Effect Of Bromoacetic Acid On Oocyte Maturation In Vitro And Granulosa Cell Activity In Mouse

During the process of chlorination of drinking water,the disinfectant reacts with natural organic matter in the water to generate disinfection by-products(DBPs).Halogenated acetic acids and trihalomethanes are the two most abundant disinfection byproducts after chlorination,with bromoacetic acid(BAA)and chloroacetic acid being the common Halogenated acetic acids.In toxicity assessment tests,BAA exhibited greater cytotoxicity and genotoxicity than chloroacetic acid.However,the effects of BAA on the reproductive aspects of females and the related toxicity mechanisms have been less studied.Therefore,in this study,mouse oocytes and granulosa cells cultured in vitro were used as models to investigate the effects of BAA on oocyte maturation and granulosa cell activity in vitro by adding different concentrations of BAA to the culture medium and using immunofluorescence staining and protein immunoblotting,as a way to reveal the possible toxic effects of BAA on female fertility and its mechanisms.The main findings were as follows:1.Effects of BAA on oocyte maturation in vitro in mice(1)In this study,mouse oocytes were cultured in vitro with gradient concentrations of BAA(0,10,20 and 30 μmol/L)and the first polar body extrusion(PBE)of oocytes in each group was counted after 14 h.It was found that the PBE rate of oocytes in the 30μmol/L BAA-treated group was significantly lower than that in the control group(P <0.0001).(2)BAA induced a sustained activation of the spindle assembly checkpoint(SAC),which resulted in the stalling of oocyte meiosis at mid-meiotic division(Metaphase I,MI).Meanwhile,BAA treatment significantly increased the proportion of oocytes with abnormal spindle assembly and disordered chromosome arrangement(P < 0.001,P <0.0001)and affected the localization of actin in oocytes.This suggests that BAA may lead to failure of oocyte polar body expulsion by disrupting the dynamics of the cytoskeleton.(3)BAA altered H3K9me3 and H3K27me2 levels in oocytes and led to a significant increase in DNA methylation levels in oocytes(P < 0.05).In addition,BAA treatment of GV-phase oocytes for 8 h resulted in a significantly stronger γ-H2 AX signal in oocytes than in controls(P < 0.0001).The above results suggest that BAA may reduce the quality of oocyte maturation in vitro by altering epigenetic modifications and inducing DNA damage.2.Effect of bromoacetic acid on granulosa cell activity in mice(1)In this study,granulosa cells were co-cultured with different concentrations of BAA(0,1,5,10,15 and 20 μmol/L)for 24 h.It was found that 5 μmol/L BAA caused granulosa cells to exhibit wrinkling and rounding and a significant decrease in cell viability(P < 0.001).Meanwhile,the protein immunoblotting results showed that BAA treatment significantly reduced the PCNA level of granulocytes(P < 0.0001),indicating that BAA could reduce cell viability and inhibit the proliferation of granulocytes.(2)BAA induced a decrease in the mitochondrial membrane potential of granulosa cells(P < 0.001)by decreasing the level of anti-apoptotic protein Bcl-2(P < 0.001)and increasing the level of pro-apoptotic protein Bax(P < 0.0001),which led to apoptosis of granulosa cells.(3)BAA treatment significantly increased the level of ROS in granulosa cells(P <0.0001),and the protein immunoblotting results showed that BAA treatment significantly increased the level of γ-H2 AX in granulosa cells compared with the control group(P <0.01),which indicated that BAA induced oxidative stress by increasing the level of ROS in granulosa cells,and then led to DNA damage in granulosa cells.In summary,30 μmol/L BAA may disrupt cytoskeletal dynamics,alter epigenetic modifications and induce DNA damage in oocytes,resulting in a significant decrease in the in vitro maturation rate and maturation quality of mouse oocytes during oocyte maturation.In addition,5 μmol/L BAA caused an increase in ROS levels in granulosa cells,which in turn induced DNA damage to block granulosa cell proliferation and activated the mitochondrial apoptotic pathway via Bcl-2 family proteins,ultimately leading to granulosa cell apoptosis.