Int J Reprod BioMed Vol. 15. No. 8.

pp: 509-520, August 2017 Original article

The effects of propolis extract on ovarian tissue and

oxidative stress in rats with maternal separation stress
Atefeh Arabameri1 M.Sc., Hamidreza Sameni2 Ph.D., Ahmadreza Bandegi3 Ph.D.

1. Department of Biology, Faculty Abstract

of Basic Sciences, Islamic Azad
University, Damghan, Iran. Background: Stress in infancy has dramatic effects on different systems, including
2. Nervous System Stem Cells the nervous system, endocrine, immune, reproductive and etc.
Research Center, Department of Objective: The purpose of this study was to investigate the effects of extract of
Anatomical Sciences, Faculty of Iranian propolis (EIP) on ovarian tissue and oxidative stress in rats with maternal
Medicine, Semnan University of
Medical Sciences, Semnan, Iran. separation stress.
3. Nervous System Stem Cells Materials and Methods: 48 immature female rats were divided randomly into six
Research Center, Department of groups. 1) Control group, 2) Control group+saline, 3) Stress group, includes infants
Biochemistry, Faculty of that were separated from their mothers 6 hr/day, the 4th, 5th and 6th groups consisted
Medicine, Semnan University of
Medical Sciences, Semnan, Iran. of infants who in addition to daily stress received 50, 100 and 200 mg/kg of EIP,
respectively. Then serum corticosterone, 17-beta-estradiol, malondialdehyde, total
superoxide dismutase, glutathione peroxidase and ferric reducing antioxidant power
levels were measured. The ovarian sections were stained by H&E, PAS, and
TUNEL methods and were studied with optical microscopy.
Results: Stress increased the blood serum corticosterone levels and 17-beta-estradiol
reduced significantly (p<0.001) and EIP prevented from this changes (p<0.01). EIP
significantly increased the number of ovarian follicles, oocytes and oocytes diameter
Corresponding Author: in neonatal rat following stress (p<0.01). EIP also significantly decreased the
Hamidreza Sameni, Nervous number of atretic follicles, TUNEL+granulosa cells, malondialdehyde levels and
System Stem Cells Research
Center, Department of Anatomical increased ferric reducing antioxidant power, total superoxide dismutase and
Sciences, Faculty of Medicine, glutathione peroxidase serum levels in neonatal rats following stress. The dose of
Semnan University of Medical 200 mg/kg EIP was more effective.
Sciences, Semnan, Iran. Postal Conclusion: This Study showed that the Iranian Propolis significantly could prevent
Code: 3513138111
Email: [email protected]
oxidative stress and histopathological changes in the ovary of the neonatal rat the
Tel: (+98) 23 33354218 following stress.
Received: 29 October 2016 Key words: Propolis, Stress, Ovary, Corticosterone, Oxidative stress, Rats.
Revised: 19 April 2017
Accepted: 23 July 2017 This article extracted from M.Sc. thesis. (Atefeh Arabameri)

Introduction development (4). In the stressed conditions,

two systems are active in the body: one is a

S
tress is any threatening event that sympatho-adreno-medullary system that
causes to reveal the behavioral and releases catecholamine's (epinephrine and
physiological responses in an norepinephrine), and the other is
individual (1). The stresses during pregnancy hypothalamus-pituitary-adrenal axis (HPA)
and after the birth are related to many mental, that releases glucocorticoids (corticosterone in
behavioral and cognitive abnormalities in rodents and cortisol in humans) by stimulating
humans and animals (2). The types of stress the adrenal cortex (5-8).
include, physical (motion limitation, foot shock, Glucocorticoid receptors are found in
exercise, cold, intense light, etc.), metabolic, hypothalamic gonadotropin-releasing
immunological, cardiovascular and mental hormone (GnRH) neurons and cells of
stress and so on can be pointed (3). It has pituitary gonadotropin. Glucocorticoids with
been reported that female reproductive the same concentration as stress state block
system is very sensitive to stress and effect of the level of GnRH in the pituitary gland and
stress on gonads in adults may be reversible subsequently, the responsiveness of
but it may not be true during initial stages of gonadotropins to GnRH is also reduced, thus
development. Thus, stress experienced during the reduction of LH level and consequently the
neonatal or pre-pubertal phases might have lack of ovulation and the defect of the
serious side effects on the ovarian follicular menstrual cycle happen (9). Glucocorticoids
 Arabameri et al

