Mechanism Investigation Of Bap Or Aging Induced Oxidative Stress On The Quality Of Porcine Oocytes

The proper development of manmalian oocytes is a prerequisite for the normal oogenesis,fertilization and embryo development,which is the basis for the initiation of life.Because of the environmental pollution and aging,the decreased quality of oocytes seriously affects the animal fecundity and restricts the development of animal husbandry.Accumulating studies have demonstrated that many molecules which play the key roles in the process of mitosis are involved in the oocyte maturation and fertilization.The function of some molecules is not conservative and it is unique during meiosis.In addition,the mechanisms regarding how external factors such as environmental toxins and aging affect the egg quality to cause the fertilization failure are still elusive.Therefore,the systematical investigation of internal regulation mechanisms and external influencing factors during oocyte meiotic maturation could contribute to enrich and improve the theoretical basis for the oocyte quality eontrol,Providing a reliable basis for improving the animal fecundity.In the present study,porcine oocytes were selected as the research subject to explore the influencing factors and regulation mechanisms of oocyte quality under different eonditions through in vitro culture,in vitro fertilization,embryo culture,fluorescent staining and western blot.The study was divided into two parts.Firstly,we found that environmental toxins benzo(a)pyrene(BaP)impaired the porcine oocyte maturation and fertilization ability via induction of oxidative stress and early cell apoptosis,and demonstrated that melatonin,as a natural antioxidant,can effectively ameliorate the low oocyte quality that was induced by benzopyrene exposure.Secondly,we clarified the mechanisms underlying the compromised oocyte quality in the process of postovulatory aging,involving the key e’vents and regulatory factors during oocyte maturation and fertilization.The main results are shown as follows:Experiment 1.Mechanism of meiosis abnormality induced by benzopyrene in porcine oocytes and protective effect of melatoninIn this study,different concentrations of BaP were supplemented into the culture medium to observe and record the maturation rate and morphological changes of oocytes.We found that 50μM BaP significantly reduced the polar body extrusion rate and cumulus cell expansion of oocytes.Immunofluorescence staining results showed that exposure to BaP significantly increased the spindle assembly abnormalities and activated spindle checkpoint protein BubR1,thereby inhibiting the first polar body extrusion and reducing the maturation rate of oocytes.In addition,BaP exposure also impaired the localization pattern of cytoskeleton and mitochondria,as well as the distribution and release of cortical granules.After BaP treatment,cortical granues in the oocyte prematurely released the content out of the oocytes including ovastacin,a component of the cortical granules,that cleaved the sperm binding site in the zona pellucida ahead of time,leading to the decreased ability of sperm binding to the zona pellucida and failure of fertilization.In the meantime,Juno,a key molecule responsible for sperm-egg fusion on the egg plasma membrane,was also damaged by BaP exposure.By fluorescent staining and quantitative analysis,we found that the expression and localization of Juno were perturbed in different degrees,which might be another factor causing the failure of fertilization.It has been shown that BaP could induce oxidative stress to disturb cellular development,we thus analyzed the level of reactive oxygen species(ROS)in oocytes to explore the cause of damage.The results showed that BaP exposure substantially increased the oxidative stress in the oocytes.High level of ROS resulted in the early apoptosis of oocytes,leading to the abnormal localization and functional decline of the organelles in oocytes.Given that melatonin is an effective antioxidant in the follicular fluids,our findings also demonstrated that melatonin could reduce the ROS level and apoptotic rate caused by BaP exposure,thus restoring the oocyte meiotic defects.In conclusion,long-term exposure to high concentration of BaP will seriously affect the quality of female oocytes and damage the ability of fertilization.Experiment 2.Mechanism of aging induced oxidative stress damaging the quality of porcine oocytesIn the present study,pig was used as an experimental animal model to investigate the potential effects of postovulatory aging on the livestock reproduction.Porcine oocytes were divided into three groups in the experiment.The first group was used as the control group.After 44 hours of in vitro culture,the maturation rate and the morphological change of oocytes were observed.The second group was cultured in vitro for 68 hours,that is,24 hours after ovulation.The third group was cultured in vitro for 92 hours,that is,48 hours after ovulation.Then the oocyte maturation and morphological change in the aging groups were recorded with the extension of in vitro aging time.In addition,the differences in localization and dynamics of important organelles in oocytes were analyzed by the immunofluorescence and fluorescence intensity analysis.Our data showed that the cotnection between cumulus cells and oocytes became gradually loose in the postovulatory aged oocytes.Meanwhile,the fragmentation rate of oocytes increased with the extension of in vitro aging time,which also caused the morphological changes of porcine oocytes.Furthermore,postovulatory aging perturbed the function and structure of organelles,including spindle assembly,actin polymerization and mitochondrial distribution,which seriously affected the development of oocytes,leading to the decline of oocyte quality.Notably,the distribution of cortical granules also changed significantly during aging.In the postovulatory aged oocytes,cortical granules were precociously released to the extracellular space of the oocytes,including the ovastacin that can cut the sperm binding site in the zona pellucida,thereby affecting the sperm-egg binding and fertilization potential.Further investigation showed that the level of ROS was accumulated in the postovulatory aged ooceytes,displaying the significantly higher degree of oxidative stress than in control oocytes,leading to the abnormal oocyte development.In summary,our findings indicate that postovulatory aging impaired the ooeyte maturation and fertilization ability by producing various molecular and cellular changes induced by oxidative stress in porcine oocytes.In conclusion,either environmental toxin BaP or aging after ovulation can cause an increase about the level of ROS in oocytes,called oxidative stress,which cause abnormal expression and localization of various cellular organelles in the oocyte.Exposure to the BaP environment for a long time,or prolonged aging of ovulation,will seriously reduce the quality of oocyte,lead to early release of cortical granules,which cause premature resection of the sperm binding site on the zona pellucida and reduce the ability to combine sperm with oocytes,eventually reduce the fertilization rate.