The Effects of Sodium Selenite on Mitochondrial DNA Copy
Number and Reactive Oxygen Species Levels of In Vitro
Matured Mouse Oocytes
The aim of present study is to determine the effects of supplementation of oocyte maturation medium with sodium selenite (SS) on oocyte mitochondrial DNA (mtDNA) copy number and reactive oxygen species (ROS) levels.
Materials and Methods
In this experimental study germinal vesicle (GV), metaphase I (MI), and metaphase II (MII)
stage oocytes were recovered from 6-8 week old female mice after superovulation. Some of the GV oocytes were
cultured and matured in the presence and absence of SS. Then
The maturation rate of GV oocytes to the MII stage significantly increased in the SS supplemented group
(79.25%) compared to the control group (72.46%, P<0.05). The intensity of mitochondrial staining was not different
among the studied groups, whereas the mitochondria distribution in the cytoplasm of the IVM oocytes showed some
aggregation pattern. The
SS increased oocyte mtDNA copy number by decreasing oxidative stress. SS had an association with better oocyte developmental competence.
Mitochondria are multifunctional organelles with criticalfunctions in ATP production, calcium homeostasis, andcell apoptosis (1). The localization and presence of theseorganelles are critical for successful fertilization (2). Different cell types have a variety of mitochondria that havetheir own genome-mitochondrial DNA (mtDNA) (3). Thereare various reports of mtDNA copy number in oocytes fromdifferent mammalian species (3-7). It is estimated that ahuman MII oocyte contains approximately 20000 to over800,000 mtDNA (4, 5); this range is 11000-428,000 in micefertilized oocytes and early embryos (6, 7). In addition, differences exist in the copies of mtDNA, ROS levels, and integrity of the cytoskeleton between in vitro matured (IVM) and naturally collected mice oocytes (8).
Reactive oxygen species (ROS) is the byproduct of the oxidative phosphorylation chain system in mitochondria (9). Elevated levels of ROS cause oxidative stress and may lead to alterations in several redox pathways (10). The cells possess powerful enzymatic and non-enzymatic antioxidant defenses to protectagainst the damaging effects of ROS on DNA, lipids, and proteins (11). Excessive ROS or inadequate antioxidant protection within the cell results in oxidative stress. The cells can be protected against these effects by supplementation of culture media with antioxidants (12-14). Some antioxidants are synthesized by oocyte mitochondria (15). The exogenous antioxidant could improve oocyte and embryo development by decreasing ROS levels (13, 14, 16).
Selenium is a trace element present in the catalytic site
of antioxidant enzymes such as glutathione peroxidase. In
the form of sodium selenite (SS), it is used as a supplement
in culture media and protects cells from oxidative damage
(17). Abedelahi et al.
(13, 16) have demonstrated that
SS can improve the
Little is known about mtDNA copy number changes
Materials and Methods
Unless otherwise indicated, allchemicals were purchased from Sigma Aldrich (Germany). This experimental study used National Medical Research Institute (NMRI) female mice (n=48). The mice were housed in the Animal House at Tarbiat Modares University. The Ethical Committee of the Tarbiat Modares University approved this study (Ref No. 52.1637).
Germinal vesicle oocyte collection
Adult female mice 6-8 weeks old (n=38) were superovulated by intraperitoneal injection (i.p.) of 10 IU pregnant mare serum gonadotropin (PMSG, Folligon, Intervet, Australia). Female mice were killed by cervical dislocation 48 hours after the PMSG injection and the dissected ovaries were placed in a-minimal essential medium (α-MEM, Gibco, UK) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Gibco, UK). Antral follicles were punctured with needles to release the oocytes and the cumulus cells were mechanically removed. Oocytes that had a prominent germinal vesicle (GV) and clear ooplasm with 90 µm diameter were selected and collected (n=817).
Some of GV oocytes (n=778) were subjected to in vitro maturation in SS supplemented and non-supplemented groups. The other GV oocytes were analyzed by mitochondrial staining (n=10) and mtDNA copy number analysis (n=29).
