PCOS is a major cause of anovulatory infertility in women of childbearing age; however, its pathogenesis is unclear, and there are no clinically effective therapeutic agents. Recently, BPA has been demonstrated to impair reproductive function and is potentially involved in PCOS development. In addition, vitamin D deficiency may be involved in PCOS pathogenesis [19]. Ovulatory function improves in some patients after vitamin D supplementation; however, the exact mechanism is unclear. In our study, BPA inhibited KGN cell viability, induced apoptosis, significantly increased ROS production, and significantly decreased mitochondrial function. BPA-induced apoptosis in KGN cells resembled the degenerative changes observed in ovarian granulosa cells in PCOS patients, confirming that BPA considerably impairs mitochondrial function in these cells. After intervention with vitamin D, the apoptosis rate of KGN cells decreased, mitochondrial function improved considerably, and the expression of molecules in the mitochondrial Cyt c apoptotic pathway significantly downregulated, confirming that vitamin D exerts a protective effect on KGN cells by improving mitochondrial function and inhibiting the mitochondrial Cyt c apoptotic pathway. For the first time, we found that 1,25(OH)2VD3 intervention improved the mitochondrial function and apoptosis of ovarian granulosa cells under the influence of BPA. These findings provide basic theoretical support for the clinical phenomenon by which vitamin D improves ovulatory function in PCOS patients.
In addition to their key role in cellular energy metabolism, mitochondria regulate apoptosis, and the mitochondrial pathway is the most important part of the endogenous apoptosis pathway. The regulatory roles of mitochondria are potentially linked to ROS production. Mitochondria are not only a source of ROS but also a target of oxygen radicals, which are associated with mitochondrial DNA mutations, cellular senescence, and cell death and lead to the release of Cyt c and other pro-apoptotic proteins into the cytoplasm [12]. Excess ROS can reduce mitochondrial membrane potential, alter mitochondrial membrane permeability, and ultimately contribute to apoptosis by releasing apoptotic factors [13]. In vitro, BPA can directly affect human sperm activity by inducing mitochondrial dysfunction [14]. After BPA treatment, we found that KGN cell viability decreased and the apoptosis rate and ROS production levels significantly increased.
Bcl-2 family proteins control the permeability of the outer mitochondrial membrane; these proteins are pro-apoptotic and anti-apoptotic (e.g., Bax and Bcl-2, respectively) and play a key role in apoptosis [21]. The endogenous mitochondrial Cyt c pathway is critical for apoptotic cell death. The insertion of Bax protein molecules into the mitochondrial membrane causes it to become porous and permeable, and Cyt c is released into the cytoplasm, where it binds to Apaf-1, which is increased in expression. Activated caspase-9 then activates the downstream protein caspase-3, and the cell undergoes programmed death. However, Bcl-2 binding to Bax inhibits the Bax-induced decrease in mitochondrial transmembrane potential, maintaining the integrity of the mitochondrial membrane and antagonizing apoptosis [21]. Our results indicate that BPA treatment significantly downregulated mitochondrial transmembrane potential changes in KGN cells, with a significant upregulation of Bax, Cyt c, Apaf-1, caspase-9, and caspase-3 expression and a significant downregulation of Bcl-2 expression. 1,25(OH)2VD3 treatment ameliorated these changes and exhibited antioxidant and anti-apoptotic protective effects, consistent with the findings of previous studies [26].
PGC-1α is a critical mitochondrial biogenesis gene linked to oxidative stress, inflammation, and apoptosis [22]. It protects against oxidized LDL and luteinizing hormone-induced granulosa cell injury via the ROS-p38 pathway [23]. In this study, the expression of PGC-1α was also assessed to clarify mitochondrial function in KGN cells. PGC-1α was significantly downregulated, and the mitochondrial structure was clearly disrupted, indicating that BPA likely caused KGN cell apoptosis by affecting the mitochondrial function. Mitochondrial function improved after 1,25(OH)2VD3 treatment. These results further confirm that 1,25(OH)2VD3 exerts a protective effect by improving the mitochondrial function of KGN cells and inhibiting the endogenous mitochondrial Cyt c pathway.
One study demonstrated that 1,25(OH)2VD3 regulates the effects of sublethal concentrations of BPA on mitochondrial function in rat ovarian granulosa cells by activating the PI3K-Akt pathway [16]. BPA dose-dependently decreased cell viability, increased ROS, negatively regulated SOD expression levels, decreased mitochondrial DNA deletions, and significantly increased mitochondrial ATP production after 1,25(OH)2VD3 pretreatment, which is consistent with the results of this study. In contrast, no significant effect of BPA treatment on the expression of PGC-1α was observed, which contradicts our findings. This may be because the cell species and drug concentrations used were different between the two studies.
This study has some limitations. First, we could not test our hypothesis in human ovarian granulosa cells due to technical and ethical issues. Second, animal experiments are needed to further test our hypothesis. Third, some studies have shown that there may be an association between BPA and vitamin D [28], which was not explored in this study. These shortcomings should be considered in future studies.
In summary, in this study, BPA induced apoptosis in KGN cells by damaging the mitochondria and activating the mitochondrial Cyt c pathway. 1,25(OH)2VD3 pretreatment improved mitochondrial function and significantly decreased the apoptosis rate. These findings suggest that 1,25(OH)2VD3 may alleviate BPA-induced apoptosis in KGN cells by improving mitochondrial function and inhibiting the mitochondrial Cyt c pathway. Our hypothesis that vitamin D improves the mitochondrial function of ovarian granulosa cells in PCOS patients was initially validated at the cellular and molecular levels. Thus, we should pay attention to vitamin D levels in PCOS patients of childbearing age.