Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women (
Since oocyte quality is a crucial factor for conception in
PCOS patients and that depends on mitochondrial function and structure, prescription of an appropriate medication may improve fertility rate as a consequence of improved oocyte quality (
This is an experimental study that the effect of vitamin D3 on mitochondrial biogenesis in a PCOS mouse model was investigated. Androgen excess and other symptoms of PCOS were induced by the injection of DHEA (Sigma, Austria), 6 mg/100 g body weight. DHEA was dissolved in 95% ethanol (0.01 mL) and mixed with sesame oil (0.09 mL). Subsequently, it was injected subcutaneously into female BALB/C mice (25 days old) for 20 consecutive days before reaching puberty (PCOS group, n=20). As a vehicle control, 0.1 mL of sesame oil (Sigma, Austria) and 0.01 mL of 95% ethanol (Sigma, Austria) were injected into another group of the same mouse strain for 20 consecutive days (n=20). A Control group of the same mouse strain without any treatment was also considered (n=20). The mice were kept at room temperature (25 ± 1°C, RT), with enough food and water, and under diurnal modulation by daily light. All the animal trials were performed in agreement with the Institutional Animal Care Committee of Iran University of Medical Sciences and Health Services for animal welfare. (ethics code: IR.IUMS.REC 1396.29969). The weight changes in mice were measured every day. Vaginal smears were also taken every day over the 20-day course of treatment. The mice were sacrificed by cervical dislocation. For histological assessments, the ovaries were subsequently fixed with 10% formalin (Merck, Germany). Next, 5-μm sections were made with a microtome, and the sections were immersed in xylene and ethanol with different grades for deparaffinization and rehydration, respectively (Merck, Germany). The ovaries were then stained with hematoxylin and eosin (DAKO, USA). For morphology assessment, the ovaries assessed by a Nikon microscope (Nikon, Japan), and photographs were taken.
For the analysis of sex hormones, cardiac blood samples were collected using needles. Blood serum was subsequently separated using a centrifuge machine at (300 rpm, 4°C, 10 minutes) and follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2) and progesterone levels were measured by an ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's guidelines.
The ovaries of 45-day BALB/C mice (DHEA-reated and the vehicle group) were removed after the mice were sacrificed via cervical dislocation. For aspiration of the follicles, 25-gauge needles were used, and the follicles were aspirated in a solution made of phosphate buffer saline (PBS) and 1.0% bovine serum albumin (BSA) (Invitrogen, USA). 70-μm cell strainers (BD Falcon, MA, USA) were used to isolate granulosa cells from the other cells and tissues. Subsequently, granulosa cells were separated from the oocytes with a 40-μm cell strainer (BD Falcon, MA, USA). Blood cell contamination was removed by RBC lysis buffer after centrifugation at 1000 rpm (4°C, 10 minutes). Then, the pellet was mixed with phenol red-free DMEM/F12 medium containing 10% fetal bovine serum (Sigma, Austria). The medium was centrifuged at 1000 rpm (4°C, 10 minutes). Next, the pellet was removed and transferred to cell culture dishes containing DMEM-F12, 10% FBS (Sigma, Austria), 100 mg/ mL streptomycin (Sigma, Austria), 100 IU/mL penicillin (Sigma, Austria), 2 mM glutamine (Sigma, Austria), 1 mM sodium pyruvate (Sigma, Austria). The culture dishes were then incubated at 37°C, with 5% CO2 and 95% humidity.
To identify granulosa cells, an antibody against FSHR, a specific marker of granulosa cells, was used. Affinitypurified rabbit anti-follicle stimulating hormone receptor (FSHR) polyclonal antibody was purchased from antibodies-online (ABIN1872743). First, the cells were spread on a slide using a cytospin centrifugation device, and the slides were then immersed in a cold normal buffered formalin (NBF) solution to be fixed. Subsequently, the cells were washed with PBS and blocked using PBSTriton/BSA. Afterward, the primary FSHR antibody was added to granulosa cells overnight. The following morning the cells were washed three times with PBS and were subsequently treated with the secondary FSHR antibody for 30 minutes. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Preparations were washed in PBS before mounting on glass slides. Slides were viewed on an epifluorescence microscope and captured with a digital camera.