also cause to reduce estrogen secretion by including its anti-cancer, anti-inflammatory,

blocking aromatase activity in granulosa cells antibacterial and antiviral effects, anti-
influencing not only steroid-making but also Parkinson’s disease and antioxidant
reduces the expression of estrogen and properties and attributed these characteristics
progesterone receptors and may cause to to its various chemical compounds (10, 12-
damage the ovary and leads to disruption or 15).
delay in pregnancy (9). Our earlier studies have shown that the
There exists a reciprocal relationship Iranian propolis extract could enhance the
between HPA and the hypothalamic-pituitary- antioxidant levels and histopathological
gonadal axes. For instance, both testosterone changes in the kidneys and control blood
and oestrogen modulate the response of the glucose and modulate some of the
HPA axis, while activation of the stress axis, biochemical factors of diabetic rats, also
especially activation that is repeating or prevent from increasing serum corticosterone
chronic, has an inhibitory effect upon and brain MDA levels and from decreasing
oestrogen and testosterone secretion. ferric reducing antioxidant power (FRAP),
Alterations in maternal care can produce superoxide dismutase (SOD) and glutathione
significant effects on both hypothalamic- peroxidase (GPx) levels in brain under
pituitary-gonadal and HPA physiology and prenatal stress (16-18). Propolis is one of the
behavior in the offspring at adulthood (6). strongest natural antioxidants. The measured
Stress, excessive secretion of antioxidant activity of propolis extract in units
glucocorticoids can cause cellular oxidative of oxygen radical absorbance capacity is four
stress (10). In the state of oxidative stress, times greater than vitamin E (19). These
high and abnormal level of Reactive oxygen effects are mainly due to of high
species (ROS), such as free radicals concentrations of polyphenols and flavonoids
(hydroxyl, nitric oxide, superoxide) or non- that are the most important active medicinal
radicals (H2O2 and lipid peroxide), can and antioxidant compounds in propolis. Since
damage to the certain molecules and the cell many disorders during puberty and adulthood
components such as lipids, nucleic acids and are caused by the various stresses which
proteins, leading to the cell death as necrosis occur in prenatal and infancy periods.
and apoptosis (10, 11). After lipid Therefore, finding the compounds that can
peroxidation, intermediate compounds that control the harmful effects of the stress on the
are the main characteristic of the oxidative different organs, including the ovaries has the
stress process, have been created in the cell; extremely high importance.
Malondialdehyde (MDA) can be referred as Regarding the crucial and decisive role of
one of these compounds. Naturally, in the the ovary in the fertility and the powerful
cells, there are the enzymes such as antioxidant properties of propolis, the aim of
Superoxide Dismutase, Catalase, Glutathione this study was to investigate the protective
peroxidase and Glutathione Reductase that effects of hydro-alcoholic extract of Iranian
are responsible for antioxidant defense (11). propolis (EIP) on ovarian tissue and oxidative
Reports have shown that the oxidative stress stress in rats with maternal separation stress.
have the important negative effects on female
fertilities and health of gametes and the Materials and methods
pharmacological or nutritional interventions as
an effective strategy protecting female Animals and experimental design
fertilities from the negative effects of ROS and All procedures were carried out in the
oxidative stress (9). laboratory of biochemistry and stem cells
Propolis is a red or brown resinous research center of Semnan University of
substance that honey bee’s collect from the Medical Sciences, Semnan, Iran. A total of 48
tree buds, leaves, sop flows and other female Wistar rats at the age of 15 days and
botanical sources and then the bees add wax the approximate weight of 20±5 were obtained
and their other secretions to it. It is used as a from the laboratory animal center of Semnan
sealant for unwanted open spaces in the hive University of Medical Sciences. The rats were
and is used as a traditional herbal medicine in maintained under constant conditions: light
many countries. Many studies confirmed the period (12 hr light/dark), relative humidity of
biological and pharmacological activities 45-55%, controlled temperature of 22±2oC

