Study On The Effects Of Perfluorooctane Sulfonate On In Vitro Maturation And Fertilization Of Porcine Oocytes

The quality of mammalian oocytes is one of the prerequisites for animal fertility.Low oocyte quality will lead to oocyte maturation disorders,fertilization failure and abnormal embryo development,which seriously restricts animal reproductive performance,reduces the production efficiency of livestock,and thus limiting the development of animal husbandry.With the continuous development of science and technology,animal husbandry is facing great opportunities and challenges.Oocytes,as the important research object of animal reproduction science,directly affect the production capacity of livestock,and therefore have been widely studied.In addition,as the basis of individual development,oocyte quality determines the fertilization ability and subsequent embryonic development.Although some environmental factors have been to reported damage the oocyte quality,there are many other unclear factors.Therefore,the exploration of regulatory mechanism and external factors of oocyte meiotic maturation would contribute to extend the theoretical basis of livestock oocytes quality control,and increase the rate of in vitro maturation of oocytes as well as reproductive performance.Perfluorooctane sulfonates(PFOS)is a widely used synthetic surfactant with potential toxicity.Due to its biodegradability and bioaccumulation,PFOS is commonly found in many areas of biome and environment.Studies have shown that omnivorous pigs,with their high body fat content,may accumulate large concentrations of persistent organic compounds,such as halogenated hydrocarbons,and thus may have adverse effects on human and livestock health.Among of them,PFOS,as an organic pollutant that is difficult to break down,binds to albumin and accumulate in different organs,such as the liver,spleen,kidneys,brain,testicles and ovaries,resulting in corresponding body damage.It is reported that long-term exposure to PFOS influences the reproductive health of animals.PFOS can delay monarchy,disrupt the menstrual cycle,cause early menopause and premature ovarian failure,and alter circulating sex steroid levels.It has been also found that PFOS affects oocyte maturation during the early stage of follicular development by interfering with gap junctional intercellular communication(GJIC)in the cumulus-oocyte complex.However,the underlying mechanism by which PFOS compromises oocyte quality in animals has not been clearly investigated.As a critical animal husbandry resource,pigs are of great importance to livestock production and social life,meanwhile,their physiological features are more similar to humans than rodents,which contribute a lot to the biomedical research.The present study chose the porcine oocytes as the research model,and applied the methods such as porcine oocyte in vitro culture system,fluorescence staining and in vitro fertilization,to investigate the effects of PFOS on in vitro maturation and fertilization of porcine oocytes and explore the potential regulatory mechanisms by which environmental factors,impacts the oocyte quality.This study is divided into three parts,and the main experimental contents and results are shown as follows:Experiment 1 PFOS disrupts the nuclear maturation of porcine oocytesTo investigate the effects of PFOS on the porcine oocyte meiotic maturation,increasing concentrations of PFOS(0,10,25,50,100 μM)were supplemented to the maturation medium to observe the cumulus cell expansion and polar body extrusion rate in each group.The results showed that 100μM PFOS significantly reduced the polar body extrusion rate and cumulus cell expansion of oocytes,indicating that PFOS hindered the in vitro maturation of oocytes.Meiotic arrest in porcine oocytes is closely related to abnormal spindle/chromosome structure.Therefore,we studied the effect of PFOS on the spindle assembly and chromosome alignment in oocytes.The data revealed that the abnormal rate of spindle apparatus and chromosome alignment in oocytes exposed to PFOS was significantly increased compared to controls.Taken together,PFOS impairs the spindle/chromosome structure to hinder the first polar body extrusion,and thus disrupting the meiotic progression and nuclear maturation of porcine oocytes.Experiment 2 PFOS perturbs the cytoplasmic maturation of porcine oocytesThe cytoplasmic maturation of oocytes includes serials of molecular changes as well as cytoskeleton and organelle reorganization,which confers oocytes to acquire developmental competency.The results displayed that actin filaments in oocytes exposed to PFOS had a discontinuous distribution with weak signals,suggesting that PFOS disrupts the dynamic polymerization of actin.In addition,by fluorescent staining and quantitative analysis,we found that PFOS treatment markedly increased the rate of mis-located mitochondria and reduced the mitochondrial signals,indicating that mitochondrial distribution and function are damaged.We also assessed the mitochondrial membrane potential(ΔΨm)of different groups by JC-1 staining.The data showed that the ratio of red to green fluorescence of PFOSexposed oocytes decreased significantly in comparison with the controls.Moreover,most of the cortical granules did not migrate to the subcortical region of PFOS-exposed oocytes,and a large number of cortical granules still retained in the cytoplasm by showing diffused localization with relative faded fluorescent signals.Consistent with above observations,the distribution of ovastacin was disrupted after PFOS treatment,which showed discontinuous localization and much lower fluorescence intensity than those in control group.Collectively,PFOS perturbs the cytoplasmic maturation of porcine oocytes by compromising actin polymerization,mitochondrial integrity,as well as distribution of cortical granules and ovastacin,and thereby impairing the fertilization ability of porcine oocytes.Experiment 3 PFOS attenuates fertilization potential of oocytes by inducing oxidative stress to cause DNA damage and apoptosisThe correct recognition and binding of oocyte and sperm is an important prerequisite for successful fertilization.Therefore,the damage to cortical granules may cause the premature exocytosis of ovastacin after PFOS treatment,which affects the sperm binding ability of oocytes.To this end,we tested the fertilization potential of oocytes by sperm-oocyte binding assay and in vitro fertilization experiment.The results showed that the number of sperm binding to the zona pellucida of unfertilized oocytes in PFOS-exposed treatment is considerably reduced compared to the controls.Moreover,the rate of 2-cell embryos in PFOS-exposed group was significantly decreased in comparison with the control group,validating that PFOS disturbs the fertilization potential of porcine oocytes.It has been known that PFOS can induce the oxidative stress and damage the normal physiological functions in a variety of cells.Thus,we further evaluated the reactive oxygen species(ROS)level,DNA damage level and apoptotic rate in PFOS-exposed oocytes.The data displayed that the fluorescence intensity of ROS and DNA damage signals increased remarkably in PFOSexposed group,accompanied by the elevated apoptotic rate.In summary,PFOS induces oxidative stress in porcine oocytes to induce the production of excessive ROS,accumulated DNA damage and occurrence of early apoptosis,which impacts oocyte maturation and fertilization in vitro.