Sodium selenite improves folliculogenesis in radiation-induced ovarian failure: a mechanistic approach.
ABSTRACT Radiotherapy is a major factor contributing to female infertility by inducing premature ovarian failure (POF). Therefore, the need for an effective radioprotective agent is evident. The present study investigated the mechanism of potential radioprotective effect of sodium selenite on radiation-induced ovarian failure and whether sodium selenite can stimulate in-vivo follicular development in experimental rats. Immature female Sprague-Dawely rats were either exposed to gamma-radiation (3.2 Gy, LD(20)), once and/or treated with sodium selenite (0.5 mg/kg), once daily for one week before irradiation. Follicular and oocyte development, apoptotic markers, proliferation marker as well as oxidative stress markers were assessed 24-h after irradiation. In addition, fertility assessment was performed after female rats became completely mature at two months of age. Sodium selenite significantly enhanced follicular development as compared to the irradiated group. Sodium selenite significantly reversed the oxidative stress effects of radiation that was evidenced by increasing in lipid peroxide level and decreasing in glutathione level, and glutathione peroxidase (GPx) activity. Assessment of apoptosis and cell proliferation markers revealed that caspase 3 and cytochrome c expressions markedly-increased, whereas, PCNA expression markedly-decreased in the irradiated group; in contrast, sodium selenite treatment prevented these alterations. Histopathological examination further confirmed the radioprotective efficacy of sodium selenite and its in-vivo effect on ovarian follicles' maturation. In conclusion, sodium selenite showed a radioprotective effect and improved folliculogenesis through increasing ovarian granulosa cells proliferation, estradiol and FSH secretion, and GPx activity, whilst decreasing lipid peroxidation and oxidative stress, leading to inhibition of the apoptosis pathway through decreasing the expressions of caspase 3 and cytochrome c.
- [Show abstract] [Hide abstract]
ABSTRACT: The deleterious damage of reproductive function following radiotherapy is of increasing importance. In the present study, we investigated the impact of long-term sodium selenite (SS) treatment on radiotherapy-induced ovarian injury in a rat model. Two-week after radiation exposure vaginal cyclicity was arrested, and serum FSH level was elevated in irradiated female rats. SS significantly ameliorated ovarian and uterine oxidative stress induced by irradiation through decreasing the lipid peroxide level and increasing the glutathione level, and glutathione peroxidase activity. In the presence of SS, ovarian cytochrome c and caspase 3 expressions triggered by radiotherapy were decreased. SS significantly counteracted radiation-induced a widespread loss of ovarian follicles and caused further stimulation of follicular proliferation through enhancing PCNA expression. Despite such alteration in ovarian function, serum estradiol level didn’t change after irradiation, whereas SS significantly increased it. In conclusion, long-term SS treatment improved reproductive development, which was impaired by radiotherapy.Reproductive Toxicology 01/2013; · 3.14 Impact Factor
Sodium Selenite Improves Folliculogenesis in Radiation-
Induced Ovarian Failure: A Mechanistic Approach
Riham S. Said1, Ahmed S. Nada1, Ebtehal El-Demerdash2*
1National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt, 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain
Shams University, Cairo, Egypt
Radiotherapy is a major factor contributing to female infertility by inducing premature ovarian failure (POF). Therefore, the
need for an effective radioprotective agent is evident. The present study investigated the mechanism of potential
radioprotective effect of sodium selenite on radiation-induced ovarian failure and whether sodium selenite can stimulate in-
vivo follicular development in experimental rats. Immature female Sprague-Dawely rats were either exposed to gamma-
radiation (3.2 Gy, LD20), once and/or treated with sodium selenite (0.5 mg/kg), once daily for one week before irradiation.
Follicular and oocyte development, apoptotic markers, proliferation marker as well as oxidative stress markers were
assessed 24-h after irradiation. In addition, fertility assessment was performed after female rats became completely mature
at two months of age. Sodium selenite significantly enhanced follicular development as compared to the irradiated group.
Sodium selenite significantly reversed the oxidative stress effects of radiation that was evidenced by increasing in lipid
peroxide level and decreasing in glutathione level, and glutathione peroxidase (GPx) activity. Assessment of apoptosis and
cell proliferation markers revealed that caspase 3 and cytochrome c expressions markedly-increased, whereas, PCNA
expression markedly-decreased in the irradiated group; in contrast, sodium selenite treatment prevented these alterations.
