Growth And Differentiation Of Porcine Oocyte Maturation And Early Embryonic Development And Study Of The Impact Factor Of Nine Pairs Of Mechanism

Animal ovaries from slaughterhouses can be used effectively to produce embryos in vitro, which is beneficial for fast breeding and reproduction. However, to date, the efficiency of in vitro porcine oocyte maturation and in vitro embryo production is low. GDF9, an important paracrine factor secreted by oocyte could stimulate follicle development towards female reproduction and enhance subsequent oocyte development. In our research, the effect of GDF9on porcine oocyte in vitro maturation and early embryo development is studied by adding GDF9or GDF9antibody alone to in vitro maturation medium, which can lay the foundation for porcine embryo in vitro production. This paper contains three parts as below.Part one:The effect of GDF9on porcine oocyte in vitro maturation and development of early embryosPorcine ovaries were collected from a local slaughterhouse. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles and cultured in vitro. After44h of IVM, nuclear maturation was evaluated. After denuding the cultured COCs, maturated oocytes were parthenogenetic activated by chemistry method. Cleavage embryos were evaluated on day2. Blastocyst (BL) formation and the number of nuclei in blastocyst were determined on day7. In addition, after44h of IVM, oocytes were collected for evaluated cytoplasmic maturation by cortical staining. In this research, GDF9(0,100,200,300ng/mL) or GDF9antibody (0,2.5,5,10μg/mL) was added alone to in vitro maturation medium. Parameters mentioned above were recorded for statistical analysis. The results showed that there was no significant difference in nuclear maturation, cytoplasmic maturation, cleavage and total number of nuclei in blastocyst, but GDF9could significantly stimulated BL formation in dose-dependent and the highest rate of BL formation was gotten on concentration of200ng/mL.Part two:The effect of GDF9on microenvironment of porcine oocyte in vitro maturation and developmentPorcine ovaries were collected from a local slaughterhouse. COCs were aspirated from antral follicles and cultured in vitro. Cumulus expansion was classified as described by published paper. Cumulus cells and maturation medium were collected for counting and detecting progesterone content with hemocytometer and ELISA respectively. In this research, optimal concentration of GDF9(200ng/mL) or GDF9antibody (10μg/mL) was selected to add alone into in vitro maturation medium. Our results showed that200ng/mL GDF9could simulate cumulus cell expansion and proliferation, but inhibit progesterone production. We supposed that GDF9improving embryonic development was possibly by creating a suitable microenvironment for oocyte development.Part three:The effect of GDF9on gene expression of cumulus cellsPorcine ovaries were collected from a local slaughterhouse. COCs were aspirated from antral follicles and cultured in vitro. After culturing44h, cumulus cells were collected for gene expression detection. Total RNA were isolated and through reverse transcript to get cDNA. HAS2(related to cumulus expansion), CSTB (related to BLs development) and FST(related to maturation and development) were amplified together with housekeeping gene ACTB for QPCR. Ct values were recorded. In this research, GDF9(200ng/mL) or GDF9antibody (lOμg/mL) was added alone into in vitro maturation medium. The results showed that concentration of200ng/mL GDF9can simulate HAS2expression and inhibit CSTB and FST expression in cumulus cells.Conclusion:GDF9created a suitable microenvironment for oocyte development, maybe through stimulating cumulus cell expansion and proliferation, improving HAS2expression, and inhibiting progesterone production and expression of CSTB and FST, which eventually elevated embryonic developmental competence.