Document Type : Original Article
Authors
1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Clinical Laboratory Sciences, School of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran
Abstract
Keywords
Hypoxic conditions can be found in many situations such
as high altitude, diving, and chronic obstructive pulmonary
disease (COPD). Globally, COPD is considered as a leading
cause of death and disability (
It is generally accepted that chronic systemic hypoxia,
whether due to high altitude or imposed experimentally by a
hypoxic or hypobaric chamber, induces physiological adaptations
that help to compensate the impaired O2
transport to tissues.
Enhancing red blood cell production (e.g.by administration
of erythropoietin (Epo) has been shown to modulate the
ventilatory response to reduced oxygen supply and critically
help the organism to cope with increased oxygen demand (
Weight loss due to exposure to chronic hypoxia may
reflect multiple changes in cardiovascular function, hormone
production, energy metabolism, and other aspects
of cellular and systemic physiology (
Furthermore hypoxic condition increases the levels of inflammatory
cytokine such as IL-1ß, IL-18 and tumor necrosis
factor-alpha (TNF-α) (
Improvement of vascular endothelial cell function, enhancement
of vascular reactivity and compliance, modulation
of lipid metabolism and reduction of inflammatory
cytokine production have been noted as the underlying
mechanisms through which poly unsaturated fatty acid
(PUFA) exert their beneficial effects (
In this experimental study, 24 male Wistar albino rats
(270-300 g,
The rats were randomly divided into 4 groups: control (Co),
sham (Sh), hypoxia (Hx) and hypoxia+flaxseed (Hx+Fx).
Hypoxic rats were kept in a hypoxic chamber with a reduced
pressure (oxygen 8% and nitrogen 92% for 4 hours/day for
30 days). The reason for using 8% oxygen is that the rats are
capable to survive at this level of hypoxia which allows us to
measure the patho-physiologic variables in them (
Control group (Co) was kept under normoxia and had free access to standard food and water. Sham group (Sh) was maintained in a hypoxia chamber (but not under hypoxia) receiving normal oxygen and food. Hypoxia group (Hx) was exposed to hypoxia 4 hours/day and fed with normal food. Hx+Fx group: 10% Fx was added to the normal food of Hx+Fx group after the first hypoxic exposure.
At the end of the experimental period, each rat was weighed and sacrified. Then, the right testis was removed and weighed. The testicular mass relative to body weight was determined on day 42 using the following equation: (testicular/body weight ratio)*100=(%).
At the end of each experiment, blood samples were collected from the left ventricle. Blood was centrifuged at 1000 g for 15 minutes and serum was separated for biochemical analysis. IL-18 levels in serum samples were quantified by an ELISA kit (zell Bio-GmbH, Germany) according to the manufacturer’s instructions.
At the end of the experiment, rats were weighed and
sacrificed and their right testis was removed. The right
testicular (internal spermatic) vein drained directly into
the right common iliac vein in 77.4%, and into the inferior
vena cava in 22.6% of the animals. The left testicular vein
drained into the left common iliac vein in all animals, but
in 90.3% of rats there was also an accessory branch of
the testicular vein draining into the left renal vein (
The caudal epididymis was used for sperm analysis. Briefly, epididiymal sperms were collected by slicing the caudal epididymis in 1 ml of Minimum Essential Medium-a (MEM-a) medium (P/N 22561-021, Gibco, CA, USA) after that 9-ml medium was added and samples were incubated for 10 minutes to allow the sperms to swim into the medium. The epididymis was then processed for further analysis.