Histopathological examination further confirmed the radioprotective efficacy of sodium selenite and its in-vivo effect on
ovarian follicles’ maturation. In conclusion, sodium selenite showed a radioprotective effect and improved folliculogenesis
through increasing ovarian granulosa cells proliferation, estradiol and FSH secretion, and GPx activity, whilst decreasing lipid
peroxidation and oxidative stress, leading to inhibition of the apoptosis pathway through decreasing the expressions of
caspase 3 and cytochrome c.
Citation: Said RS, Nada AS, El-Demerdash E (2012) Sodium Selenite Improves Folliculogenesis in Radiation-Induced Ovarian Failure: A Mechanistic Approach. PLoS
ONE 7(12): e50928. doi:10.1371/journal.pone.0050928
Editor: Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America
Received July 25, 2012; Accepted October 25, 2012; Published December 6, 2012
Copyright: ? 2012 Said et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: No source of funding.
Competing Interests: The authors have declared that no competing interest exist.
* E-mail: firstname.lastname@example.org
Premature ovarian failure (POF) is a heterogeneous disorder
defined as cessation of ovarian function with elevated gonadotro-
pins and low estrogen levels before or at the age of 40 . It affects
approximately one in 10,000 women by age of 20; one in 1,000
women by age of 30; one in 100 women by age of 40 .
Unexplained POF occurs in up to 1% of the world’s female
population ; it is associated with loss of fertility, which in most
cases is due to the absence of follicles, and in other cases, to the
inability of remaining follicles to respond to stimulation . It is
well known that the ovaries contain a limited number of follicles;
this number is about 200,000 at puberty and the progressive
decrease with aging leads to only 400 follicles at the time of
menopause . Nevertheless, the currently used cancer therapies
are often detrimental to fertility .
Radiotherapy, one of the most important cancer treatment
modalities, relies on the generation and use of reactive oxygen
species (ROS) to eradicate tumors , and in the process, non-
target tissues are also damaged. The increase in ROS production
in granulosa cells seems to have a deleterious effect on oocyte
fertilization, embryo quality and implantation rate. Moreover, it
seems that germ cells, in comparison with somatic cells, are more
susceptible to oxidative stress [8,9]. Additionally; radiotherapy is
known to result in oocyte loss  and ovarian atrophy, combined
with reduce follicle stores, leading subsequently to menstrual
irregularities, ovarian failure and associated infertility.
Currently, the search for more effective radioprotectors has
been intensified due to increased use of ionizing radiation in
radiotherapy for the treatment of malignant tumors. Selenium
(Se); an essential trace element; It is among the most well-known
radioprotectors, and is necessary for the maintenance of various
physiological processes . Se is incorporated into the catalytic
site of antioxidant enzymes, such as glutathione peroxidase (GPx),
and is involved in cell growth and development by protecting cells
against the toxic and damaging effects of ROS . It is assumed
that radiotherapy induces Se deficiency which possibly enhances
radiation side-effects [13,14]. As well, Paszkowski et al.  found
a significant depletion of Se in the follicular fluid of women with
unexplained infertility. In addition, Barrington et al.  demon-
strated that idiopathic miscarriage is associated with Se deficiency.
However, the biochemical mechanism through which Se prevents
female reproductive disorders is not clear. One of the possibilities
that needed further studying is modulation of oxidative stress and
enhancement of the antioxidant system in the body. It was found
that the synthesis of glutathione; an intracellular antioxidant, is a
critical part of oocyte cytoplasmic maturation . Indeed, the
PLOS ONE | www.plosone.org 1December 2012 | Volume 7 | Issue 12 | e50928
evidence from in-vitro studies indicated that Se is capable of
improving the in vitro growth and maturation of the mouse
preantral follicles . Additionally, sodium selenite improved the
in-vitro follicular development of immature mouse ovaries by
reducing the ROS level and increasing the total antioxidant
capacity and GPx activity . Further, in-vitro studies using
bovine granulosa cells demonstrated that Se significantly stimu-
lated the proliferation of cells and enhanced estradiol production
. At the same time, it was registered that selenite had a
radiosensitizing effect and increased the therapeutic index of
radiation therapy for cancer cell lines [19,20].
From the above, the effectiveness of Se in enhancing
folliculogenesis in-vivo and the exact cellular mechanisms are not
defined. Therefore, the present study was designed to examine
whether Se has any significant role on radiation-induced ovarian-
uterine dysfunction in vivo by studying its effects on different
markers of oxidative stress, apoptosis, proliferation and folliculo-
Materials and Methods
Sodium selenite, reduced glutathione (GSH), Ellman’s reagent
[5,5-dithio-bis (2-nitrobenzoic acid); DTNB], and thiobarbituric
acid (TBA) were purchased from (Sigma Chemical Co., St Louis,
(K2HPO4), potassium di-hydrogen phosphate (KH2PO4), and
trichloroacetic acid (TCA) were purchased from El-Nasr Chemical
Co. (Egypt). Glutathione peroxidase kit was purchased from
(Randox Laboratories, UK). All other chemicals and solvents were
of the highest grade commercially available.