In vivo metaphase I and metaphase II oocyte collection
For harvesting the ovulated
The OV-MII oocytes were analyzed for ROS concentration and mitochondrial staining using MitoTracker green (n=10) and for mtDNA copy number (n=15). These oocytes were individually stored at -80°C.
In vitro maturation
The GV oocytes were cultured in two groups, SS+ and SS-. The SS+ group (n=317 in 10 repeats) was cultured in α-MEM medium supplemented with 100 mIU/ml rFSH (Sereno, Switzerland), 10 IU/ml hCG, 10% FBS, and 10 ng/ml SS (13) under mineral oil at 37°C in 5% CO2 and air for 14 hours. The second group, or the media without SS supplementation (n=461 in 10 repeats), was considered to be the non-treated control group. After 14 hours, we morphologically assessed the oocyte maturation rate. Absence of GV within the ooplasm was used as the criteria for MI oocytes whereas extrusion of the first polar body was considered to be the criterion for MII oocytes. The matured MI and MII oocytes were classified as IVM-MI and IVMMII. These experiments were performed for at least 10 times and we assessed the collected oocytes as follows.
Visualization of the mitochondria using MitoTracker green
The presence of viable mitochondria was identified by MitoTracker green (Molecular Probes, Invitrogen, Eugene, OR, USA) staining. We prepared a stock solution of MitoTracker green at a concentration of 1 mmol in DMSO and stored the solution at -20oC. The in vitro MII oocytes from both experimental groups and in vivo collected oocytes at the GV, MI, and MII stages (n=10 for each group and developmental stage) were stained with 0.2 mmol MitoTracker green in PBS at 37oC for 10 minutes. After washing in PBS, the oocytes were mounted on glass slides and observed under fluorescent microscope at the 490 wavelength (21). Then, a micrograph of each oocyte was prepared and imported into ImageJ software (National Institutes of Health, Bethesda, MD, USA). Next, we analyzed and compared the fluorescence intensity in different groups of oocytes.
Reactive oxygen species analysis
DNA extraction from individual oocytes
We extracted DNA from completely denuded individual oocytes from all studied groups (n=15 for each developmental stage per group). A total of 10 µl of lysis solution that contained 50 mM tris-HCl (pH=8.5), 0.1 mM EDTA, 0.5% Tween-20, and 200 µg/ml proteinase K (Roche, Germany) were added to each tube followed by an overnight incubation at 55ºC. The samples were heated to 95ºC for 10 minutes to inactivate proteinase K. Each sample was used directly as template DNA for polymerase chain reaction (PCR).
We sought to identify the unique regions of the mouse
mitochondrial genome with no pseudogene in the nuclear
DNA. The entire sequence of mouse mitochondrial DNA
was obtained from NCBI (NC_005089.1). The FASTA
format of this sequence was split into 200 bp fragments
with 50 bp overlaps. These fragments were searched
against the mouse nuclear genome using NCBI Blast.
The unique regions of the mitochondrial genome that had
no duplicate in the nuclear genome were identified and
used for primer design. Specific primers (Table 1,) were
design using Primer3Plus (
|Primer code||Primer sequence (5´-3´)||Length (bp)|
Preparation of standard dilutions
In order to obtain standard curves we constructed standard DNA by cloning the PCR products. These products were amplified using the primer sets presented in Table 1 into the pTZ57R/T vector (Thermo Scientific Bio, USA). We used the MTF and MTR primers to amplify a 68 bp unique fragment of mtDNA. After electrophoresis, the amplified product was extracted from agarose gel by the ExpinTM Combo GP kit (GeneAll Biotechnology, Korea) according to the manufacturer’s protocol. The extracted product was cloned into the vector pTZ57R/T (Thermo Scientific, USA), purified, and sequenced. The recombinant plasmid was linearized and cleaned up by a GeneAll kit (General Biosystem, Korea). The product underwent spectrophotometry. The concentration of recombinant plasmid was calculated and diluted to 3×105 copies/5 µl. We prepared four serial dilutions of standard DNA with at 1/10 standard concentration. These standard dilutions were kept at 4ºC until analysis and used in real- time PCR for mtDNA copy number quantification.
