Effect of Vitamin D3 on Mitochondrial Biogenesis in Granulosa Cells Derived from Polycystic Ovary Syndrome

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:

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 mtDNA copy number, quantitative polymerase-chain-reaction (qRT-PCR) was used. Forward primer and reverse primer were used to analyze mtDNA. SYBR Green I Master Mix (10 μl) (Sigma, Austria), which contains 10 pmol of reverse primer and 10 pmol of forward primer was mixed with DNA (10 ng). The qPCR set-up consisted of 4 segments: 50°C for 2 minutes, 95°C for 10 seconds followed by 40 cycles of denaturation at 95°C for 5 seconds, annealing at 59°C for 35 seconds, and extension at 72°C for 1 minutes. For each qPCR reaction, the copy number of the mtDNA and the threshold cycle number (Ct) of the β-actin gene were measured. The runs were replicated at least two times, and the normalization was performed against the housekeeping gene, β-actin. For the quantification of the mtDNA copy number, the double delta Ct analysis was applied.

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 (19) for three independent biological replicates. Statistical significance between different groups was evaluated and analyzed by on-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The level of statistical significance was set at P<0.05. The SPSS software (version 21.0) was utilized for the data analysis.

In the PCOS group induced by DHEA, the serum level of estradiol and LH were higher when compared to control and vehicle groups, respectively (Table 1). The lower amount of FSH serum was detected in the PCOS group versus the control group and vehicle group due to estradiol negative feedback. The ratio of LH/FSH was significantly increased in the PCOS group in comparison to the vehicle group (Table 1). The estrous cycle was irregular in the PCOS group and ultimately stopped, whereas, in the control and vehicle group, normal cycles (nearly 5-7 days) continued as normal. Using mice in the control and vehicle groups that were just in the estrous cycle to exclude the influence of the estrous cycles on other measurements.

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 (Fig .1A). Due to the seizure of the estrus cycle, no corpus luteum was detected in the PCOS group (Fig .1B).

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 (Fig .2). Thus, for the treatment of granulosa cells, vitamin D3 (100 nM) was used for 24 hours. Subsequently, RNA was extracted, and Reverse transcription- polymerase chain reaction (RT-PCR) performed to measure the expression of mitochondrial biogenesis gene (TFAM) in different groups. Vitamin D3 increased the expression of TFAM in the PCOS group (Fig .3) by 5-fold compared to the PCOS group without any vitamin D3 treatment. This indicates that vitamin D3 might stimulate mitochondrial biogenesis in PCOS-induced granulosa cells.

For the analysis of the mtDNA, qPCR was performed. Our results revealed that in the PCOS group treated with vitamin D3, the mtDNA copy number increased significantly in comparison to the non-treated PCOS group (Fig .4). Data analysis by the quartile distribution of mtDNA copy number in the non-treated PCOS group showed an association between mtDNA copy number and PCOS risk.

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 (Fig .5).