The study was conducted according to the ethical guidelines
(Ain Shams University, Egypt). Immature female Sprague-Dawely
rats (23 days of age) were obtained from Nile Co. for
Pharmaceutical and Chemical industries, Egypt. Rats were housed
in an air-conditioned atmosphere, at a temperature of 25uC with
alternatively 12-h light and dark cycles. Animals were acclimated
for two weeks before experimentation. They were kept on a
standard diet and water ad libitum. Standard diet pellets (El-Nasr,
Egypt) contained not less than 20% protein, 5% fiber, 3.5% fat,
6.5% ash and a vitamin mixture.
Whole body gamma-irradiation was carried out using a Cesium
(137CS) source, Gamma Cell-40 biological irradiator, at the
National Centre for Radiation Research and Technology
(NCRRT), Cairo, Egypt. The animals were exposed to a single
dose of (3.2 Gy) gamma ray with a dose rate of 0.48 Gy/min. This
dose represents the LD20according to the study of Lee et al. .
The plastic boxes containing rats were positioned in a chamber
fixed to the irradiation equipment.
Animals were divided into four groups (twelve animals per
group) and treated for one week as follows; the first group acting as
a control received saline (0.5 ml/100 g B.W., i.p.) once daily. The
second irradiated group received saline (0.5 ml/100 g B.W., i.p.)
once daily then exposed to a single dose of (3.2 Gy) whole-body
irradiation with gamma ray. The third group was given sodium
selenite (0.5 mg/kg, i.p.) once daily. The dose was chosen
according to the study of Pontual et al.  and Cekan et al.
. The fourth group was administered sodium selenite (0.5 mg/
kg, i.p.) once daily, and then exposed to a single dose whole-body
irradiation. The second and the fourth groups were subjected to
irradiation after 24-h of the last saline or sodium selenite injection.
The control, irradiated, sodium selenite, and treated animals were
tested daily for estrus cycles throughout the experiment using
vaginal lavage techniques. Briefly, vaginal lavage was performed in
the morning by flushing the vagina with 10 ml of distilled water
and subsequently aspirated, then smeared onto a glass slide. The
flushed vaginal fluid was fixed with 70% ethanol and examined
microscopically using methylene blue stain. Twenty-four h after
irradiation, blood samples were collected from the retro-orbital
plexus and allowed to clot. Afterwards, rats were sacrificed;
ovarian and uterine tissues were dissected, washed with ice-cold
saline, and then, weighed.
Tissue Collection and Processing
Serum was separated by centrifugation at 3000 g for 15 minutes
and kept frozen at 280uC until assessment of 17 b-estradiol (E2)
and follicle-stimulating hormone (FSH). Samples of ovarian and
uterine tissues were homogenized at 1:10 (w:v) in saline (PH 7.4)
with an Ultra Turrax homogenizer after that the supernatant was
obtained by centrifugation at 10,000 g for 15 minutes then, stored
at –80uC until analysis of oxidative stress markers, including
reduced glutathione (GSH), GPx activity and lipid peroxidation.
In addition, further ovarian and uterine tissues were fixed in an
appropriate buffer for light microscopical examination as well as
immunohistochemical detection of proliferation marker, prolifer-
ating cell nuclear antigen (PCNA), and apoptotic markers, caspase
3 and cytochrome c.
Circulating Levels of Serum E2 and FSH
To determine whether the fall in the number of growing follicles
altered the functional maturation of the ovary, serum E2 and FSH
were measured during the prepubertal period. An ELISA kit
(DRG International, Inc., USA) was used for estimating the
circulating levels of serum E2. As well, serum FSH measurements
were performed by commercially available radioimmunoassay kit
(rat FSH IRMA C.T., IBL International GMBH, Germany). The
intra- and inter-assay coefficients of variation were found to be less
than 9% and 10%, respectively for E2, and less than 3% and 8%,
respectively for FSH. The minimum detectable concentration for
E2 and FSH was 3.6 pg/ml and 0.2 ng/ml, respectively.
