The Effects Of Fluorene-9-Bisphenol On Mouse And Porcine Oocyte In Vitro Maturation

There is a big threat to animal and human health with the advance of modernization,and it has become a worldwide issue.Environmental pollutants can enter animals and humans through a variety of ways(drinking water,etc.),resulting in abnormal physiological functions.Fluorene-9-bisphenol(BHPF),a substitute of bisphenol A(BPA)used in the production of “BPA-free” plastics,has been proofed that can enter humans and mice through water bodies,and shows anti-oestrogenic activity.All these suggest that BHPF may be harmful to living organisms.However,the effects of BHPF on oocyte maturation is unknown.The objective of this study is to evaluate the toxic effects of BHPF treatment on mouse and porcine oocyte in vitro maturation.Our study firstly reports the toxic effects of BHPF on oocyte maturation in female animals and raises concerns about the safety of BPA substitutes,provides a theoretical basis for revealing the relationship between BHPF and animal as well as human reproductive health,and also provides a reference for explaining whether BHPF can be reasonably applied to daily consumer products.This study is divided into two parts and the results are as follows: 1.The effects of BHPF on mouse oocyte in vitro maturationMouse oocyte maturation medium was supplemented with different concentrations of BHPF(0,50 ?M,100 ?M and 150 ?M),and meiotic resumption(GVBD)and first polar body extrusion(PBE)rates were observed and counted respectively at 2 h and 12 h in vitro culture.Results showed that BHPF significantly reduced GVBD and PBE rates(P < 0.01).Cell viability was measured after treatment of mouse oocytes with BHPF(100 ?M and 150 ?M)for 2 h and 12 h,and results showed that BHPF significantly reduce intracellular ATP levels in oocytes(P < 0.05 and P < 0.01).After treated with BHPF for 7.5 h,the key regulatory factors and events in meiotic progression were detected by using Immunofluorescence staining(IF),Real-time PCR,fluorescence intensity measurement and western blot(WB)analysis,and results showed that BHPF led to abnormal spindle assembly(P < 0.01),disturbed actin dynamics(P < 0.01),mitochondrial dysfunction(abnormal mitochondrial distribution and reduced mtDNA copy number,P < 0.01),mitophagy(up-regulation of PINK1,Beclin1 and LC3 B,P < 0.01;down-regulation of TOMM20 and TIMM17 A,P < 0.01),increased ROS level(P < 0.05),early apoptosis(P < 0.01)and increased H3K9me3 as well as H3K27me3 level(P < 0.01)in mouse oocytes.2.The effects of BHPF on porcine oocyte in vitro maturationDifferent concentrations of BHPF(0,25 ?M,50 ?M and 75 ?M)were added to porcine oocyte maturation medium,and cumulus expansion of porcine COCs was observed after 44 h in vitro culture and the first polar body extrusion rates of oocytes were counted.Results showed that BHPF(50 ?M and 75 ?M)significantly inhibited cumulus expansion,and decreased the first polar body extrusion(PBE)rate in porcine oocytes(P < 0.05).After treatment of porcine COCs with BHPF for 27 h,cumulus cells were removed and the key regulators and events in meiotic progression were detected by using cell viability assay,Immunofluorescence staining(IF)and Fluorescent probe staining.Results showed that BHPF(50 ?M)resulted in abnormal spindle assembly(P < 0.05),decreased ATP level(P < 0.05),increased ROS level(P < 0.05),early apoptosis(P < 0.05)and abnormal CGs distribution(P < 0.05)in porcine oocytes.In order to explore whether melatonin has a mitigating effect on BHPF-induced PBE failure,we used a co-addition treatment of different concentrations of melatonin(10-500 ?M)and 50 ?M BHPF,cultured COCs for 44 h in vitro,then observed and counted PBE in oocytes.Results showd that melatonin(10-500 ?M)supplement did not alleviate the BHPF-induced PBE failure in porcine oocytes(P > 0.05).In summary,BHPF significantly inhibited mouse and porcine oocyte meiotic maturation in vitro,causing abnormal spindle assembly,disturbed actin dynamics,mitochondrial dysfunction,decreased ATP level,increased ROS level and early apoptosis in oocytes,which are all negative to the PBE.In addition,BHPF also reduced oocyte quality by altering histone modifications and CGs distribution.