510 International Journal of Reproductive BioMedicine Vol. 15. No. 8. pp: 509-520, August 2017
 Effects of Stress and Propolis on Ovary

and free enough access to standard diet and evaporated using a rotary vacuum evaporator
water in plastic cages. to obtain the purified propolis extract. The EIP
In this experimental study, the animals concentrations were measured and diluted to
were randomly divided into six groups the required dilutions (weight to volume) using
(n=8/each) as follows: 1) Control group (C) 10% ethanol. The extracts were stored at 2-
included 21-day pups without any intervention, 8oC under protective light conditions and
2) Sham group included 15-day pups that warmed to room temperature just before
were beside their mothers during this period injecting (22, 23).
and received 0.1 ml saline solution daily, 3)
Stress group (S), included 15-day pups that Assay of serum concentration
were separated from their mothers 6 hr/day corticosterone and 17-beta-estradiol
during this period, 4) the S+P50 group which The corticosterone and 17-beta-estradiol
were under stress and received 50 mg/kg EIP serum concentration were determined by
daily, 5) the S+P100 group which were under enzyme linked immunosorbent assay (ELISA)
stress and received 100 mg/kg EIP daily, and using the kits Corticosterone, ELISA, DRG,
6) S+P200 group which were under stress and Marburg, Germany and 17-beta estradiol,
received 200 mg/kg EIP daily (16-18). ELISA, Bolden, England. The sensitivity of
The animals in groups C and Sham didn't these kits is 0.39 nanograms per milliliter. To
have any stress and had enough diet and do this, the blood was collected from the
water, and the animals in the third to sixth hearts of the animals under complete
groups were separated from their mothers 6 anesthesia 24 hr after the last injection at the
hr/day, were kept in separate cages with dose of 80/20 mg/kg Ketamine/Xylazine and
suitable beds and their injections were done after centrifugation at 300 rpm for 20 min; the
intraperitoneally daily. The timing of obtained serum was stored at -20oC until
separation from the mothers (6 hr/day) was measuring the hormone levels (24).
randomly changed every day to avoid
habituation (for example, as the periods from Assay of antioxidant activity
8AM-14PM, 9AM-15PM, and so forth). The After EIP treatment, all the pups were
EIP was injected to the animals of the fourth, anesthetized by administering ketamine/
fifth and sixth groups intraperitoneally for xylazine (80/20 mg/kg) intraperitoneally. After
seven days (from 15th day to 21st day after opening the abdominal cavity, the left ovary
birth) before stress. The right ovaries were was removed, washed with physiological
fixed in 4% paraformaldehyde, embedded in saline and was homogenized (10% w/v) in
cold saline (1.15 M KCl) to prepare for the
paraffin. They were then serially sectioned (5
assay for activity of antioxidant enzymes. The
µm), mounted on glass slides for histological
homogenates were centrifuged at 20,000× gr
and immunohistochemical studies. The blood
for 10 min at 4oC. The supernatants were
sample was collected, serum was separated,
collected and used for assessment of Ferric
stored at -20oC until corticosterone, and 17- reducing antioxidant power, MDA levels, T-
beta-Estradiol concentration was determined SOD (Total superoxide dismutase), and GPx,
(20, 21). activities (Randox Laboratories, Shanghai,
china) (25-26).
Preparation of propolis extract
In this investigation, propolis was collected Histological evaluation
from the bee-hives located in different parts of To study the Histomorphometry and to
the Semnan province and verified by the count of oocytes and ovarian follicles, the
agricultural organization. Extracts were ovaries were removed from the body and their
prepared according to the method of weights were determined after blood
Greenaway (23). In summary, the major collection. The right ovaries fixed in 4%
components of propolis were chopped into paraformaldehyde, were processed according
small pieces, mixed (25 gr) with 250 ml of to the standard histological method and 5μm
80% ethanol, and incubated at room thick serial paraffin sections were cut and
temperature for 48 hr with shaking (150 rpm). stained with hematoxylin and eosin. Different
The extract was clarified twice by Whatman categories of ovarian follicles were counted
grade 42 filter paper and ethanol was according to Pedersen and Peters methods