Measurement of Oxidative Stress
To determine GSH, 0.5 ml homogenate was added to a
centrifuge tube with 0.5 ml of 10% TCA. The tubes were shaken
gently and intermittently for 15 min, followed by centrifugation at
3000 g for 10 min. Aliquots of the resulting supernatant were
added to a tube containing phosphate buffer and Ellman’s reagent
then the absorbance was read at 412 nm within five min,
according to Ellman’s method . The results were expressed
as mmol of GSH/g wet tissue. Lipid peroxidation was determined
by estimating the level of thiobarbituric acid reactive substances
(TBARS) measured as malondialdehyde (MDA), according to the
method of Mihara and Uchiyama . Briefly, the reaction
mixture (0.5 ml homogenate +2.5 ml 20% TCA +1.0 ml 0.6%
TBA) was heated for 20 min in a boiling-water bath, followed by
cooling and addition of n-butanol with vigorous shaking.
Afterward, the alcohol layer was separated by centrifugation at
2000 g for 10 min and the absorbance was measured at 535 nm.
The results were expressed as nmol of MDA/g wet tissue using
1,1,3,3- tetraethoxypropane as standard.
Furthermore, ovarian and uterine GPx activities were deter-
mined spectrophotometrically based on that of Paglia and
Se Restores Folliculogenesis in Ovarian Failure
PLOS ONE | www.plosone.org2December 2012 | Volume 7 | Issue 12 | e50928
Valentine . GPx catalyses the oxidation of glutathione (GSH)
by Cumene Hydroperoxide; in the presence of glutathione
reductase and NADPH, the oxidized glutathione is immediately
converted to the reduced form with a concomitant oxidation of
NADPH to NADP+. The decrease in absorbance at 340 nm is
measured. Specific activities were expressed as a unit/g wet tissue.
The ovaries or uterus was fixed in 10% formalin overnight and
embedded in paraffin. Serial sections of 4 mm thick were stained
with hematoxylin and eosin for light microscopic histological
examination. In all ovarian samples, the fifth cut was chosen to
count the number of follicles and to evaluate follicular develop-
ment using a digital video camera mounted on a light microscope
(CX21, OLYMPUS, JAPAN). Follicles were classified as primor-
dial if they contained an oocyte surrounded by flattened
pregranulosa cells. Follicles were classified as pre-antral if they
contained an oocyte with a visible nucleolus, more than one layer
and less than five layers of granulosa cells and lacked an antral
space. Follicles were classified as antral if they contained an oocyte
with a visible nucleolus, more than five layers of granulosa cells
and/or an antral space as described previously . Atretic
follicles were identified due to the presence of a degenerating
oocyte or granulosa cells’ pyknosis .
tive capacity of granulosa cells and thus the subsequent
development of the growing follicles were modified in irradiated
ovaries, immunohistochemical analysis of PCNA  was carried
out. Paraffin embedded tissue sections of 3 mm thick were
rehydrated first in xylene and next in graded ethanol solutions.
The slides were then blocked with 5% bovine serum albumin in
Tris buffered saline (TBS) for 2-h. Immunohistochemical analyses
were performed by a standard streptavidin-biotin-peroxidase
procedure. The sections were incubated with a mouse anti-PCNA
monoclonal antibody (Thermo Fisher Scientific, Cat. No. MS-
106-R7) overnight at 4uC. After rinsing thoroughly with TBS, the
sections were incubated with a biotinylated goat anti-rabbit
secondary antibody for 10–15 min, after that, the horseradish-
peroxidase-conjugated streptavidin solution was added and
incubated at room temperature for 10–15 min. Sections were
then washed with TBS and incubated for 5–10 min in a solution of
0.02% diaminobenzidine (DAB) containing 0.01% H2O2. Counter
staining was performed using hematoxylin, and the slides were
visualized under a light microscope. The number of PCNA
positively-stained cells over the total number of granulosa cells was
counted in seven high-power fields (406) using a digital video
camera, then, the percentage of PCNA positive cells was
To determine whether the prolifera-
Table 1. Effect of sodium selenite injection (SS, 0.5 mg/kg, i.p.; once daily for 1 week) and/or whole body-irradiation on ovarian
and uterine weights.
Ovarian weightUterine weight
Groupsmgmg/100 g body weightmgmg/100 g body weight
Data expressed as Mean 6 SD.
a or b: Significantly different from control or radiation group, respectively at P,0.05 using one-way ANOVA followed by Tukey–Kramer as a post-hoc test.
Figure 1. Circulating hormone levels. Changes in serum levels of FSH (A) and Estradiol (B), expressed as a percentage of control, after sodium
selenite administration in c-radiation subjected rats. Values are given as mean 6 SD. a or b: Statistically significant from control or radiation group,
respectively at P,0.05 using one-way ANOVA followed by Tukey–Kramer as a post-hoc test.
