluted with 95µl saline solution. The diluted suspension
according to standard protocol described by Erenpreisa
criteria: light blue (good chromatin) and dark blue
Abnormal chromatin int egrity 100
Good abnormal chromatin int egrity
×
+
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1480
Meiotic Chromosome Aberrations Analysis: followed by Duncan's multiple range test for multiple
Chromosomes from primary spermatocytes were prepared comparisons. Statistical significance was set at p 0.05.
according to technique of Evans et al. [10]. Briefly, the Pearson’s correlation coefficient (r) was calculated in
tunica of the testis was removed and the tubules were order to evaluate correlations between the different
transferred to a small Petri dish containing isotonic parameters analyzed. The percentage of abnormal
solution (2.2% sodium citrate) and teased out with curved chromatin integrity was calculated according to
forceps on a piece of mesh. The cell suspension was Erenpreisa et al. [8] using the following formula
transferred into a conical tube and centrifuged at 1000 rpm
for 10 min at room temperature. The supernatant was
removed and the pellets were re-suspended in hypotonic
solution (1.1% trisodium citrate) for 20 min at 37°C and
fixed in cold Carnoy's fixative. The cells were centrifuged RESULTS
and re-suspended in a small volume of Carnoy's fixative.
To prepare slides, 3–5 drops of the fixed cell suspension Routine Sperm Analysis: Sperm analysis showed that
were dropped on a clean slide and air-dried. The slides PEG6000 exhibited significant dose dependent decrease
were stained in 10% Giemsa solution in phosphate buffer (p= 0.01) in the percentage of sperm motility and count
(pH 6.8) for 5 min. A total 100 well spread diakinesis- when compared to negative control group (Table 1).
metaphase I cells per mice were examined under light However, such reduction in motility and number of sperm
microscope at 1000X magnification. was more in the positive control value. As shown in
Fetal Chromosome Aberrations Analysis: significant increase in the frequencies of abnormal sperm
Chromosomes from fetal liver were prepared according to morphology in both head and tail regions when compared
Romagnano et al. [11]. Fetal liver tissue was placed in a with negative control. The increase in sperm abnormalities
conical centrifuge tube with 2ml RPMI-1640 medium and in PEG6000-treated mice was dose-dependent manner but
dissociated manually using Pasteur pipette. The liver lower than the positive control value. Sperm with
sample was incubated with 0.02ml 0.05% colchicine at amorphous head shape and coiled tail represent a major
37°C for 90 min. The samples were centrifuged for 5 min part of sperm abnormalities induced after oral repeated
and then incubated in 2 ml of a prewarmed hypotonic KCL treatment with different doses of PEG6000. They reached
solution (0.56%) for 15 min at 37° C. Cells were fixed twice their maximum percentages 9.20% and 22.20% respectively
Carnoy's fixative and slides were prepared and air- after treatment with the highest tested dose of PEG6000.
dried, stained with 10% Giemsa in phosphate buffer Sperm with small and big head were observed only in
(pH 6.8). Good-quality metaphases (n = 100) per each positive control.
fetus were examined under light microscope at 2000X
magnification. Sperm Chromatin Integrity: As shown in Table 1, the
Histological and Histochemical Study: Right testis from integrity) was significantly increased after repeated oral
each mouse were excised and fixed in 10% formal saline treatment with all doses of PEG6000 when compared with
followed by dehydration in ascending grades of alcohol, negative and positive control groups. It reached its
clearing in xylene and embedding in paraffin wax. Paraffin maximum 16.36±0.14 at the highest tested dose of PEG6000
sections (5 ìm thicknesses) were stained with hematoxylin compared with 2.58±0.15 for the negative control. Such
and eosin (H&E) for the histological examination and percentages represent 6.34 fold increases compared with
Feulgen method for demonstration DNA content [12]. The control group. These data suggested that PEG6000 has
DNA contents were elucidated from Feulgen stained prominent impact on chromatin proteins inhibition
sections subjected to measurement of optical density especially protamine.