International Journal of Reproductive BioMedicine Vol. 15. No. 8. pp: 509-520, August 2017 511
 Arabameri et al

(27). Briefly, every fifth ovary section was Statistical analysis

scanned under a dot side-digital virtual All data were expressed as mean±SEM.
microscope, and the number of follicles in the The quantitative data have been analyzed
entire section was counted. To avoid multiple using the SPSS software (Statistical Package
counts of the same follicle, only those with a for Social Sciences, version 20.0, SPSS Inc,
visible oocyte nucleus were included. Chicago, Illinois, USA). Data from
In this manner, in a slice of the ovary, the experiments with more than two independent
primitive follicles were counted from each 4 variables have been analyzed using the
slices and the unilamellar primary follicles of analysis of variance (ANOVA) followed by the
every 6 slices. But counting multilamellar Tukey–Kramer post-hoc tests. The significant
primary follicles, pre-antral and antral follicles difference was considered between the
is in this way that the follicles which contain groups at a significance level of p<0.05.
only oocytes with the perfect size in each slice
of the ovary are counted and so the repeated Results
counting of mentioned follicles can be avoided
in other slices (28, 29). Atretic follicles were Effect of postnatal stress and EIP on body
also counted and identified according to weight
Greenwald's described morphological method The mean body weight and percent body
using hematoxylin-eosin staining, serial weight gain in S (stress) group were
sections and allocated terminal significantly lower than the control groups.
deoxynucleotidyl transferase dUTP nick end Treatment with EIP at doses of 50, 100 and
labeling (TUNEL) method (30, 31). 200 mg/kg were significantly increased body
weight (F=6.428, p=0.001, Figure 1).
TUNEL assay
The follicular atresia, determined by the Effect of postnatal stress and EIP on
presence of more than 5% pycnotic granulosa serum corticosterone and 17-beta-estradiol
cells (apoptotic cells), was further levels
characterized by conducting TUNEL kit (In situ The mean serum concentration of
cell death detection kit, POD, Roche corticosterone was significantly higher in
Germany) was used in order to identify the stressed rats compared to healthy and control
apoptotic cells. In this way, to determine the groups. Serum corticosterone levels
cell apoptosis, TdT enzyme is able to label 3‫ـ׳‬ significantly reduced in the neonates that had
OHs which have achieved the free form by stress and received EIP at doses of 100 and
breaking down the DNA during apoptosis. 200 mg/kg (F=6.211, p=0.001, Figure 2).
These sites were labeled with conjugated Stress also reduced the amount of 17-beta-
dUTP by fluorescein, and then the sites estradiol in the blood serum of the neonates in
containing fluorescein were detected by anti- comparison with the sham and control groups.
fluorescein antibody connected to POD. Serum 17-beta-estradiol levels significantly
If there is a breakage in the DNA and the increased in the neonates that had stress and
fluorescein is connected, fluorescein antibody received EIP at doses of 100 and 200 mg/kg
is attached to these sites and POD in this (F=6.729, p=0.01, Figure 2).
antibody can make the brown color in reaction
with the di-amino benzidine substrate. The Effect of postnatal stress and EIP on
cores of apoptotic cells become brown in this follicular and oocyte development
method. The number of TUNEL positive and The mean number of primordial, primary
negative granulosa cells were counted in and secondary follicles in the stressed rats
randomly selected areas in each section and was significantly lower than others groups.
expressed in terms of percentage of TUNEL The treatment of stressed pups with EIP (100
positive and negative cells (32). & 200 mg/kg) significantly increased the
number of different types of the follicles
Ethical consideration (F=4.235, p=003; F=9.527, p=001; F=9.097,
All experimental protocols were approved p=001 respectivly; Table I). The mean number
by the ethics committee of Semnan University and diameter of oocytes were decreased
of Medical Sciences, Semnan, Iran significantly in stressed rats in comparison
(IR.SEMUMS.REC.1392.126), with the others groups. The treatment of

512 International Journal of Reproductive BioMedicine Vol. 15. No. 8. pp: 509-520, August 2017
 Effects of Stress and Propolis on Ovary

stressed pups with EIP (100 and 200 mg/kg) 100, and 200 mg/kg) in the animals were
significantly increased the number and under stress reduced the number of these
diameter of the oocytes (F=2.627, p=0.032; cells (F=4.456, p=0.002, Figure 3, 4).
F=3.100, p=0.016 respectively; Table I).