The treatment groups for granulosa cells were as follows:
PCOS granulosa cells treated with vitamin D3 (100
nM) for 24 hours (
PCOS granulosa cells without any treatments
Non-PCOS granulosa cells (control group)
The Trizol reagent (Sigma, Austria) was used to extract the total RNA of granulosa cells in all groups. Then, chloroform was added to the mixture of granulosa cells and the Trizol reagent. Afterward, the mixture was centrifugated at 1000 rpm (4 C, 10 minutes). The upper phase containing the total RNA was collected. Next, the total RNA was washed with 75% ethanol, allowed to air dry, and then reconstituted in diethylpyrocarbonate (DEPC) water. Using a cDNA synthesis kit (Thermo Scientific, USA), the total RNA was reverse-transcribed according to the manufacturer’s guideline. In summary, a mixture of the random hexamer, first-strand buffer (all from Fermentas), DNase- (Fermentas Inc, MD, USA) treated RNA, RiboLockTM RNase inhibitor, dNTP Mix, Dithiothreitol (0.1M) and SuperScriptTM II Reverse Transcriptase was made for reverse transcription of each sample. The thermocycler (company) was set at 25°C for 10 minutes, 43°C for 40 minutes, and 75°C for 15 minutes. Quantitative PCR was performed using 1 μl of cDNA in a reaction consisting of ROXTM Reference Dye, SYBR Premix EX TaqTM (Takara, Japan), and 1 μl of the desired primer. The β-actin gene was utilized as a housekeeping gene. The reactions were amplified with StepOne™ Real- Time PCR System (Applied Biosystems, MA, USA) as following: denaturation at 95 °C for 10 seconds, 35 cycles of amplification (95°C for 5 seconds and 60°C for 30 seconds), separation stage at 95°C for 15 seconds, 60°C for 1 minutes, and 95°C for 15 seconds. Using oligo 7.60 software to design primers. The TFAM forward primer was CCG AGC TCC TCC TCC TTT GC and the TFAM reverse primer was CCT ACA ACG CAG CGA CCG AG.
For the measurement of the
Isolated granulosa cells were fixed using 2.5% glutaraldehyde in PBS and then treated with 1.0% osmium tetroxide in the same buffer for the post-fixation procedure. For performing the dehydration process, ethanol and propylene oxide were used. Then inserted in epoxy resin, and sectioned. Using ethanolic uranyl acetate to contrast the sections and lead citrate and observed under a transmission electron microscope (Zeiss LEO 906 (TEM), 100 kV, Germany).
The data in this experiment are expressed as the means
and standard error of the mean (
In the PCOS group induced by DHEA, the serum level
of estradiol and LH were higher when compared to control and vehicle groups, respectively (
|Estradiol (pg/mL)||132 ± 9.10||142 ± 8.52||3786 ± 13.1**|
|FSH (IU/L)||6. 59 ± 0.82||6.84 ± 0.29||4.11 ± 0.64|
|LH (IU/L)||5.60 ± 0.11||6.13 ± 0.38||18.58 ± 0.82*|
|LH/FSH (IU/L)||0.84 ± 0.13||0.89 ± 1.31||4.52 ± 1.28**|
|Progesterone (pg/mL)||3.647 ± 0.69||3.268 ± 0.54||2.369 ± 0.19|
Data are presented as mean ± SD. *; P<0.05, **; P<0.005, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, DHEA; dehydroepiandrosterone.