Quantification of mitochondria DNA copy number using real-time polymerase chain reaction
Real-time PCR was performed to determine the total amount of mtDNA of each single oocyte in all study groups. Each reaction contained 10 µl of SYBR green master mix (Applied Biosystems, USA), 2 µl primer mix (MTF and MTR), 3 µl of sterile water, and 5 µl of oocyte DNAextract (5 µl of each total DNAsample). Each oocyte DNA extract was divided into two wells as duplicates. All real-time runs included four concentrations of serial standard dilutions in triplicate (R2≥0.99). To rule out cross contamination a "no template control" (NTC) was added to each single real-time run. The reactions were performed with an ABI 7500 instrument (Applied Biosystems, CA, USA). Each PCR reaction included an initial denaturation step of 95ºC for 10 minutes, followed by 40 cycles of 95ºC for 15 seconds, 60ºC for 30 seconds, and 72ºC for 30 seconds. A melting curve stage was included at the end of the run to confirm the absence of non-specific products and primer dimerization. The copy number of mtDNA for each oocyte was calculated from both duplicate wells.
Statistical analysis was performed using SPSS software (IBM SPSS statistics 22). All data were presented as mean ± SD. The normality of data was tested by the Kolmogorov-Smirnov test and the data of developmental rates of oocytes were compared by the t test. The mtDNA copy number and ROS level of oocytes were assessed by one-way ANOVA and Tukey’s HSD was used as the post hoc test. Statistical significance was P<0.05 for all analyses.
Maturation rate of oocytes
Table 2 summarizes the maturation rates of GV oocytes. The percent of oocytes which matured in the presence of SS were 6.85 ± 1.28 for the MI stage and 79.25 ± 0.52 for the MII stage. In the absence of SS, the maturation rate for GV oocytes was 6.36 ± 1.50 for the MI stage and 71.32 ± 3.78 for the MII stage. These rates were significantly higher in the SS supplemented group compared to the non-treated control group (P<0.05).
|Group||Sodium selenite||Total number||Number of arrested GV (mean% ± SE)||Number of MI(mean% ± SE)||Number of MII(mean% ± SE)||Number of degenerated (mean% ± SE)|
|Control||-||461||73 (16.67 ± 2.14)||32 (6.36 ± 1.50)||335 (71.32 ± 3.78)||21 (5.63 ± 1.86)|
|Experiment||+||317||38 (11.97 ± 1.54)||22 (6.85 ± 1.28)||251 (79.25 ± 0.52)*||6 (1.90 ± 0.93)|
* ; There was significant difference with the control group in the same column(P<0.05), GV; Germinal vesicle, MI; Metaphase I oocytes, and MII; Metaphase II oocytes.
We observed mitochondrial distribution of oocytes at different developmental stages in the study groups with a fluorescent microscope using MitoTracker green. The representative micrographs of these oocytes were shown in Figure 1 and 2. The mitochondrial distribution in the cytoplasm of in vivo obtained oocytes consisted of a homogenously diffused pattern (Fig .1,). There were some aggregations of mitochondria within the IVM oocytes (Fig .2,). We observed similar patterns of mitochondrial distribution in all IVM oocytes with and without SS supplementation (Fig .2A-D,).
The florescent intensities with regards to mitochondrial staining (Fig .3A,) in GV oocytes was 38.21 ± 0.40. In the presence of SS, it was 37.62 ± 1.24 for IVM-MI and 41.02 ± 0.72 for IVM-MII. In the absence of SS, this finding was 36.99 ± 1.13 for IVM-MI and 39.32 ± 1.12 for IVMMII. Fluorescence intensity for OV-MI was 39.22 ± 0.72 and 41.69 ± 2.64 for OV-MII. There was no significant difference between the groups.