Effect of postnatal stress and EIP on Effect of postnatal stress and EIP on the
apoptosis of granulosa cells and atretic Lipid peroxidation and antioxidant activity
follicles The EIP showed a strong effect on lipid
The mean number of ovarian atretic peroxidation and antioxidant parameters. A
follicles in stressed rats was significantly significant increase in MDA and reduction in
higher than the normal groups, control and T-SOD, GPx, and FRAP concentration were
under treatment with EIP. The treatment with observed in the stress rats as compared to the
EIP reduced the number of atretic follicles that control group (F=3.324, p=0.012) (Figure 5
this reduction was significantly in the treated and 6). The increase in MDA concentration in
groups with doses of 100 and 200 mg/kg in the ovary of stress animals indicated an
general (F=6.544, p=0.001, Table II). The exasperated oxidative stress. The treatment of
number of TUNEL positive granulosa cells in rats with EIP (100 and 200 mg/kg) resulted in
the ovaries of rat pups in the normal and a significant reduction in MDA and
control groups was significantly lower than upregulation of FRAP, T-SOD, and GPx levels
stress group. The neonatal stress increased in the ovarian tissue compared to vehicle-
the number of TUNEL positive granulosa cells treated stress rats (F=2.879, p=0.028) (Figure
in the ovarian follicles and the use of EIP (50, 5, 6).

Table I. Effect of postnatal stress (maternal separation stress) and Iranian propolis extract on the number and diameter of oocytes and
the number of different kinds of ovarian follicles in the rat neonates.
Normal follicles in the ovaries (n) Diameter of the
Variables Number of
Primordial Primary Secondary oocyte
Groups Antral follicles oocytes (at level)
follicles follicle follicle (micrometer)
Control 119.7 ± 7.05 40.5 ± 2.96 ** 11.4 ± 1.19 ** 9.7 ± 1.32 # 153.25 ± 23.63 # 22.25 ± 2.6
Sham 116.6 ± 6.15 38.7 ± 5.67 ** 9 ± 1.27 * 8.6 ± 1.21 164.25 ± 26.19 * 21.5 ± 1.75
Stress + Saline 67.2 ± 4.38 12 ± 1.24 3.5 ± 0.40 5.1 ± .060 71.75 ± 16.45 15.25 ± 1.6
Stress + Propolis 50 82.7 ± 4.18 31.8 ± 4.98 * 9.5 ± 1.1 * 8.5 ± 1.30 119.75 ± 16.66 19.5 ± 2.03
Stress + Propolis 100 120.8 ± 7.52 # 33.1 ± 3.14 * 11 ± 1.01 ** 8.1 ± .085 140 ± 21.32 # 24.5 ± 2.1 #
Stress + Propolis 200 124.8 ± 8.97 * 42.6 ± 1.33 ** 12 ± .097 ** 9.9 ± 1.20 # 166.75 ± 25.56 * 25.25 ± 2.15 *
All data are presented as Mean ± SEM.
#: The difference between the desired group and the stress group is significant at p<0.05 level.
*: The difference between the desired group and the stress group is significant at p<0.01 level.
**: The difference between the desired group and the stress group is significant at p<0.001 level. Different as judged by Tukey–Kramer post-hoc
tests.

