Exogenous Granulocyte-macrophage Colony Stimulating Factor Supplementation In Culture Medium Improves The In Vitro Bovine Early Embryo Development

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent hematopoietic growth factor secreted by lymphocyte T and macrophagocyte. The precursor of granulocyte and macrophagocyte can be induced by GM-CSF to form colony. GM-CSF could improve proliferation and differentiation in a variety of cell types. Previous studies shown that GM-CSF could improve the quality of preimplantation embryos and improve the pregnancy rate after transplantation as well as reduce the occurrence of spontaneous abortion. The present study was conducted to investigate the influence of exogenous GM-CSF on the development of bovine in vitro embryos and the mechanism of this process.To investigate the influence of exogenous GM-CSF on the development of bovine in vitro embryos, different concentrations of GM-CSF (0、1、10、20 ng/mL) were added in culture medium at 5 days after in vitro fertilization. To select the optimal concentration, the blastocyst rate was calculated two days after in vitro fertilization. The endogenesis ROS level, total cells number and apotosis rate of in vitro produced blastocysts were deccted between the optimal concentration and control group. To reveal the mechanism that GM-CSF increase the embryonic development ability before implantation, the real-time PCR was used to determine the expression level of genes associated with embryo development, anti-apotosis, anti-oxidate and glycometabolism/Warburg Effect.Results suggested that in the condition of 10 ng/mL, GM-CSF significantly increased blastocysts rate (30.75%) than control group and 20 ng/mL group (21.00% & 19.75%, P<0.05), the endogenesis ROS level was down regulated significantly, the total number of blastocyst was significantly increased (86.6± 4.22 vs. 66.625±4.33, P<0.05), But there was no significant difference in apotosis rate between control group and 10 ng/mL GM-CSF treated group(15.21%±1.44% vs. 17.60%±0.97%, P>0.05). In the group of 10 ng/mL GM-CSF in culture mediun, the relative expression of Bcl-2, SOD-1, GPx4, survivin and PCNA in bovine blastocyst were up-regulated, the relative expression level of G6PD, PDK-1 and PGK-1 withch releated to glycometabolism/Warburg Effect also increased (P<0.05). While the relative expres-sion level of KEAP-1 was down-regulated (P<0.05).In conclusion, these results demonstrated that exogenous GM-CSF at its optimal concentration could improve the developmental competence, cell proliferation and reduce oxidative stress of bovine preimplantation embryos in vitro. GM-CSF may via PI3K/Akt and JAK/STAT5 signaling pathways and there downstream molecules (HIF1α, Nrf2) exert its biological function as antiapoptotic, oxidation resistance, cell growth promption and increased glycolysis in bovine preimplantation embryos.