Reactive oxygen species concentration
The ROS levels in all studied MII oocytes (IVM and
Mitochondrial DNA copy number
The mean mtDNA copy number in single oocytes for all
study groups is shown in Figure 4. This copy number in GV
oocytes was 127,468.68 ± 1066.61. The copy number for
OV-MI oocytes was 199,335.58 ± 28843.67, whereas for
OV-MII oocytes it was 472,881.19 ± 28822.47. The IVMMI
oocytes in the SS supplemented groups had a mtDNA
copy number of 168,244.12 ± 3759.48. IVM-MII oocytes
in the SS supplemented groups had a mean mtDNA copy
number of 349,414.2 ± 56027.22. In the group without SS
these numbers were 137,223.5 ± 4285.05 (IVM-MI) and
238,720.16 ± 8267.06 (IVM-MII). Oocytes from the SS
supplemented group had a significantly higher mtDNA
copy number compared to the group without SS (P<0.05).
All IVM oocytes had significantly lower mtDNA copy
numbers than their respected
The present study, similar to other investigations, showed
that the developmental competence of IVM oocytes was
As our data demonstrated, all IVM-MII oocytes had
higher ROS levels than
Overall, our results revealed a significantly lower mtDNA
copy number for all IVM oocytes (MI and MII) compared
Additionally, our data showed that mtDNAcopy number of oocytes increased significantly from the GV (127,468) to the MII (472,881) stages. The average mtDNA copy number determined in the present study was close to other investigations (6, 7). In contrast, no significant increase in mtDNA copy number from GV to IVM derived MII oocytes were reported in ovines and humans (4, 31). Attempts to quantify the amount of mtDNA in oocytes using PCR-based methods showed highly variable results. This discrepancy in mtDNA within the oocytes could by mainly related to technical error, different sources of oocytes (pooled or single), and different developmental stages of oocytes in several species (3-5, 32, 33).
Our results, for the first time, demonstrated that supplementation of maturation medium with SS could increase the mtDNA copy number of MI and MII oocytes compared to the non-treated group. Perhaps, the mitochondrial biogenesis in oocytes was stimulated during IVM in the presence of SS and was associated with higher developmental competence of the oocytes. In agreement with this suggestion, it has been shown that mtDNA copy number could change in response to environmental signals such as temperature, energy deprivation, nutrients, and growth factors (34).
This study showed that the mitochondrial distribution in IVM oocytes had some aggregation in comparison with in vivo obtained oocytes; however, the intensity of mitochondrial staining did not differ in these studied groups. Similarly, Stojkovic et al. (21) showed that the mitochondrial clumps became larger after IVM of oocytes. Liu et al. (35) demonstrated that the distribution of mitochondria in IVM oocytes differed slightly from that of in vivo obtained oocytes. They concluded that this different pattern resulted in the reduced developmental potential of IVM oocytes. Insufficient culture conditions might prevent mitochondrial migration within the ooplasm and affect cytoplasmic maturation (36). Thus, proper distribution of mitochondria during IVM of oocytes is critical for further development. In this regard, Kim et al. (14) have reported that treating oocytes with antioxidant could improve cytoplasmic maturation and cause morphologically uniform distribution of mitochondria and lipid droplets in the cytoplasm.
SS increases oocyte mtDNAcopy number by decreasing oxidative stress and is associated with better oocyte developmental competence.
This work was financially supported by Tarbiat Modares University as a post-Doctorate thesis and by the Iran National Science Foundation. We thank Mr. Pour Beyranvand for providing excellent technical assistance. There is no conflict of interest in this study.
N.G.; Designed and performed the experiments, analyzed data and co-wrote the paper. M.S.; Supervised the research, designed the experiments and co-wrote the paper. M.A: Performed experiments and analyzed data. All authors read and approved the final manuscript.