Table II. Effect of postnatal stress (maternal separation stress) and Iranian propolis extract on the average number of ovarian atretic
follicles in the rat neonates.
Variables Atretic ovarian follicles (n)
Categories Primary follicle Secondary follicle Antral follicles
Control 12.3 ± 1.12 * 8.4 ± .056 ** 3.8 ± .058 **
Sham 11.25 ± 0.92 ** 7.8 ± .064 ** 4.2 ± .059 *
Stress + Saline 21.4 ± 0.86 16.6 ± .048 8.75 ± .089
Stress + Propolis 50 18.3 ± 0.67 13.25 ± .046 7.44 ± .047
Stress + Propolis 100 14.75 ± 0.74 # 10.8 ± .067 * 5.2 ± .01.05 #
Stress + Propolis 200 11.75 ± 0.64 ** 8.25 ± .064 ** 3.4 ± .076 **
All data are presented as Mean ± SEM
#: The difference between the desired group and the stress group is significant at p<0.05 level.
*: The difference between the desired group and the stress group is significant at p<0.01 level.
**: The difference between the desired group and the stress group is significant at p<0.001 level. Different as judged by Tukey–Kramer post-hoc
tests.

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 Arabameri et al

Figure 1: Effects of maternal separation stress and Iranian propolis extract on body weight in the rat neonates. Control group
(Control), control group+saline infusion (Sham), stress group + saline infusion (stress), stress group+50 mg/kg propolis extract
(SP50), stress group+100 mg/kg propolis extract (SP100) and stress group+200 mg/kg propolis extract (SP200). * P<0.001,
compared to the Control and Sham groups, # P<0.001compared to the Stress group. All data are presented as Mean ± SEM, n=8.

Figure 2. Effects of maternal separation stress and Iranian propolis extract on corticosterone level (ng/mL) and 17 beta-estradiol
level (ng/mL) in the rat neonates. Control group (Control), control group + saline infusion (Sham), stress group+saline infusion
(Stress), stress group+50 mg/kg propolis extract (SP50), stress group+100 mg/kg propolis extract (SP100) and stress group+200
mg/kg propolis extract (SP200). * P<0.001, compared to the Control and Sham groups, # P<0.001, compared to the Stress group. All
data are presented as Mean ± SEM, n=8.

Figure 3. Effect of propolis on the number of TUNEL positive granulosa cells (in 0.022 mm2 of the ovary surface) in the ovaries of a
rat with neonatal stress. Control group (Control), control group+saline infusion (Sham), stress group+saline infusion (Stress), stress
group+50 mg/kg propolis extract (SP50), stress group+100 mg/kg propolis extract (SP100) and stress group+200 mg/kg propolis
extract (SP200). * P<0.01, compared to the C and Sham groups, # P<0.001, compared to the S group. All data are presented as
Mean±SEM, n=6.

514 International Journal of Reproductive BioMedicine Vol. 15. No. 8. pp: 509-520, August 2017
 Effects of Stress and Propolis on Ovary

Figure 4. Effect of propolis on the number of TUNEL positive granulosa cells (in 0.022 mm2 of the ovary surface) in the ovaries of a
rat with neonatal stress. (A1, A2) Sham group, (B1, B2) stress group, (C1, C2) stress+100 mg/kg propolis extract group and (D1, D2)
stress+200 mg/kg propolis extract group. The arrows indicated by TUNEL positive granulosa cells in the ovarian tissue. TUNEL
positive granulosa cells in the stressed group (B1, B2) compared to other groups increased and propolis extract (C1, C2, D1, D2)
these cells is reduced. Magnification 100X and 400X, a bar in figures A1, B1, C1, and D1=250 µm and bar in figures A2, B2, C2,
and D2=100 µm.

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 Arabameri et al

Figure 5. Effects of propolis on MDA (Malondialdehyde) content in ovarian tissue of rats with neonatal stress. Control group
(Control), control group+saline infusion (Sham), stress group+saline infusion (Stress), stress group+50 mg/kg propolis extract
(SP50), stress group+100 mg/kg propolis extract (SP100) and stress group+200 mg/kg propolis extract (SP200). * P<0.01, compared
to the C and Sham groups, # P<0.05, compared to the S group, ## P<0.01, compared to the S group. All data are presented as
Mean±SEM, n=8.