To enumerate the spermatozoa, the heads of spermatozoa
were counted. For sperm counting, a hemocytometer device
was used. Here, 50 µl of the suspension was mixed with an
equal volume of 2% formalin. Then, 10 µl of this diluted suspension
was transferred to a Neubauer chamber. The sperms
were counted under light microscopy at ×400 (
A part of sperm sample was used for preparing smears
to evaluate the sperm morphological abnormalities. For
this purpose, 10 µL of suspension was spread onto a glass
slide and allowed to air-dry at room temperature to prepare
a smear. The smears were then stained with Diff-
Quik stain and 200 sperms were then examined under
light microscopy at ×400 (
In order to study the sperm viability, 10 µl of sperm suspension
was mixed with 2 µl Eosin-y 0.05%. Slides were prepared
and incubated for two minutes at room temperature before
evaluation at ×400 magnifications using light microscopy. Two
hundred sperms were counted for each sample. Dead sperms
appeared pink and live sperms were not stained (
One to two drops of the sperm suspension were placed
on a glass slide and motile sperms were counted immediately
using light microscopy (
Rat testes were rapidly removed and manually homogenized
in cold phosphate buffer (pH=7.4) and debris was
removed by centrifugation at 3500 g for 10 minutes. Then,
50 mg of supernatant was homogenized in 10 volumes of
KH2PO4 (100 mmol) buffer and was centrifuged at 12,000
g for 30 minutes at 4ºC. The supernatant was collected and
used for enzymes and MDA levels studies (
Total antioxidant capacity was measured based on the
absorbance of the 2,2'-azinobis-3-ethylbenzothiazoline-
6-sulfonic acid (ABTS+) radical cation. The pre-formed
radical monocation ± of 2,2'-azinobis-(3-ethylbenzothiazoline-
Data were statistically analyzed using SPSS-22 (IBM crop., Armonk, NY, USA) software. All data were expressed as mean ± standard errors of mean (SEM), median and interquartile range (IQR). At first, the normality of variables was checked using the Kolmogorov-Smirnov test. Then, for analyzing the differences among four groups of study, one way-ANOVA test and Tukey-post hoc test were chosen if the distribution of data were normal (for sperm parameters, testicular/ body weight ratio, diameter of seminiferous tubules, MDA level and TAC). Otherwise, nonparametric test of Kruskal-Wallis was carried out (for thickness of the germinal epithelium). The statistical significance level was set at 0.05.
Using one way-ANOVA test, serum levels of IL-18
were compared to confirm state of hypoxia. Tukey post
hoc test showed a significant difference in serum levels of
IL-18 in rat exposed to 30-days hypoxia (0.08 ± 0.05 pg/
ml) compared to control (0.51 ± 0.08 pg/ml, P=0.0001)
and Sham (0.52 ± 0.08 pg/ml, P=0.0001) groups (
Effects of hypoxia on serum levels of IL-18 (pg/ml) in rats following hypoxia. *; P<0.05 compared to control and sham groups, Co; Normal group that received normal oxygen levels and normal food, Sh; Sham group maintained in hypoxia chamber with normal oxygen levels and food, and Hx; Animals were exposed to hypoxia and received normal food.
The effect of oral Fx on the testicular/body weight ratio was evaluated in rats after hypoxia. According to the
ANOVA test, the testicular mass/body weight were significantly
different in the studied groups (P=0.0001,
Effects of oral flaxseed on testicular mass/body weight ratio in rats following hypoxia. *; P<0.05 compared to control and sham groups, Co; Normal group that received normal oxygen levels and normal food, Sh; Sham group maintained in a hypoxia chamber with normal oxygen levels and food, Hx; Animals were exposed to hypoxia and received normal food, and Hx+Fx; Animals were exposed to hypoxia and treated by normal food supplemented with 10% Fx.
The effects of oral Fx on sperm parameters were evaluated
in rats after hypoxia. The mean sperm count was
significantly different in the studied groups (P=0.0001,
Effects of oral flaxseed on sperm parameters of rats following hypoxia. A. Sperm count, B. Sperm motility, C. Sperm viability, and D. Sperm abnormality. *; P<0.05 compared to control and sham groups, #; P<0.05 compared to HX group, Co; Normal group that received normal oxygen levels and normal food, Sh; Sham group maintained in hypoxia chamber with normal oxygen levels and food, Hx; Animals were exposed to hypoxia and received normal food, and Hx+Fx; Animals were exposed to hypoxia and received normal food supplemented with 10% Fx food.