using Image Pro Plus image analysis software (Media Staining treated sperm with AO exhibited that
Cybernetics Inc, 2002). PEG6000 induced significant increase in the denaturation
Statistical Analysis: Statistical analysis was performed relationship. The proportion of red spermatozoa reached
using SPSS for Windows (Version 11). Data were 8.06 ±0.21, 10.90±0.23 and 16.36±0.14 at the three different
compared by one-way analysis of variance (ANOVA) doses 50, 100 and 200 mg/kg, respectively compared with
Table (2), the three tested doses of PEG6000 induced
percentage of dark blue sperm (abnormal chromatin
DNA strand (red spermatozoa) with dose-dependent
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1481
Table 1: Effect of treatment with PEG6000 on mouse sperm motility, sperm count and chromatin integrity
Sperm with abnormal chromatin integrity
Sperm motility Total sperm count × 10 (Mean% ± SE)
6
------------------------------- ------------------------------------- -----------------------------------------------------
Treatment groups Mean ± SE % Mean ± SE % AO orange sperm TB dark sperm
I-Negative control 82.00 ±1.23 82 549.60 ±13.96 78.5 2.58±0.15 2.66 ±0.051
a a e e
II-Positive control 52.00 ±1.23 52 196.40 ±4.29 28.05 25.90±0.19 27.6±0.93
e e a a
III-PEG-6000
50 72.00 ±1.23 72 432.20 ±8.49 61.74 7.44±0.35 8.06 ±0.21
b b d d
100 68.00 ±1.23 68 354.60 ±21.39 50.65 10.62±0.30 11.90±0.23
c c c c
200 64.00 ±1.87 64 270.20 ±12.59 38.6 16.36±0.14 18.46±0.28
d d b b
5000 sperm were examined for chromatin integrity in 5 mice per each experimental group
Mean values followed with different superscript letters within the same column are significantly different from one another (P= 0.05).
Table 2: Effect of treatment with PEG6000 on the mouse sperm plasma membrane integrity
Type of head abnormalities
---------------------------------------------------------------------------------------------------------------------------
Treatment groups Amorphous Hookless Banana Forked Small Big Coiled tail Total
I-Negative control 0.60±0.25 2.20±0.20 0.00±0.00 0.00±0.00 0.00 ±0.00 0.00 ±0.00 2.60±0.25 5.40±0.25
e c b b b b e e
II-Positive control 12.80 ±1.07 9.40±0.87 1.20 ±0.37 2.80 ±0.49 1.80±0.49 1.00±1.03 27.00±1.76 56.00±3.98
a a a a a a a a
III-PEG-6000
50 5.00±0.32 4.60 ±0.25 0.40±0.25 0 .40±0.25 0.00±0.00 0.00±0.00 10.20±0.86 20.60±0.51
d c ab b b b d d
100 7.20±0.37 7.40±0.40 0.60±0.25 0.40±0.25 0.00±0.00 0.00±0.00 15.20±0.66 30.80±0.86
c b ab b b b c c
200 9.20±0.37 8.20±0.37 1.20±0.37 0.80±0.37 0.00±0.00 0.00 ±0.00 22.20±.860 41.40±1.21
b b a b b b b b
5000 sperm were examined in 5 mice per each experimental group
Data were expressed as mean% ± S.E. Mean values followed with different superscript letters within the same column are significantly different from one another
(P 0.05)
Table 3: Effect of treatment with PEG6000 on mouse meiotic chromosomal aberrations
Structural aberrations Numerical aberrations
--------------------------------------------------------------------------------------------------------- -------------------------------------
Treatment groups X-Y Uni. Auto.Uni X-Y Break Fragment Chain Aneuploidy Polyploidy Total
I-Negative control 3.00±0.45 1.80±0.20 0.20±0.20 0.40±0.25 0.00±0.00 0.00±0.00 0.00±0.00 5.4± 0.250
c d a a b c be
II-Positive control 6.60±1.03 9.00±0.84 0.80±0.20 2.00±1.05 0.60±0.25 2.60±0.40 1.20±0.37 22.80±0.58
ab a a b a a a a
III-PEG6000
50 5.80 ±0.80 3.00 ±0.55 0.40±0.25 0.40±0.24 0.00+0.00 0.00+0.00 0.00±0.00 9.60±0.98
b cd a a b c b d
100 7.00 ±1.04 4.20 ±0.66 0.60±0.25 0.60 ±0.40 0.20±0.20 0.80±0.37 0.20±0.20 13.60±0.93
ab bc a a a ab bc b c
200 8.60±0.68 5.60 ±0.51 0.80±0.37 0.80±0.37 0.40 ±0.25 1.20±0.49 0.40±0.20 17.80±0.58
a b a a ab b b b
500 metaphases were examined in 5 mice per each experimental group. Auto = Autosomal; Uni= Univalent
Data were expressed as mean % ± S.E. Values within the same column followed by different letters are significantly different form one another (p 0.05).