Figure 6. Effects of propolis on antioxidant enzyme activity in ovarian tissue of rats with neonatal stress. (A) T-SOD activity in the
ovarian tissue, (B) GPx activity in the ovarian tissue, and (C) Ferric reducing antioxidant power (FRAP) in ovarian tissue. Control
group (Control), control group+saline infusion (Sham), stress group+saline infusion (Stress), stress group+50 mg/kg propolis extract
(SP50), stress group+100 mg/kg propolis extract (SP100) and stress group+200 mg/kg propolis extract (SP200). * P<0.01, compared
to the C and Sham groups, # P<0.05, compared to the S group, ## P<0.01, compared to the S group. All data are presented as
Mean±SEM, n=8.

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 Effects of Stress and Propolis on Ovary

Discussion Propolis is rich in antioxidants such as

polyphones and flavonoids (13-34). The
In this study, we showed that the maternal composition of propolis depends upon the
separation stress increased serum vegetation of the area from where it was
corticosterone level and decreased 17-beta- collected. Colorimetric methods are
estradiol level and EIP prevented these convenient and appropriate for the routine
changes. Moreover, the maternal separation analysis of phenolics (35). Antioxidant activity
stress reduced the number of primordial, has also been demonstrated in propolis. It’s
primary and secondary follicles, the number, proposed that strong antioxidant activity
and diameter of the oocytes and treatment of occurs in propolis with high amounts of
stressed pups with EIP (100 & 200 mg/kg) phenolic compounds and weak activity
significantly increased the number of ovarian happens with low amounts (36). Total
follicles, number, and diameter of the oocytes. polyphone content of ethanol extracts of this
Our findings also showed that neonatal stress sample was (6.4 gr/100g±0.03). According to
increases the number of atretic follicles and the other studies, total polyphone content of
apoptotic granulosa cells (TUNEL positive) by this extract is more of them.
intensifying atresia process and the treatment The several reports have shown that the
of the pups with EIP reduces the number of ovaries of the female rats contain only the
atretic follicles and apoptotic granulosa cells oocytes immediately after birth and are
by decreasing the intensity of follicular atresia without any follicles, and this means that
process. follicular development and evolution process
Increased serum corticosterone level in occurs after the birth in the rats (29, 33). As
young rats due to the separation from the well, it is known that primordial follicles are
mother represents one of the important created three days after the birth and lead to
conditions of stress (31). In addition, loss of the formation of primary follicles immediately
body weight is expected in stressed young after their growth and differentiation process
rats as stress inhibits feeding behavior. The (32). Our studies showed that stress causes
treatment of the stressed neonates with EIP at reduction in the different kinds of ovarian
all used doses prevented the body weight loss follicles including, primordial, primary and
of the animals. These protective effects of secondary follicles that the findings reported
propolis were associated with the decrease of by Bhatt and et al confirm it (19, 20). On the
corticosterone level and an increase of the of other hand, the maternal separation stress
the 17-beta-estradiol level. Previous findings significantly increased the number of atretic
have shown that the stress reduces follicles in the ovaries of the neonates and this
gonadotropins which are as an apoptotic is along with previous reports (37, 38). It
inhibitor factor in granulosa cells (9). seems that the reduction of ovarian follicles
Thus, the apoptosis induction by stress and and increase of atretic follicles are probably
subsequently the reduction in the number of resulting from the impaired secretion of
granulosa cells reduces the biosynthesis of gonadotropins and steroids (17-beta-estradiol)
17-beta-estradiol and creates hypo-estrogenic followed by oxidative stress induced by the
conditions in the ovary (32). The reduction of chronic stress conditions.
17-beta-estradiol level decreases the ovum In the present study, propolis, as one of the
quality through its apoptosis induction. Our most powerful natural antioxidants (its
hypothesis is that the neonatal chronic stress antioxidant power is four times vitamin E), is
induces the apoptosis of granulosa cells and likely able to prevent the destruction and the
may reduce 17-beta estradiol biosynthesis, follicular atresia and ultimately protects the
and consequently reduce the quality of the reduced number of ovarian follicles. Previous
ovum. Our results are in agreement with the findings have shown that when vitamins E and
findings reported by Okamoto et al, showed C are used as antioxidants to cure the animals
that oral administration of propolis induces treated with Methidathion (an agent of lipid
estrogenic activity in estrogen target organs in peroxidation in the ovary and an agent of
vivo suggesting that propolis is a useful increasing atretic follicles), they significantly
dietary source of phytoestrogens and a reduce the number of atretic follicles (39).
promising treatment for post-menopausal The chronic stress reduces the production
symptoms (33). and secretion of gonadotropins FSH and LH

