Effects of Saffron (Crocus sativus L.) Aqueous Extract
on In vitro Maturation, Fertilization and Embryo
Development of Mouse Oocytes
Lower pregnancy rates of
Materials and Methods:
In this experimental study ,cumulus-oocyte complexes (COCs) were collected from 6-8 weeks old novel medical research institute (NMRI) female mice ovaries. COCs were cultured in IVM medium supplemented with 0 (control), 5, 10, 20 and 40 µg/ml of SAE in 5% CO2 at 37℃. The rates of maturation, fertilization and development were recorded. ANOVA and Duncan's protected least significant test, using the SAS program was applied for all statistical analysis.
The maturation rate was significantly higher in all groups treated with different
concentrations of SAE compared with the control group (p<0.05). However, the lower
concentrations of SAE (10 and 5 µg/ml) in maturation medium respectively increased the
fertilization rate of oocytes and
The results of this study indicate that lower concentrations of SAE are more appropriate to be added to maturation medium when compared with other experimental and control groups. Generally, we conclude that addition of appropriate amounts of natural extracts such as SAE to maturation medium improves oocyte maturation and embryo development.
Identification of the optimal condition is the
most important subject for IVM techniques used
in basic agricultural and biotechnology research.
Embryo physiology and viability are affected by
the culture conditions; however nuclear and/or
cytoplasmic maturation are not supported properly
by current culture systems (7). Additionally,
unfavorable media conditions in mammals leads
to declines in developmental competence. Oocyte
viability is reduced by the effects of heat stress,
oxygen concentration and glucose content (8,9).
It has been shown that the therapeutic properties
of plants are due to their phytochemicals,
which have antioxidative effects (12).
Volatile factors (safranal), bitter principles (picrocrocin) and dye materials (crocetin and its glycoside, crocin) (14) are agents of the therapeutic effects of saffron. Saffron or its active constituents have demonstrated anticonvulsant (15), antidepressant (16), anti-inflammatory, antinociceptive (17) and antitumor activities (18, 19). Radical scavenger effects as well as learning and memory-improving properties (20, 21) and promotion of the diffusion of oxygen in different tissues (14) have also been reported. In addition, the carotenoids components of saffron, known as biological antioxidants, play important roles in human health via the protection of cells and tissues from damaging effects of free radicals and singlet oxygen (22, 23) Crocin and crocetin, two main chemical components of saffron, protect cells from oxidative stress by scavenging free radicals such as superoxides (24). Since antioxidants influence oocyte maturation capacity and by considering the ability of saffron to clean free radicals, this study evaluates the effects of saffron aqueous extract (SAE) on IVM and subsequent embryo development of mouse oocytes.
Materials and Methods
Unless indicated, all chemicals were purchased from Sigma,Germany. All procedures performed on animals received the prior approval of the Ethics Board at Royan Institute.
Saffron stigmas were collected from Ghaen (Khorasan Razavi Province, Northeast Iran). The plant was authenticated and voucher specimen coded 408 was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran.
Preparation of the plant extract
About 10 g of stigmas were ground to a powder and dried in the shade at room temperature. Dried stigmas were decocted in water for 30 minutes. Thereafter, the extract was filtered and concentrated using a rotary evaporator apparatus (Heidolph, Germany). The final weight of the crude extract was 2 g. The SAE was maintained at 4℃ throughout the experiments. Before adding SAE to maturation medium, it was filtered by 0.22 µm filters.
Collection of immature cumulus-oocyte complexes
Oocytes were obtained from 6-8 week old novel medical research institute (NMRI) female mice (purchased from Pasture Institute, Tehran, Iran). Animals were kept under controlled conditions (12 hours light/12 hours dark) and fed with water and pellets ad libitum. They were killed by cervical dislocation and their ovaries transferred into dissecting medium that contained minimum essential medium (MEM-α) supplemented with 5% fetal bovine serum (FBS), 100 IU penicillin and 100 IU streptomycin. Germinal vesicle-stage oocytes (GV) of the ovarian follicles were released by puncturing with a 26-gauge sterile needle under a stereomicroscope and acquired oocytes were used for the IVM procedure.
In vitro maturation
IVM medium consisted of MEM-α supplemented
with 100 IU penicillin, 100 IU streptomycin, 5%
FBS, 7.5 IU/ml recombinant human follicular stimulating
hormone (rhFSH; Organon, Holand) and
100 IU/ml human chorionic gonadotrophin (hCG)
Organon, Holand(. Lethal dose (LD50) values of
aqueous stigma extract were reported to be 6.67 g/
kg in rats (16). Therefore, the doses employed in the
present study (5, 10, 20 and 40 µg/ml) were much
lower than the reported LD50. Different concentrations
of SAE (0, 5, 10, 20 and 40 µg/ml) were added
to the maturation medium. The 10-15 COCs were
transferred to 25 µl drops that were covered with
mineral oil and cultured for 16-18 hours at 37℃ and
5% CO2. At various intervals from the onset of incubation,
oocytes were observed by invert microscope
(Nikon,Japan) and observation of nucleus morphological
changes [GV and germinal vesicle break
down (GVBD)] or the extrusion of first polar body
(Metaphase II: MII) were used as the criterion for
nuclear maturation of GV-stage oocytes. Oocytes
with extruded first polar body (PB1) were used for
IVF and in vitro development
Epididymal sperm suspentions were prepared
from 6-8 weeks old adult NMRI male mice and
incubated for 1 hour in IVF medium to ensure
ANOVA and Duncan's protected least significant test, using SAS program (Statistical Analysis System version 1.9) was used for all statistical analyses. All percentages of values were subjected to arcsine transformation prior to analysis. All data was expressed based on mean ± SEM. A probability of p<0.05 was considered statistically significant.
