Investigation Of The Effects Of Brefeldin A On The Porcine Oocyte Meiotic Maturation

The quality of mammalian oocytes is closely related to the fertility of livestock.The normal development of oocytes is a prerequisite for ensuring maturity,fertilization and embryo development,and is essential for the sustainable development of animal husbandry.The maturation of oocytes is a complex and rigorous process,including important events such as maintenance of the germinal vesicle,germinal vesicle breakdown,cortical particle transport,spindle formation,chromosome alignment,and first polar body extrusion.In order to ensure the success of this process,a series of organelles are involved.The organelles related to nuclear maturation have chromosomes and spindles.The organelles associated with cytoplasmic maturation have microfilament skeletons,mitochondria,cortical particles,and protein transport oocytes.The maturation process plays an important role in regulating the dynamic assembly and meiosis of various organelles.In this experiment,pigs were used as research objects,and the effects of protein transport on oocyte development were studied by in vitro culture and immunofluorescence of oocytes.In order to explore the influencing factors and mechanism of regulation of oocyte maturation during oocyte development,there are two main aspects of the research:(1)the effect on the nuclear maturation of the oocyte;and(2)the effect on the cytoplasmic maturation of the oocyte.The main findings are as follows:An experimental model for the inhibition of Golgi’s functional construction of protein transport abnormalities by adding the Brefeldin A(BFA).In this experiment,different concentrations of BFA inhibitors were added during the maturation of oocytes to observe the effects on oocyte development and various organelles in the cells.It was found that the maturation of oocytes exposed to 2 μg/ml BFA was significantly affected,mainly manifested in the change of the degree of cumulus cell diffusion and the significant decrease of the first polar body extraction rate.Immunofluorescence staining of the indicator chromosome and spindle of oocyte nuclear maturation showed that the abnormal rate of chromosome and spindle of oocytes exposed to BFA was significantly higher than that of the control group in the middle of the first meiosis.Since the nuclear maturation and cytoplasmic maturation of oocytes were not synchronized,some organelles related to cytoplasmic maturation were observed by immunofluorescence,including microfilaments,mitochondria,cortical granules and their components ovastacin.The study found that after BFA treatment,the microfilament skeleton and mitochondrial localization of oocytes and the distribution and release of cortical particles were affected,mainly manifested that the fluorescence signal intensity of the microfilament skeleton and mitochondria is significantly weaker than that of the control group,and the localization is incomplete.Compared with the control group,the cortical particles were located in the thin layer beneath the plasma membrane.After the exposure of BFA,the localization of cortical particles appeared intracellularly dispersed,and a large number of cortical vesicles had not migrated to the cortical area and had been released.The expression of ovastacin,which is the inclusion of cortical granules,is also affected,and its number is significantly lower than that of the control group,which may affect subsequent fertilization processes and early embryo development.In conclusion,our experiments have found that inhibition of protein transport by the use of the inhibitor during oocyte maturation affects the nuclear maturation and cytoplasmic maturation of oocytes,thereby impeding oocyte development process.