Study On The Effect Of Progesterone On The Proliferation In Endometrial Stromal Cells Of Goat

Postpartum uterine infection is a common reproductive disease in dairy cows,including pyometra,hysteritis or endometritis etc,resulting in an increase in the elimination rate of cows and a huge economic loss to the breeding industry.Under normal circumstances,the postpartum uterus undergoes self-repair under the control of various factors to restore the normal structure and function of the uterus.Postpartum uterine infections lead to prolonged regeneration of the endometrium in dairy cows.Escherichia coli(E.coli)is one of the main pathogens casing uterine infection via the endotoxin lipopolysaccharide(LPS).Progesterone is an important ovarian hormone that changes dynamically during the estrus and pregnancy cycle of cows,and together with estrogen regulates the proliferation and differentiation of the endometrium.It has been well-accepted that elevated levels of progesterone increase the risk of uterine infection.Though progesterone has been suggested to regulate cell proliferation,less is known about its effect on the endometrial cells.Goats and cows have similar reproductive mechanisms and undergo a similar uterine repair process.The aim of the present study was to explore the effects of progesterone on the cell viability,the cell cycle,the expression of cell proliferation related factors and pathways in LPS-stimulated endometrial stromal cells of goat(gESC).The test was divided into four groups.The cells in the control group were not treated at all;the cells in the LPS group were treated with 1μg/mL LPS;the cells in the progesterone group were treated with 1,3,or 5 ng/mL progesterone;the cells in the co-treatment group were co-treated with progesterone(1,3,or 5 ng/mL)and 1 μg/mL LPS.The test results were as follows:(1)To investigate the effects of progesterone on the proliferation and cell cycle of gESC.Cells were treated as mentioned above for 24 h,and cell viability and cell cycle were measured by CCK-8 and flow cytometry,respectively.As a result,the cell proliferation activity was up-regulated(p<0.01)in progesterone group or LPS group.Compared with LPS group,the cell proliferation was decreased(p<0.01)in the group of progesterone co-treated with LPS.Treatment of gESC with 3 or 5 ng/mL progesterone increased the number of cells in G0/G1 phase(p<0.05).Treatment of gESC with 1,3 or 5 ng/mL progesterone increased the number of cells in s phase(p<0.01).Progesterone group up-regulated(p<0.01)the cell proliferation index(PI).Treatment of gESC with 1 μg/mL LPS alone down-regulated G0/G1 phase,up-regulated(p<0.01)the number of cells in S phase and cell PI.Compared with LPS group,the number of cells increased(p<0.01)in G0/G1 phase,decreased(p<0.01)in S phase and in cell PI.Conclusion:Progesterone treatment or LPS treatment can promote the cell cycle to s phase and increase the overall proliferation of cells.Progesterone inhibited LPS-induced cell proliferation and cell cycle progression,and arrest cells in G0/G1 phase.(2)The endometrial repair process involves in various repair-related factors.To investigate the effects of progesterone the repair-related gene expressions in gESC,the cells were treated with progesterone as mentioned above.The mRNA expressions of PR,VEGF,EGFR,Cx-43 and b-FGF were detected at 3,12 and 18 h.As a result,the expressions of PR,VEGF,EGFR,Cx-43 and b-FGF increased(p<0.05)after progesterone(1,3 or 5 ng/mL)treatment.The increment of mRNA expressions was most obvious at 3 h(p<0.01).In cells treated with 1 μg/mL LPS alone,the gene expressions of PR,VEGF,EGFR,Cx-43 and b-FGF increased(p<0.05)at observed time points,except the EGFR at 18 h(p>0.05).Compared with the LPS group,the gene expressions of PR,VEGF,EGFR and b-FGF decreased(p<0.05)at 3 h in the LPS and progesterone(1,3 and 5 ng/mL)co-treatment group.The expression levels decreased(p<0.01)for EGFR,Cx-43 and b-FGF mRNA at 12 h,for PR,VEGF and Cx-43 at 18 h in cells co-treated with LPS and progesterone.Conclusion:Progesterone or LPS treatment alone up-regulated the gene expressions of PR,VEGF,EGFR,Cx-43 and b-FGF.Progesterone suppressed the LPS-induced expressions of PR,VEGF,EGFR,Cx-43 and b-FGF.(3)To investigate the effects of progesterone on Wnt/β-catenin and PI3K/Akt pathways in gESC,the cells were treated with progesterone and/or LPS for 30 min.Western blot was used to detect the protein expressions of β-catenin,c-Myc and cyclin-D1,and the phosphorylations of PI3K and Akt.The nuclear translocation of β-catenin was detected by immunofluorescence.As a result,the expressions of β-catenin and cyclin-D1 increased in the 1,3 or 5 ng/mL progesterone-treated groups(p<0.05).The expression of c-Myc increased in the 1 and 3 ng/mL progesterone-treated groups(p<0.05).The phosphorylation level of Akt increased(p<0.05)in the 5 ng/mL progesterone treatment group.The phosphorylation level of PI3K increased in the 1,3 or 5 ng/mL progesterone treatment groups(p<0.01).The expressions of β-catenin,c-Myc and cyclin D1 increased(p<0.05)in LPS group.The level of(3-catenin in the nucleus was higher in LPS group than that in the control group.Compared with LPS group,the expression of β-catenin decreased(p<0.01),and the level of β-catenin in the nucleus was lower after co-treatment with 1,3 or 5 ng/mL progesterone and LPS.The expression of cyclin D1 decreased(p<0.01)in cells co-treated with 5 ng/mL progesterone and LPS.The expression of c-Myc decreased(p<0.05)in progesterone(1 or 3 ng/mL)and LPS co-treatment group.The relative phosphorylation levels of Akt and PI3K decreased after LPS and progesterone co-treatment(p<0.05).The activation and nucleation of β-catenin could be observed by immunofluorescence,which is consistent with the protein results.In conclusion,LPS activated the Wnt/p-catenin and the PI3K/Akt pathways in gESC,which was suppressed by progesterone.However,progesterone alone stimulated the Wnt/β-catenin and the PI3K/Akt pathways.In summary,progesterone or LPS alone promoted cell proliferation and cell cycle progression,activated the cell proliferation-related factors and pathways.Progesterone suppress the LPS-induced cell proliferation,cell cycle progression,the gene expressions of PR,VEGF,EGFR,Cx-43 and b-FGF,and the activation of Wnt/β-catenin and PI3K/Akt pathways.These result suggested that progesterone could inhibit the LPS-induced cell proliferation,which may be mediated through the Wnt/p-catenin and PI3K/Akt pathways.