In this study, oocytes were cultured for 18 hours in IVM medium supplemented with various concentrations of SAE. As shown in table 1, the percentage of metaphase oocytes significantly increased in all four experimental groups compared to the control group (p<0.05). The addition of 10 µg/ml SAE extract to maturation medium significantly increased the fertilization rate compared to the group treated with 5 µg/ml and the control groups (p<0.05; Table 2,).
Addition of 5 and 40 µg/ml SAE extract during IVM significantly increased the number of 2cell embryos compared to the control group. The addition of 5µg/ml SAE extract to maturation medium significantly increased the percentage of blastocysts compared to the group treated with 40 µg/ ml and the control group. Blastocyst formation decreased when 40 µg/ml SAE extract was added to the maturation medium. As shown in table 2, no significant difference was observed between groups treated with 40 µg/ml compared to the control group.
|Different concentrations (µg/ml) of SAE||Maturation stage of oocytes (%)|
|Total COCs||GV (mean ± SEM)||GVBD (mean ± SEM)||MII (mean ± SEM)|
|0||76||14.7 ± 2.3a||24.8 ± 1.6a||61.5 ± 2.1b|
|5||94||10.8 ± 1.4a, b||13.0 ± 2.2b||73.4 ± 3.1a|
|10||96||11.7 ± 1.4a, b||17.0 ± 2.6b||70.6 ± 2.6a|
|20||102||8.3 ± 0.4b||14.1 ± 1.9b||74.0 ± 2.4a|
|40||98||8.7 ± 0.6b||13.2 ± 2.0b||75.5 ± 2.3a|
Percentage of metaphase
|Different concentrations (µg/ml) of SAE||No. of matured oocytes||Fertilized ova with 2PN (%)||24 hours||96 hours|
|0||59||71.06 ± 2.6b||69.1 ± 3.6b||20.0 ± 2.7bb|
|5||86||71.84 ± 1.8b||78.5 ± 1.3a||29.8 ± 2.3a|
|10||87||78.85 ± 0.9a||75.2 ± 2.6a, b||28.7 ± 2.5a, b|
|20||90||74.28 ± 2.4a, b||74.2 ± 2.7a, b||27.4 ± 4.8a, b|
|40||93||75.04 ± 1.2a, b||77.2 ± 1.9a||20.5 ± 1.7b|
Percentage of fertilized ova with 2PN (%) and percentage of embryos expressed as mean
In this study the effect of saffron supplementation
during IVM of mouse oocytes was assessed.
The major findings of this research showed that
the addition of all experimental amounts of SAE
to the maturation medium improved maturation
rate. While lower concentrations increased both
IVF and IVD. Nair et al. observed that saffron increased
the intracellular levels of reduced glutathione
and suggested that saffron had antioxidant
activity (25). It was demonstrated that the addition
of antioxidants such as β-mercaptoethanol
(26), cysteamine (27) and glutathione (28) to
maturation medium positively influenced subsequent
embryo development of mouse oocytes.
It has been shown that saffron, a medicinal plant
(29), exhibited strong radical scavenging properties.
Crocin and crocetin derivatives have been
reported to be the most abundant constituents
of saffron (14, 18, 30). Saffron components protected
cells by binding to nucleic acids, proteins
and lipids (31). Crocin, a bioactive constituent
of Crocus sativus, exhibited significant radical
scavenging activity and thus, antioxidative activity
(32). Previous studies have shown that crocin
To our knowledge, the present study is the first to demonstrate the beneficial effect of SAE supplementation of IVM medium on early mouse embryo development. The improving effect of natural extract on IVM has been shown in previous studies (43). The results of this research and previous research (43) indicated that the addition of those plant extracts to maturation medium as natural antioxidants was safe and possibly had lower side effects. According to findings of this experiment and previous reports (29), saffron may be regarded as a valuable plant source for use in traditional medicine.
Supplementation of IVM media with optimum concentrations of antioxidants such as SAE may help increase the numbers of blastocysts obtained from the IVM-IVD procedure. The improved effects might be dependent on the SAE concentrations in maturation medium.
We are grateful to Mr. M. Kamalinejad, Department of Pharmacognosy, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran for provision of SAE. The authors would like to thank Royan Institute for financial supporting of this project .
There is no conflict of interest in this article.