Table 4: Frequencies of different types of chromosomal aberrations induced in fetal liver generated from paternal mice treated with PEG6000
Metaphases with different types of chromosomal aberrations
--------------------------------------------------------------------------------------------------------------------------------------------- Abnormal metaphases
Structural ab errations Numerical aberration s ---------------------------------------
----------------------------------------------------------------------------------------- ---------------------------------------------- Including Excluding
Treatment groups Ch. Gap Chro. gap Ch. Break C.T Del. &Fr. Total Prediploidy Polyploidy Total gaps gaps
I-Negative control 0.12±0.07 0.00±0.00 0.12±0.07 0.12±0.07 0.00±0.00 0.36±0.11 0.16±0.08 0.00±0.00 0.16±0.08 0.48±0.12 0.40±0. 10
dd e d c e d d d e e
II-Positive control 5.04±0.23 0.80±0.41 4.72±0.16 4.12±0.19 1.60±0.21 16.28±0.40 1.84±0.14 1.64±0.10 3.48±0.15 19.72±0.38 13.88±0.40
aa a a a a a a a a a
III-PEG6000
50 0.72±0.09 0.20±0.08 0.56±0.10 1.24±0.15 0.36±0.10 3.28±0.24 0.56±0.10 0.44±0.10 1.00±0.15 4.28±0.30 3.16±0.21
c cd c c c d c c c d d
100 1.64±0.10 0.40±0.10 1.00±0.12 1.60±0.10 0.92±0.13 5.60±0.22 1.32±0.14 1.04±0.14 2.32±0. 19 7.92±0.24 5.88±0.23
b bc b c b c b b b c c
200 1.80±0.08 0.56±0.10 1.20±0.14 2.08±0.11 1.24±0.13 6.88±0.27 1.84±0.08 1.48±0.10 3.32±0.11 10.16±0.29 7.84 ±0.24
b b d b ab b a a a bb
Data w ere expr essed as mean% ± S.E. Values within the same column follow by different superscript letters are significantly different from one another (P 0.05). Ch=chromatid;
Chro=chr omosomal; C.T= Centromeric attenuation; Del=Deletion; Fr=Fragment 25 fetus were analysis for chromosomal aberrations per each experimental group. 100 metaphases were
examined per each fetus
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1482
Table 5: Effect of treatment with PEG6000 on DNA content in testes of male mice
Control 50mg/kg PEG 100mg/kg PEG 200mg/kg PEG
Spermatogonial DNA 0.432+0.004 d 0.193+0.007 a 0.275+0.003 b 0.300+0.003 c
Spermatocytes DNA 0.341+0.003 d 0.180+0.005 a 0.254+0.002 b 0.273+0.003 c
Data were expressed as mean% ± S.E. Values within the same column follow by different superscript letters are significantly different from one another
(P= 0.05).
2.66±0.051 for control group (Table 1). Such percentages PEG6000. However, metaphases with fragments and X-Y
represent 3.03, 4.09 and 6.15 fold increases at three doses breaks were insignificantly increased with all three doses
of PEG6000 when compared with the value of control of PEG6000. Estimation of aneuploidy depends on number
group, respectively. This result implies that PEG6000 has of hyperhaploid metaphases, while hypohaploids were
effect on disturbance of oxidation process of thiols of completely excluded because an artificial loss of a single
cyteines in protamine molecules leading to abnormal chromosome may be occur during preparation. The two
sperm with poor disulphide bonds. highest doses of PEG6000 were induced a statistically
The proportion of spermatozoa with abnormal significant and insignificant increase in metaphases with
chromatin conformation detected by AO was strongly hyperhaploid and polyploidy respectively compared with
correlated with the proportion of spermatozoa detected by controls group. It is noteworthy that, some types of
TB (r=0.972, p 0.0001). Pearson's correlation analysis aberrations including chain, aneuploidy, polyploidy were
showed positive significant correlations between not recorded at low dose of PEG6000 and control group.
abnormal sperm morphology and abnormal chromatin Pearson's correlation analysis illustrated high significant
integrity detected by TB and AO. TB assay was strongly positive correlation between meiotic CAs and total
correlated with abnormal sperm morphology in head abnormal sperm morphologyinduced in PEG6000 (r=0.877,
(r= 0.911, p 0.0001) and tail (0.941, p 0.0001) regions. p 0.001).
