Adv Pharm Bull. 2016 Mar; 6(1): 65–69.
Published online 2016 Mar 17. doi: 10.15171/apb.2016.010
PMCID: PMC4845541
Abstract
Purpose:
Cumulus cells have a critical role in normal oocyte development and
fertilization. Prunus cerasus is an anthocyanin rich berry and performs
strong antioxidant activity. The present study set to determine if
Prunus cerasus can affect expression of HAS2 (hyaluronan synthase 2) and
progesterone receptor in Cumulus cells and its consequences outcome of
the in vitro fertilization.
Methods:
60 female and 15 male adult mice were used for mating and IVF (in vitro
fertilization). Prunus cerasus extraction was added to the diet of
female mice for 30 days. Ovulation induction and oocytes collection were
done as routine. The cumulus cells were dissected apart, and the
expression of progesterone receptor and HAS2 was detected using RT-PCR
(real-time polymerase chain reaction). Fertilization rate was evaluated
by IVF. All data were analyzed using t-test.
Results:
Data was showed that expression of progesterone receptor and HAS2 in
cumulus cells of mice that received prunus cerasus increased. Moreover,
oocyte fertilization rate also increased significantly.
Conclusion:
Prunus cerasus as an antioxidant natural can become an important
medication for improving oocyte quality and opening new opportunities
for infertility treatment. It is concluded that Prunus cerasus
consumption could improve fertility rate by increasing progesterone
receptor and HAS2 activity in cumulus cells.
Keywords: Cumulus cells, HAS2, Infertility, PGR, Prunus cerasus
Introduction
Infertility is defined as inability to obtain fertility after one year of unprotected regular intercourse.1-4
It has been reported that about 10% -15% of young couples suffer from
infertility. Of these, 40%-55% are due to female factors, 20%-30% are
due to male factors, and 15%-17% are unexplained infertility.1 Over the years, ART (assisted reproductive technology) has been a method for infertility treatment with different causes.5,6 In order to increase the success rate of fertilization, several herbal and chemical supplemental factors have been used.7 Because of less invasiveness and costliness nature of traditional medicine, recent research has focused on herbal use.8,9 Anthocyanin as secondary metabolite group of flavonoids has attracted much attention.10 Strong antioxidant activity was the best known property of flavonoids.11
Antioxidant activity of anthocyanin is associated with a variety of
health benefits including inflammation, cancer, atherosclerosis, and
diabetes.12
Recent studies have shown that reproductive processes may be influenced
by hypothalamic-pituitary-gonadal axis via scavenging free radicals.13-15
However, interest in antioxidant has recently been intensified because
of their possible effect on egg quality, fertilization, and pregnancy
rates.16,17
Antioxidant therapy has been attended to alleviate infertility, and
dietary antioxidant has been beneficial for female reproductive
disorder.18
Fruits including Sour cherry (Prunus cerasus), containing anthocyanin
and strong antioxidant activity, have attracted much attention.19
Prunus cerasus anthocyanins have a vast range of biochemical and
pharmacological effect, and have been recommended as nutritional
supplement.20
Granulosa cells consist of mural and cumulus cells in preovulatory
follicles. Mural cells are in the follicle wall and cumulus cell in
vicinal of oocyte.21
In the midcycle, increased level of LH result in ovulation by inducing
resumption of meiotic maturation, transformation of granulosa cells, and
expansion of cumulus-oocyte complex. The latter, is associated with
accumulation of hyaluronic acid-rich extra cellular matrix in cumulus
cells.22
During the process of cumulus expansion, the most important genes
involved in the formation of matrix hyaluronic are hyaluronic synthase2
(HAS2).23 HAS2 mRNA is necessary for cumulus expansion and signaling during ovulatory response.24
Cumulus expansion facilitates the fertilization, and the failure of
this process can lead to fertilization problem and is associated with
low potential for implantation and decline in the quality of oocyte.25,26
Paracrine factor under the influence of progesterone is required for
preimplantation embryonic development, but direct or indirect effect of
progesterone is not clear.27
Previous studies in our laboratory showed that the presence of
Progesterone receptor is essential for female fertility and ovulation
control.28,29
In this study, Prunus cerasus effect on progesterone receptor
expression, HAS2 in cumulus cells and the rate of in vitro fertilization
are evaluated.
Materials and Methods
Preparation of extract
Sour cherry were collected from the market and authenticated by a
botanist (School of Pharmacy, Tabriz University of Medical Sciences).
The extract, according to World Health Organization (WHO) protocol was
prepared. 500g of Sour cherry was shed-dried powdered and added to 5 Li
of 70°C ethanol (v/v) and left to macerate for 20 hours at room
temperature. The basin was rotated slowly during this time. After
filtration, ethanol at low pressure at 30 °C. The samples were stored
frozen at –20 °C until used.
Study population
A total of 60 female BALB/c mice (4-5weeks) and 15 adult male BALB/c
mice were obtained from the animal house of Tabriz University of Medical
Sciences (TUMS). For a period of 2 weeks prior to the experiment, the
mice were kept in animal room for adapting. 1.5 kg of mice food was
added to 1000ml of Prunus cerasus extract and mixed together completely.
The mixed diet let to dry in 2 days. Female mice were randomly
divided into two equal groups. In the control group, each animal
received diet without Prunus cerasus. In the experimental group, each
animal received 25g diet with Prunus cerasus for 4 weeks. After 4 weeks
for induction of ovulation 10 IU HMG/PMSG (pregnant mare serum
gonadotropin) and after 48 hours, 10 IU hCG (human chorionic
gonadotropin) was injected intraperitoneally.
