Vitamin D3 regulates mtDNA copy number, mitochondrial biogenesis and membrane integrity in granulosa cells through mitogen activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK1/2) pathway in a mouse model of polycystic ovary syndrome

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women. Menstrual dysfunction, anovulation, hyperandrogenism, hirsutism and polycystic ovaries are regarded as signs and symptoms of PCOS. Hyperandrogenism intensify follicular atresia by apoptosis in granulosa cells. So, apoptosis and oxidative stress can disturb follicular growth in PCOS women. Since apoptosis are regulated by mitochondria, these organelles may be affected in PCOS by high rate of follicular atresia. Therefore, granulosa mitochondrial function disturbance causes some problems in oocyte function and might decrease fertility and pregnancy rate. The aim of study was investigation the effect of vitamin D3 on mtDNA copy number, mitochondrial biogenesis and membrane integrity via the MAPK pathway in granulosa cells of PCOS mouse model.Objective: The effect of vitamin D3 on mtDNA copy number, mitochondrial biogenesis and membrane integrityin granulosa cells derived from mouse model of poly cystic ovarian syndrome (PCOS) via mitogen-activated protein kinase (MAPK-ERK1/2).Materials and Methods: In this study, the PCOS model was triggered by injection of dehydroepiandrosterone (DHEA). Then, isolated granulosa cells were treated with vitamin D3, MAPK activator and inhibitor. Granulosa cells isolated from PCOS ovaries after identification by FSHR (granulosa cell marker) and CD45 (leuckocyte marker) were cultured in six groups: (1) granulosa cells treated with vitamin D3 (100 nM for 24 hr); (2) granulosa cells treated with MAPK activator (10 μM for 4 hr); (3) granulosa cells treated with MAPK inhibitor (10 μM for 4 hr); (4) granulosa cells treated with vitamin D3 and MAPK inhibitor; (5) granulosa cells treated with MAPK inhibitor and MAPK activator; (6) non-treated granulosa cells (control group). Mitochondrial biogenesis gene expression was compared between different groups using real-time PCR. mtDNA copy number was investigated by qRT-PCR. Mitochondrial membrane integrity was evaluated by transmission electrone microscopy (TEM).Results: Mitochondrial biogenesis (TFAM, NRF2) were downregulated in granulosa cells of PCOS mice when compared to normal mice, but treatment with vitamin D3 and MAPK activator increased mRNA expression levels of these genes. To evaluate the alterations of mitochondrial structure, transmission electron microscopy was used. Most of the mitochondria in PCOS group without any treatments and in PCOS group that treated with vitamin D3 and MAPK inhibitor and also the group that treated with MAPK activator and MAPK inhibitor were spherical with almost no cristae. Our results showed that in PCOS group that treated with vitamin D3 and MAPK activator, the mtDNA copy number increased significantly in comparison to control group (non-treated PCOS group) but in PCOS group that treated with vitamin D3 and MAPK inhibitor and also the group that treated with MAPK activator and MAPK inhibitor the mtDNA copy number decreased.Conclusion: This study suggests that vitamin D3 improve mtDNA copy number, mitochondrial biogenesis and membrane integrity through MAPK pathway in granulosa cells of PCOS mice that may improve follicular development and oocyte quality.

Keywords:

MAPK-ERK1/2 pathway , Granulosa cell , Polycystic ovary syndrome , Mitochondrial biogenesis , mtDNA , Mitochondrial membrane integrity , Vitamin D3