Furthermore, AO was highly correlated with abnormal
sperm morphology in head (r= 0.941, p 0.0001) and tail Fetal Chromosome Aberrations (CAs) Analysis:
(0.954, p 0.0001) regions. Analysis of mitotic CAs from fetuses generated from
Meiotic Chromosome Aberrations (CAs) Analysis: All with PEG6000 showed that PEG6000 caused
the tested doses of PEG6000 induced significant increase precocious increase in the occurrence of CAs with dose-
in the incidence of CAs in mouse spermatocytes with dependent relationship even after excluding gaps (Table
dose dependent relationship (Table 3). Such proportions 4). All types of numerical and structural CAs were
were arisen from dramatically increase in frequency of X-Y significantly increased with all three doses of PEG6000
and autosomal univalents with all doses of PEG6000. when compared with negative control group. However,
Metaphases with translocations in the form of chain IV such increase was lower than the value of the positive
were significant increase by two highest doses of control group.
mating untreated female mice and paternal mice treated
Fig. 1: Section of control testis of mice showing normal architecture of testis, seminiferous tubules (S) show a clear
lumen and all cell types are represented including sertoli cells (arrow head), spermatogonia, primary
spermatocytes and spermatids (Hx& E A, ×200; B, ×500).
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1483
Fig. 2: Section of testis treated with 50 ml/Kg PEG6000 showing (A):degeneration in some seminiferous tubules,
detachment of spermatogenic cells and vacuolation areas (area lacking of spermatogenic activity; arrow head).
B: pyknosis in spermatogenic cells (arrow), depletion of germ cells layers (A-B Hx & E. x200).
Fig. 3: Section of testis treated with 100 ml/Kg PEG600 showing (A): marked degeneration, necrosis of germ cells
(arrows), sloughing of germ cells into tubular lumen. (B): higher magnification of another section showing
depletion of long spermatids ( weave arrow) and the presence of gaps (arrow head) inbetween the spermatogenic
cells (Hx. & E. X 200 & 400).
Fig. 4: Section of testis mice treated with 200 mg/kg PEG6000 showing (A): extensive exfoliation of germ cells into tubular
lumen (arrow), while the germ cells degeneration, vacuolation, disorganization were generally seen. B-higher
magnification of the same section showing depletion of most germ cells layers and long spermatids (Hx & E x 200
& 400).
Histological and Histochemical Study: Histological detachment of spermatogenic cells and cytoplasmic
examination of testes from control mice revealed normal vacuolation, pyknosis in spermatogenic cell nuclei and
spermatogenesis, with nearly all tubules showing sings of depletion of germ cells layers (Figure 2). The
evidence of endogenous spermatogenesis (Figure 1). On testis of mice treated with 100mg/kg bw appeared marked
the other hand, testicular degeneration and atrophy were degeneration, necrosis of germ cells, sloughing of
observed in the testis of groups treated with PEG600 with spermatogenic cells into tubular lumen, depletion of long
a dose dependent manner. Testis of dose of 50mg/k PEG spermatids and the presence of gaps between the
6000 revealed degeneration in some seminiferous tubules, spermatogenic cells (Figure 3). Also,200mg/kg of PEG600
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1484
treated mice showed extensive exfoliation of germ cells late spermatocytes and spermatids, decreased numbers of
into tubular lumen, while disorganization of sperm and increased numbers of immature germ cells,
spermatogenic layers, vacuolation and germ cells decreased sperm motility, peritubular membrane damage
degeneration were still present (Figure 4). and germinal epithelial distortion of the seminiferous
The mean value of DNA per nucleus was measured epithelium [18].