The effects of oral Fx on the diameter of seminiferous tubules
and thickness of the germinal epithelium were evaluated after
hypoxia in rats. According to ANOVA test, the mean diameter
of seminiferous tubules was significantly different in the studied
groups compared to control and sham (P=0.0001,
Effects of flaxseed on diameter of seminiferous tubules and thickness of the germinal epithelium in rats exposed to hypoxia. Comparing A. The diameter of seminiferous tubules and B. Thickness of the germinal epithelium in different groups. *; P<0.05 compared to Control and Sham groups, #; P<0.05 compared to Hx group, Co; Normal group that received normal oxygen levels and normal food, Sh; Sham group maintained in hypoxia chamber with normal oxygen levels and food, Hx; Animals were exposed to hypoxia and received normal food, and Hx+Fx; Animals were exposed to hypoxia and received normal food supplementated with 10% Fx food.
No significant difference was observed in the mean MDA
among studied groups (control=7.78 ± 0.11 nmol/mg and
sham=7.13 ± 0.09 nmol/mg, Hx=8.57 ± 0.28 nmol/mg and
Hx+Fx=6.7 ± 0.81 nmol/mg) (P=0.075,
Effects of oral flaxseed on MDA and TAC concentrations in rats exposed to hypoxia. A. MDA and B. TAC concentrations of rats following hypoxia. MDA; Malondialdehyde, TAC; Total antioxidant capacity, *; P<0.05 compared to Control and Sham groups, #; P<0.05 compared to Hx group, Co; Normal group normal oxygen and normal food, Sh; Sham group maintained in hypoxia chamber with normal oxygen and food, Hx; Animals were exposed to hypoxia and received normal food, and Hx+Fx; Animals were exposed to hypoxia and received normal food supplementated with 10% Fx food.
Hypoxia is a condition can result in overproduction of ROS which along with a decrease in the level of antioxidants, may give rise to oxidative stress. Oxidative stress as an imbalance between generation of ROS and ability of endogenous antioxidant systems to scavenge ROS has adverse influence on testes structure and sperm parameters.
In this study, we found that hypoxia leads to reduction
in the germinal epithelial thickness and some changes in
the serum, testes and sperm parameters in rats also hypoxia
results in excessive formation of ROS. We also
observed that hypoxia increases interstitial space of the
testes, which extends the oxygen diffusion distance and
impairs oxygen delivery to germ cells. It makes germ
cells more susceptible to damage, which was confirmed
by our observation concerning degeneration of germ cells
in hypoxic rats. A similar outcome was reported by other
researchers (
Spermatogenesis is vulnerable to hypoxia because spermatogenesis
has a high proliferation rate, damanding notable
oxygen levels in the testes and it has been reported
that breathing 10% O2/90% N2 results in a 24% decrease
in testicular blood flow, but a 23% increase in cerebral
blood flow. These characteristics may attribute to the
morphological changes of spermatogenesis induced by
hypoxia. Besides, a significant decrease in testicular mass
followed by adverse effects on reproductive hormones
such as testosterone was observed (
In our study there was significant reduction in body
weight of Hx+Fx group in comparison to the control
and sham groups. Researchers have observed that doses
of 5 and 10 g of flaxseed fibers result in prolonged
decrease in the levels of ghrelin a hunger-signaling gut
peptide (
Dissimilar to many other cell types, sperm lipid membranes contain an exceptionally high percentage of polyunsaturated fatty acids (PUFAs) that provide the fluidity to the membrane contraction events associated with fertilization. However, PUFAs are readily oxidized and produce malondialdehyde.
We reported that lipid peroxidation assessed by MDA
levels in all groups exposed to hypoxia was increase but
the differences among different groups were not significant.
The hypoxia-induced changes in lipid metabolism
were mediated via hepatic stearoyl coenzyme A desaturase
(
This study shows an increase in serum inflammatory
markers (i.e.IL-18) only in group who expose to hypoxia
and higher levels of lipid peroxidation and reduces antioxidant
activity. In addition, we found flaxseed could
effectively counteract peroxidation damage, mediated by
the attenuation of systemic and tissue oxidative stress induced
by Hypoxia. This is reflected by an increase in TAC
values in Hx+Fx group as compared to the Hx group. This
is in agreements with previous studies (
A high rate of death was observed among animals during the last time of hypoxia procedure.
To confirm the results of this study, we suggest to evaluate the testicular tissue superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRD), and glutathion-S-transferase (GST) activities to confirm the obtained findings.
The conclusion the present study revealed that flaxseed as an antioxidant drug can reduce hypoxia-induced damages in the testes.