Oocyte collection
One day post hCG injection, the mice were sacrificed and ovaries were
removed from the female mice. Mice ovaries (n=100) were obtained and
placed in sterile PBS and transported to the Tissue Engineering
Laboratory in two groups. The stromal tissue surrounding the ovaries was
removed, and oocyte were collected under flashing method and cultured
in dishes with Universal IVF culture medium (Origio-Medicult, 10300060)
under sterile mineral oil.
Isolation of cumulus cells
The cumulus cells surrounding the oocyte (COCS) were separated from
oocyte using hyaluronidase enzyme. Hyaluronidase enzyme causes the
connection of oocyte and cumulus cells to be released and facilitates
separation. Cumulus cells were transferred to another dish containing
medium. PBS was added for washing and centrifuged 2 times for 5 min.
After centrifuge, the pellet of cumulus cells was centrifuged for 1 h at
-20 °C and then stored at -80 °C until RNA isolation.
In vitro fertilization
Male mice were killed by cervical dislocation, and the sperm were
collected from the cauda epididymis by incubating the pieces of
epididymis in 37°C Co2 incubator for 20 min. The sperm sample was added
to the collected oocyte in the control group and experimental case
group. The fertility success rate was assessed based on the formation of
embryo.
Real time RT – PCR
RNA expression was indicated using real time RT-PCR assay to measure
HAS2 and PGR mRNA. The primers, used in PCR are presented in Table 1.
Internal control gene (GAPDH) was used for normalization of the result.
Total RNA of HAS2 and progesterone receptor were extracted using RNeasy
Micro kit from cumulus cell. The cDNA were synthesized, and primer
design was done. Real time- polymerase chain reaction (RT-PCR) was
performed for gene expression analysis.
process of primer for Real-time RT PCR
Statistical analysis
All statistical analysis was done using SPSS software version 22 and
independent sample T-test. P values≤0.05 were considered statistically
significant.
Results
Effect of Prunus cerasus on HAS2 and PGR expression in cumulus cells
Expression of HAS2 and PGR mRNA in cumulus cells, isolated from mice
oocyte, was assessed by quantitative real time PCR. There was a
significant increase in HAS2 mRNA in the experimental group compared to
the control (Figure 1). However, the expression level of PGR gene in experimental group was increased compared to the control group (Figure 2).
Effect of prunus cerasus on fertilization rate
IVF was performed in both control and experimental groups, and 100
oocytes were used per group. Counting the number of embryos revealed
that in the control group which was not received Prunus cerasus, 77% of
oocytes were developed into embryos, while in the experimental group
receiving Prunus cerasus for 4 weeks, 89% of oocytes were developed into
embryos (P≤0.0001). Developed embryos in the experimental group are
shown in Figure 3.
Discussion
The results revealed that the addition of Prunus cerasus to laboratory
diet resulted in increase of expression of HAS2 and PGR gene in cumulus
cells and fertility success rate. Since Prunus cerasus contain
significant levels of anthocyanins, strong antioxidant activity can
influence fertility.10
Significant increase in the expression of HAS2 and PGR gene in the case
group compared to the control group could be due to the loss of
oxidative stress produced by antioxidant property of Prunus cerasus,
which can also be justified for increasing fertility rate. Follicular
fluid contains a lot of antioxidants that protect oocyte against free
radicals. The disorder in pro oxidant/antioxidant system in follicular
fluid can damage oocyte′s DNA, and cytoskeleton of membrane can lead to
impaired fertility.30,31 Use of antioxidant rich food prevents progression of diseases related to oxidative stress.32
Free radicals that are indication of oxidative stress in many diseases
are highly reactive, toxic, and short-lived molecules that hurt DNA,
protein, lipid, and carbohydrates within the tissue.32
Oxidative stress specially affects microenvironment of oocyte, which
has a detrimental influence on follicular development, ovulation, oocyte
quality, implantation, and early embryonic development.33In the study of Damar and Eksi and other studies, anthocyanin and antioxidant capacity of Prunus cerasus is evidenced.11,34,35 Showell MG et al.’s study on the effects of antioxidants on fertility was contradictory, needing further investigation.32 Unlike the present study, Jozwick et al. did not report any relationship between free radical marker and pregnancy rate.36
The results of the present study is in agreement with Polak et al. who
state that the concentration of antioxidant in peritoneal fluid of
patients with unexplained infertility was significantly lower than the
its concentration in fertile patients.37
Study of Agawarl et al is in line with the results of current study
demonstrating that pregnancy rates in patients undergoing IVF, using
antioxidant-supplemented media, were higher than standard media without
antioxidants.32
Since studies have shown that antioxidants affect oocyte quality, high
degree of expansion of cumulus cells is directly related to oocyte
quality and expression of HAS2 and PGR gene is necessary for cumulus
expansion. According to the results it could be postulated, it is
antioxidant capacity of prunus cerasus that resulted in increased gene
expression and fertilization rate.
Conclusion
It is concluded that Prunus cerasus consumption improves fertility rate
by increasing progesterone receptor and HAS2 activity in cumulus cells.
Acknowledgments
This article is resulted from a research proposal leading to the thesis
of Fatemeh Namvar, MSc student of anatomical sciences and was approved
by research deputy of Tabriz University of Medical Sciences, Tabriz,
Iran.
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