by image analyzer and the results was expressed as In the current report, the reduction in sperm count
optical density values (Table 5). The mean value for may be reflected impact of PEG6000 on spermatogenesis
nuclear DNA content of spermatogonial nuclei was causing severe erosion and necrosis of the germinal
significantly lower in all PEG600 treated groups epithelium of the testes [13]. Testicular atrophy and
(0.300 ±0.003,0.275 ±0.003 and 0.193±0.00715, respectively) dege neration of germinal ep ithelium caus ing oligo or
than control (0.432±0.004). Also the mean value for azoospermia has been recognized consequence of
nuclear DNA content of spermatocytes nuclei were treatment male rat with Ethylene glycol monomethyl ether,
significantly lower in all treated groups compared to EGME [19]. Additionally, occupational exposure for male
control group. workers to EGs derivative caused an increased prevalence
DISCUSSION ratio for a lower sperm count per ejaculate [20, 21]. The
The present study clearly illustrated that repeated occur as a consequence of endogenous factors like the
oral administration of PEG6000 for six weeks caused machinery for motility (flagellum) and alterations in the
marked reduction in number and motility of sperm and available energy and oxidative damages. Similar finding
obvious increase in occurrence of sperm with abnormal were reported that induction of free radical by EG may be
morphology and abnormal chromatin integrity. In affected on sperm motility by peroxidation of
addition, dramatically increase in the incidence of CAs in polyunsaturated fatty acid in sperm or by destruction of
mouse spermatocytes and fetal liver generated from sperm mitochondria which is responsible for the energy
mating untreated female mice with treated paternal mice production necessary to maintain spermatozoa movement
with PEG6000. Histological examination revealed that in some species [22, 23]. Recently, oral administration of
PEG6000 caused testicular alteration that characterized by EGME at 600 mg/kg/daily for 5 weeks significantly
seminiferous tubules degeneration, detachment of decreased total and progressive motility of rat
spermatogenic cells, cytoplasmic vacuolation, necrosis spermatozoa [24].
and pyknosis of germ cells, sloughing of spermatogenic Cytochemical experiments were undertaken to
cells into tubular lumen, depletion of long spermatids and validate impact of PEG6000 on sperm plasma membrane
extensive exfoliation of germ cells into tubular lumen. and chromatin integrity. Sperm analysis revealed that the
These findings may be attributed to accumulation of proportion of sperm with abnormal morphology or
PEG6000 in testis that causes detrimental effects to the abnormal chromatin integrity detected by TB and AO
male reproductive performance [13-15]. The observed were dramatically increased in PEG6000 treated mice.
effects are attributed to the mode of action of PEG6000. These finding suggested that PEG6000 has prominent
The precise mechanisms of PEG6000 remain unknown. effect on deficiency of protamines and disturbance the
However, the reproductive toxicity of PEG6000 is probably formation of disulphide bonds which responsible for tight
due to mode of action of one or more of its metabolites packaging of sperm chromatin. This view is explained by
rather than to the parent compound. This view supported the fact that sperm chromatin condensation during the
with the earlier studies reported that ethylene gylycols final steps of spermatogenesis in mammals is a multistep
(EGs) are biologically activated by enzyme alcohol process that includes the sequential replacement of the
dehydrogenase into alkoxyacetic acid metabolites that chromatin proteins (especially histones) by protamines,
interfered with the formation of nucleic acids [16]. Thus, allowing a different structural organization to take place in
toxicity may be more apparent in tissues undergoing rapid the sperm nucleus [25]. Protamine is rich in cysteine and
cell proliferation, such as during spermatogenesis, also other basic amino acids. During the epididymal
embryogenesis and fetal development [17]. Similar results passage, the thiols of cysteines in protamine are oxidized
were obtained when the animals exposed to ethylene to disulfide bonds (S-S). This type of organization
glycol monoethyl ether (EGME) showed testicular provides sperm with a highly condensed nucleus (rich in
atrophy, abnormal sperm morphology, degeneration of S-S) and also protects sperm DNA against the enzymatic
of oligospermia and azzospermia and an increased odds
reduction in sperm motility in PEG6000-treated mice might
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1485
attack of nucleases and polymerases. Due to the tight these data may be due to differences in treatment
packaging afforded by the protamines, any modification
or absence of these proteins leads to an anomaly in the
packaging process of sperm nucleus and influence sperm
quality and fertilizing capacity [26, 27]. Furthermore,
abnormal sperm chromatin structure is thought to arise
from many sources including deficiencies in
recombination during spermatogenesis, absence of
protamine, failure in epididymal maturation and chromatin
stability during ejaculation [27, 28]. It is remarkable that,
the high correlations between AO and TB assays
suggested that the two assays were similarly efficacious
in identifying chromatin alternations. Erenpreiss et al. [29]
found high correlation between the primary sperm
morphological abnormalities and sperm chromatin
alterations. The substitution of histones by protamines
that occurs during spermiogenesis is responsible for the
high sperm chromatin compactation [7, 29]. Interestingly,
the percentage of total sperm with abnormal morphology
was higher than the percentage of sperm with abnormal
chromatin integrity which determined by AO or TB. This
finding demonstrated that abnormalities in chromatin
condensation can influence the head morphology in
different ways, although chromatin abnormalities are not
always followed by evident morphological alterations.