Se Restores Folliculogenesis in Ovarian Failure
PLOS ONE | www.plosone.org3December 2012 | Volume 7 | Issue 12 | e50928
assay, immunohistochemical analysis of apoptosis was carried
out. Ovarian and uterine sections were incubated with primary
antibody, which was a rabbit anti-active caspase 3 polyclonal
antibody (abcam, Cat. NO. ab 2302) or a mouse anti-cytochrome
c monoclonal antibody (Thermo Fisher Scientific, Cat. No. MS-
1192-R7), using a biotinylated goat anti-rabbit, as secondary
antibody. For negative controls, primary antibody was omitted.
Fractions of ovarian caspase 3 and cytochrome c DAB-positive
immunoreactive areas were calculated automatically in seven
high-power fields (206), representing the percentage of immuno-
positive cells to the total area of the microscopic field using a
digital video camera mounted on a light microscope (CX21,
OLYMPUS, JAPAN). All steps for immunohistochemical evalu-
ation were carried out using image analysis software (Image J,
1.46a, NIH, USA).
As previously-mentioned in PCNA
Female rats from all groups (n=5 in each group) were mated
with age related, and sexually-experienced males at 60-day post
natal (dpn) for up to 3 sexual cycles (3 estrus). One male was
housed with a group of two or three females. Pregnant females at
14–15 d of gestation were isolated. Number of pregnant females
and newborn pups were counted and kept with their mother until
2 dpn to check breastfeeding and eventual lethality.
Data are presented as the mean 6 SD and were analyzed by
one-way analysis of variance (ANOVA) followed by Tukey–
Kramer as a post-hoc test. In addition, number of pups was
compared using Kruskal-Wallis’s test followed by Dunn’s multiple
comparisons as a post-hoc test. The 0.05 level of probability was
used as the criterion for significance. All statistical analyses were
performed using Instat version 3 software package. Graphs were
sketched using GraphPad Prism (ISIH software, USA) version 5
Vaginal Smear and Organ Weight Changes
No differences in food consumption were seen between the
groups of animals throughout the experimental schedule. Weights
of ovarian and uterine tissues were compared after normalization
to 100 g body weight. Animals exposed to radiation showed
significant reduction in their ovarian and uterine weights, as
compared to control group. Pre-treatment of animals with sodium
selenite significantly counteracted effects of radiation and main-
tained sex organs’ weights comparable to that of the control group.
Animals treated with sodium selenite alone did not show any
significant difference from the control group (Table 1). Addition-
ally, selenite-treated animals with or without c-radiation exposures
were undergoing estrus cycles, similar to controls; On the other
hand, irradiated animals arrested at diestrus phase after 24-h of
irradiation. However, after two months; before pregnancy; we
Figure 2. Morphometric analysis of ovarian follicle populations. Numbers of (A) primordial, (B) preantral, (C) antral and (D) atretic ovarian
follicles was expressed as a percentage of control in each follicle type. Follicle counts were performed on histological sections as described in
Materials and Methods. Bars represent the mean 6 SD of at least three independent experiments. a or b: Statistically significant from control or
irradiated group, respectively at P,0.05. Data were analyzed by one-way ANOVA followed by Tukey–Kramer as a post-hoc test.
Se Restores Folliculogenesis in Ovarian Failure
PLOS ONE | www.plosone.org4December 2012 | Volume 7 | Issue 12 | e50928
Figure 3. Follicular proliferation. Immunohistochemical localization of PCNA in ovarian follicles was studied 24-h after irradiation. (A) Expression
of PCNA in ovaries of the control group. (B) Expression of PCNA in ovaries treated with sodium selenite (0.5 mg/kg) alone. (C) Expression of PCNA in
ovaries exposed to c-radiation (3.2 Gy). (D) Expression of PCNA in ovaries treated with sodium selenite (0.5 mg/kg) for one week before being
exposed to c-radiation (3.2 Gy). Scale bar, 10 mm. (E) Quantitative image analysis for IHC staining expressed as a percentage of PCNA positive cells
against the total number of granulosa cells across seven higher power fields (406) for each rat section. Each column represents the mean 6 SD of at
least three independent experiments. a or b: Statistically significant from control or irradiated group, respectively at P,0.05 using one-way ANOVA
followed by Tukey–Kramer as a post-hoc test.
Se Restores Folliculogenesis in Ovarian Failure
PLOS ONE | www.plosone.org5December 2012 | Volume 7 | Issue 12 | e50928