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 Arabameri et al

that are necessary for vital, growth and colon (48). It has been proven that propolis is
development of ovarian follicles and oocytes absorbed from the blood circulation and plays
by stimulating the activity of hypothalamus- a role as a hydrophilic antioxidant to absorb
pituitary-adrenal axis (40). The effect of stress vitamin C (48). In addition to flavonoids, a
on the adult gonads may be irreversible in the compound called caffeic acid phenethyl ester
early stages of follicular development (pre- (CAPE) is also responsible for the antioxidant
puberty). The increase secretion of ACTH properties of propolis; it was reported this
inhibits follicular development and ovulation compound protects the cell membrane against
(40). So, reducing the amount ACTH disrupts lipid peroxidation. Previous studies have
follicular development process and oocyte shown that CAPE reduces malondialdehyde
maturation, which eventually alters the (MDA) level by suppressing the production of
number and size of oocytes. an oxygen free radical as an antioxidant (49).
Stress as one of the factors that drastic To further investigate the mechanism of
changes in the level of glucocorticoid action of propolis, we measured the anti-
(corticosterone in rodents) and steroids oxidative capacity and the activity of FRAP.
hormones are created and played an We examined the total antioxidant activity in
important role in increasing the rate of the ovary tissue using FRAP as an indicator of
follicular atresia. The formation of free radicals the strength of non-enzymatic antioxidants.
increases by raising the levels of The results showed that in a postnatal stress
glucocorticoids in the stress state and rat ovary, there is a significant decrease in the
consequently, the production of sex steroids is antioxidant activity (FRAP) in stress group and
reduced. ROS may play an important role in treatment with the EPI (100 and 200 mg/kg)
the survival and development of ovarian enhanced the antioxidant capacity of the
follicles. Therefore, their increases may cause FRAP. SOD neutralizes the superoxide anions
drastic reduction in primordial follicles in the by converting them into hydrogen peroxide
ovaries. ROS may play an important role in and GPx reduces hydrogen peroxide to water;
beginning the apoptosis process of follicles, together SOD and GPx serve as an
including antral follicles. As well reducing the antioxidant defense mechanism (47, 48).
amount of glutathione (as a cellular
antioxidant) causes to stimulate follicular Conclusion
atresia and apoptosis in granulosa cells of the
follicles. However, previous studies have These findings suggest that propolis may
shown that estrogen has antioxidant prevent the destructive effects of
properties, and therefore its deficit during psychological stress (maternal separation of
stress may lead to the production of ROS (41- pups) as well as reduce the structural and
44). In this study, using EIP caused to developmental changes in the ovaries of rat
decrease ROS production and subsequently neonates using its strong antioxidant effects
to reduce the apoptosis of granulosa cells and
due to the compounds such as flavonoids and
follicular atresia, possibly by reducing the
polyphenols. Thus, propolis with more
levels of corticosteroids and increasing the
powerful antioxidant properties might be used
production of ovarian steroids.
as a compound with therapeutic potential
Propolis is one of the richest sources of
plant phenols (flavonoids and phenolic acids), protection against damage caused by stress.
which are widely recognized as powerful
antioxidants (12, 19, 44-47). Various Acknowledgments
flavonoids and phenolic are able to take the
free radicals and protect the lipids and other This article is derived from a student thesis
compounds such as vitamin C against approved by the Research Deputy of Semnan
Oxidation or the destruction during oxidative University of Medical Sciences and Damghan
stress (9, 45). It has been reported that the Islamic Azad University, Iran.
enzymatic and non-enzymatic activities of
antioxidants have been significantly increased Conflict of interest
using propolis; as well propolis has induced a
significant increase in the level of vitamin C in The authors confirm that this article content
plasma, kidney, stomach, small intestine, and has no conflict of interest.

518 International Journal of Reproductive BioMedicine Vol. 15. No. 8. pp: 509-520, August 2017
 Effects of Stress and Propolis on Ovary

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