Effects Of Heat Stress And LPS On Proliferation And Secretion Of Duck Follicular Granulosa Cells In Vitro

Guangdong has been a large province for waterfowl cultivation in China,in which the number of laying ducks has increased rapidly in recent years.Yet the high temperature and high humidity climate of the south facilitate proliferation of various harmful bacteria in the environment,and egg production of the laying ducks often suffer from heat stress?HS?and elevated lipopolysaccharide?LPS?concentration.Egg production of poultry is determined by follicular development,in which granulosa cells play an important role.It is speculated that heat stress and LPS may affect the function of granulosa cells and thus egg production of ducks.To investigate the mechanism how HS and LPS affect granulosa cells,this study established the granulosa cell model of the Shanma duck in vitro,and explored the effect of HS and LPS,alone and in combination,on proliferation and secretion of these cells.Experiment 1:Effect of HS on proliferation and secretion of duck follicular granulosa cells in vitro.A control group?cultured at 39^oC?and two high-temperature groups?cultured at 41^oC and 43^oC?were set for this experiment.Cell cycle progression was analyzed by flow cytometry,relative expression of proliferation and reproduction-related genes was analyzed by qRT-PCR,and secretion of relevant hormones was determined by ELISA.The results demonstrated that heat stress caused arrest of cell cycle at G1 phase,with mRNAs of IGF2,IGFBP2,Cyclin D1,CDK6 downregulated;heat shock also caused secretion of P4,E2,ACV by the granulosa cells,while INH secretion was inhibited;mRNAs of StAR and 3?-HSD were upregulated,and INH?A mRNA was downregulated.Experiment 2:Effect of LPS on proliferation and secretion of duck follicular granulosa cells in vitro.A control group?adding no LPS?and LPS-treated groups?adding 250ng/ml,500ng/ml,1000ng/ml,and 2000ng/ml LPS?were set for this experiment.After 24 hours of treatment,the culture media were collected for ELISA to determine secretion of relevant hormones,and the cells were collected for cell cycle assay by flow cytometry and qRT-PCR to determine expression of cell cycle-related genes.It was found that LPS stimulation caused cell cycle arrest of granulosa cells in G1 phase,down-regulated expression of IGF2,IGFBP2,CyclinD1and CDK6 mRNA,and up-regulated the expression of p27^kip1 mRNA.LPS also promoted secretion of P4,E2 and ACV in granulosa cells,and inhibited secretion of INH in a dose-dependent manner.Expression of FSHR and 3?-HSD mRNA were also upregulated,and GnIHR mRNA was down-regulated.Experiment 3:Effect of HS and LPS in combination on proliferation and secretion of duck follicular granulosa cells in vitro.A control group?39^oC+0 ng/ml LPS?,an LPS group?39^oC+2000ng/l LPS?,a HS group?43^oC+0ng/ml?,and an LPS+HS group?43^oC+2000ng/ml LPS?were sect for this experiment.After 12 or24 hours of treatment,the culture media were collected for ELISA to determine secretion of relevant hormones,and the cells were collected for cell cycle assay by flow cytometry,qRT-PCR to determine expression of cell cycle-related genes,and Western blot to determine expression of proteins involved in the MAPK signaling pathway and cell proliferation.The results demonstrate that the effects of HS and LPS on proliferation and secretion of granulosa cells in vitro were additive.The combined treatment further increased the ratio of cells in G1 phase compared to each treatment alone,and further decreased mRNA levels of IGF2,IGFBP2,Cyclin D1,CDK6,and further upregulated p27^kip1 mRNA,and downregulated ERK and pERK expression.The combined treatment also promoted secretion of P4,E2 and ACV,and inhibited INH secretion;upregulated mRNA expression of FSHR,StAR,3?-HSD,and downregulated mRNA and protein expression of CYP19A1.HS and LPS,alone and in combination,significantly inhibits proliferation of granulosa cells,possibly via changing expression of cell cycle-related genes,such as Cyclin D1 and p27^kip1,and expression of phosphorylation of ERK in the MAPK signaling pathway.Both treatments promoted secretion of P4,E2,ACV,and inhibited INH secretion,which may be attributed to changes in StAR,3?-HSD,CYP19A1 and possibly other reproduction-related genes and proteins.Our results provide not only reference for related scientific research,but also the scientific basis for the prevention and control of heat stress and LPS hazard in duck production.