Similarly, morphological alterations in the head were not
always followed by abnormalities in chromatin
condensation [30]. This implies sperm head with abnormal
chromatin was evaluated as normal morphology sperm
while head sperm with abnormal morphology were
appeared as sperm with normal chromatin. Meanwhile,
coiled tail and chromatin alterations detected by TB or AO
had significant positive correlations. This finding
confirmed that final chromatin compaction occurs in the
epididymus, where secondary sperm defects [31].
Meiotic CAs analysis of PEG6000-treated mice
revealed significant increase in the frequency of CAs in
mouse spermatocytes. This is implied that PEG6000 has
destructive effect on genetic material in germ cells. These
observations are validated by histochemical findings
since PEG6000 caused reduction of the nuclear DNA
content in spermatogonia and spermatocytes. Previous
studies have shown that administration of EGME to the
rats resulted in testicular damage with the spermatocyte
being the primary cellular site for toxicity [32]. However,
Foster et al. [33] reported that Sertoli and Leydig cells,
spermatogonia, prepachytene spermatocytes and
spermatids were unaffected by EGME administration from
250 to 1000 mg/kg for 11 days apart from partial maturation
depletion of early spermatid stage. The differences in
duration, dose and mode of administration and/or to
species differences. Aneugenic activity of PEG6000 was
illustrated by increase the incidence of numerical CAs in
mouse spermatocytes. This implies that PEG6000 interacts
with tubulin subunits to prevent microtubule assembly,
inducing abnormal chromosome segregation in dividing
cells and causing aneuploidy. In accordance with our
observations, earlier studies demonstrated that inhalation
exposure of rats to 300 ppm EGME for 3 days resulted in
degenerative changes in spermatocytes of pachytene and
meiotic division at spermatogenic stage in rats [34]. Such
degenerative pachytene or meiotic spermatocytes were
associated with discontinued chromosomal microtubules
with deposition of electron-dense chromatin material and
chromatin clumping around synaptonemal complexes [34].
Furthermore, it has been reported that EGEE treatment
significantly decreased populations of haploid cells and
increased diploid and tetraploid cells in spermatozoa at an
oral gavage dose of 400 mg/kg b.w in adult rats [35].
Unassociated univalents, particularly X-Y univalents are
the most frequently observed than other type of structural
CAs in PEG6000 treated mice indicating its genotoxic
effect in germ cells. Such effect is due to the prominent
effect of PEG6000 on the cohesion of two homologous
chromosomes forming unassociated univalent. It has been
documented that, homologous chromosomes are
physically held together as bivalents by crossover events
and cohesion between the DNA molecules. The gradual
loss of these physical connections between homologous
chromosomes contributes to the high rates of
unassociated univalent [36].
Comparing meiotic CAs with abnormal sperm
morphology proved significant positive correlation
between them. This correlation confirmed that induction
of meiotic CAs by PEG6000 should have trigger structural
changes in cell organelles involved in head and tail
formation, leading to a significant increase in sperm
abnormalities. Bagchi et al. [37] reported that, EGME
caused alternations in testicular DNA, by interfering
either with the integrity of the DNA itself and/or with the
expression of the genome and therefore, the
differentiation of sperm during spermatogenesis was
altered, resulting in induced sperm abnormalities.
In the current work, the observed CAs in fetal livers
generated from paternal mice exposed to PEG6000 is
attributed to induction DNA damage in male germ cells
that are kept unrepaired and transmitted to the next
generation [38]. Additional support for this view is
World Appl. Sci. J., 31 (8): 1478-1487, 2014
1486
provided by Sakkas et al. [39] who reported that sperm 9. Martins, C.F., M.N. Dode, S.N. Báo and R. Rumpf,
with protamine deficiency produce premature chromatin
condensation that is the cause of failure in fertilization
and embryo development. Besides, the majority of de
novo structural CAs in fetuses and newborns are
considered being of paternal origin that is of sperm origin
[40]. In addition, polyploidy and aneuploidy were the
major CAs observed in mouse embryo exposed to
cryoprotectant EG during vitrification [41].
We concluded that the male chronic exposure to
PEG6000 caused impairment of the reproductive functions
in male mice. PEG6000 reproductive toxicity revealed to its
potential male-mediated fetotoxicity. This is clearly of
great concern for possible polyethylene glycols impact on
human health.
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