Upon H&E staining, the specimen was analyzed under the light microscope. Normal follicles were detected at
various developmental stages in the vehicle group. Corpus luteum was also observed in the control group, which
was an indicator of normal ovulation (
Histological assessment of ovaries. A. Follicles of normal ovaries represented follicles at different stages, and corpus luteum (CL) and B. Ovaries of the polycystic ovary syndrome (PCOS) model induced by dehydroepiandrosterone (DHEA) revealed antral and pre-antral follicles and some cysts (FC) were observed in H&E staining. No corpus luteum was observed in the PCOS ovary .Scale bar: 50 μm.
To assure that the cells being experimented on granulosa cells, a granulosa cell antibody was used against
FSHR. Photograph analysis showed that the target cells
were stained with this antibody, indicating that they were
granulosa cells (
Follicle-stimulating hormone receptor (FSHR) (specific markers of granulosa cells) was investigated. The FSHR expression in isolated granulosa cells (green) was observed. Nuclei (blue) were stained by 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 100 μm.
The expression of TFAM (mitochondrial biogenesis gene) in cultured granulosa cells of DHEA-induced PCOS BALB/C mice was compared between three groups. Granulosa cells were pre-incubated in the serumfree medium in the presence or absence of vitamin D3. The expression of the mitochondrial biogenesis gene was upregulated in the vitamin D3 group. It is also revealed that the gene expression was declined in PCOS granulosa cells in comparison with non-PCOS healthy granulosa cells (control group), ***; P<0.05, DHEA; Dehydroepiandrosterone, and PCOS; Polycystic ovary syndrome.
For the analysis of the
The mitochondrial DNA copy number (mtDNA) in cultured granulosa cells of DHEA-induced PCOS BALB/C mice was compared between three groups. Granulosa cells were pre-incubated in the serum-free medium in the presence or absence of vitamin D3. The mitochondrial DNA copy number was significantly increased in the vitamin D3 group in comparison with the non-treated PCOS group (**; P<0.05). It is also revealed that the mitochondrial DNA copy number was declined in the non-treated PCOS granulosa cells in comparison with the non-PCOS healthy granulosa cells (control group). DHEA; Dehydroepiandrosterone, PCOS; Polycystic ovary syndrome.
For the evaluation of the alterations of the mitochondria structure, transmission electron microscopy was
employed. Most of the mitochondria in the PCOS group
without any treatment were spherical, with almost no cristae; however, in the PCOS group treated with vitamin D3
as well as in the non- PCOS group (control group) intact inner and outer membrane and a clear intermembrane
space was observed (
Mitochondria membrane structure (TEM).
The present study demonstrated that vitamin D3 affected
Disrupted ovulation and oocyte quality induced by hyperandrogenism can be improved by different treatments
such as metformin and spironolactone (
For the assessment of the effect of vitamin D3 on mitochondrial biogenesis, isolated granulosa cells from PCOS
ovaries were treated with vitamin D3. Mitochondrial membrane integrity and alteration in mtDNA copy numbers were
also evaluated. It has been demonstrated that mitochondria,
as the powerhouse of the cell, are of importance for optimum oocyte quality and fertilization. Poor oocyte quality
and subsequent embryonic development could be attributed to mitochondrial dysfunction (
In the present study, we demonstrated that mitochondrial biogenesis could be upregulated after 24 hours of treatment with vitamin D3. The findings showed that vitamin
D3, as a supplementation, improves the main mitochondrial biogenesis marker (
In this study, for the first time, we have shown the vitamin D3 effect on
In line with this finding, researchers showed reduced
mtDNA copy number in the PCOS patients (
According to our results, mtDNA copy number, the biogenesis might be affected by vitamin D3 in PCOS granulosa cells. We nominate that mitochondrial biogenesis genes expression might be increased by vitamin D3. Therefore, vitamin D3 can have a significant role in the alleviation of mitochondria and follicular damages in PCOS ovaries. However, extensive studies are needed to determine the optimal dose and duration of treatment with vitamin D3 in PCOS women.