| The objective of present study was to investigate the cAMP-PKA signalling pathway of short chain fatty acids(SCFAs)on sexual hormone secretion in porcine granulosa cells(PGCs).PGCs were treated with different dose(0,0.005,0.05,0.5,5,10 mM)of SCFAs,10 ?M Forskolin(PKA activator)and 2.3?M Bupivacaine(cAMP production inhibitor),then the level of progesterone(P4)and estradiol(E2)in the medium of PGCs culture,the cAMP content in the PGCs,and the mRNA abundance of G protein coupled receptors 41(GPR41),GPR43,steroidogenic factor-1(SF-1),steroidogenic acute regulatory(StAR),cytochromo P450 side chain cleavage(P450scc),3?-hydroxysteroid dehydrogenase(3?-HSD)and estradiol receptor ?(ER-?)of PGCs were analysed.1 Effects of SCFAs on the sex hormone secretion of PGCs.1.1 Effects of acetic acid on the P4 and E2 secretion of PGCs.0.005?20 mM acetic acid and 0.005?0.05 mM propionic acid did not influence the P4 secretion of PGCs,whereas 0.05 mM butyric acid significantly increased P4 secretion(P<0.05).0.5?10 mM propionic acid and 5?10 mM butyric acid significantly decreased the secretion of P4(P<0.05).0.05?20 mM acetic acid,5?10 mM propionic acid and butyric acid increased the level of E2(P<0.05).10?M Forskolin significantly increased the secretion of P4 and E2 in PGCs(P<0.05).The P4 level of the treatments of 5 mM acetic acid,propionic acid,butyric acid co-cultured withlO ?M Forskolin was higher than that of 10?M Forskolin treatment,whereas the E2 level was lower than the treatment of 5 mM acetic acid and 10?M Forskolin(P<0.05).2.3?M Bupivacaine significantly decreased the secretion of P4 in PGCs(P<0.05)without the changes of E2.The level of E2 in the treatment of 5 mM actic acid+2.3?M Bupivacaine was higher than that of 2.3 ?M Bupivacaine treatment(P<0.05),lower than 5 mM actic acid treatment(P<0.05)?The level of E2 in the treatment of 5 mM propionic acid+2.3 ?M Bupivacaine was lower than 5 mM propionic acid(P<0.05).The level of P4 in the treatment of 5 mM butyric acid+2.3?M Bupivacaine was lower than 2.3 pM Bupivacaine treatment(P<0.05)?2 The effect of SCFAs on cAMP-PKA singling pathway in PGCs2.1 The expression of GPR41 and GPR43 mRNA10 ?M Forskolin,2.3?M Bupivacaine,0.005?10 mM actic acid?0.005?0.5 mM propionic acid and 0.005?5 mM butyric acid did not influence the level of GPR41 and GPR43 mRNA in PGCS?However,5-10 mM propionic acid and 10 mM butyric acid upregulated the expression of GPR41and GPR43 mRNA(P<0.05).5 mM actic acid+10?M Forskolin significantly increased the expression of GPR41 and GPR43 mRNA(P<0.05),5 mM propionic acid+10 ?M Forskolin significantly increased the expression of GPR41 mRNA(P<0.05),significantly decreased the expression of GPR43 mRNA(P<0.05),5 mM butyric acid+10 ?M Forskolin did not influence the expression of GPR41 and GPR43 mRNA in PGCs.5 mM propionic acid+2.3 ?M Bupivacaine significantly increased the expression of GPR43 mRNA in PGCs(P<0.05).2.2 cAMP level in PGCsAs compared to the control group,10 ?M Forskolin treatment significantly increased the cAMP level(P<0.05),whereas 5 mM acetic acid,propionic acid and butyric acid and 2.3 ?M Bupivacaine treatment significantly decreased the cAMP level in PGCs(P<0.05).5 mM acetic acid,propionic acid and butyric acid co-cultured with 10 ?M Forskolin did not influence the up-regulation of Forskolin on the level of cAMP in PGCs.2.3 ?M Bupivacaine significantly decreased the cAMP level of PGCs(P<0.05).The level of cAMP in the treatment of 2.3 ?M Bupivacaine+5 mM actic acid?2.3 ?M Bupivacaine+5 mM propionic acid,and 2.3 ?M Bupivacaine+5 mM butyric acid were lower than the treatments of 2.3?M Bupivacaine,5 mM actic acid,5 mM propionic acid,5 mM butyric acid(P<0.05).3 The mRNA expression of steroidogenic genes in PGCs.In contrast to the control group,10?M Forskolin significantly reduced the ER-?mRNA expression(P<0.05),up-regulated the mRNA expression of SF-1,StAR,3?-HSD and P450scc in PGCs(P<0.05).2.3 ?M Bupivacaine significantly increased the ER-? mRNA expression(P<0.05),decreased the mRNA expression of StAR,P450scc(P<0.05).5 mM acetic acid significantly decreased the ER-P mRNA expression(P<0.05),and increased the StAR mRNA expression(P<0.05).The SF-1,StAR,3?-HSD,P450scc mRNA expression of the 5 mM acetic acid treatment were significantly lower than the treatment of 10 ?M Forskolin+5 mM acetic acid(P<0.05).The expression of ER-? mRNA of the 2.3 ?M Bupivacaine+5 mM acetic acid treatment was higher than 5 mM actic acid treatment,but lower than the treatment of 2.3 ?M Bupivacaine(P<0.05).5 mM propionic acid significantly decreased the expression of the ER-?,SF-1,StAR and P450scc mRNA in PGCs(P<0.05).The level of SF-1,StAR,3?-HSD and P450scc mRNA in the 10 ?M Forskolin+5 mM propionic acid was lower than the 10?M Forskolin treatment(P<0.05),higher than 5 mM propionic acid treatment(P<0.05),but the level of 3?-HSD mRNA was lower than 5 mM propionic acid treatment(P<0.05),the expression of ER-? and StAR mRNA was lower than 2.3?M Bupivacaine treatment(P<0.05).In contrast to the control group,5 mM butyric acid significant increased the 3?-HSD mRNA expression(P<0.05),and decreased the expression of ER-?,SF-1,StAR,P450scc mRNA(P<0.05).The StAR,3P-HSD and P450scc mRNA expression of 10 ?M Forskolin+5 mM butyric acid was significantly lower than the 10 ?M Forskolin treatment(P<0.05).The levels of ER-?,SF-1 mRNA expression of 2.3 pM Bupivacaine+5 mM butyric acid treatmeat were higher than the 5 mM butyric acid treatment(P<0.05).The 3?-HSD mRNA expression of 2.3?M Bupivacaine+5 mM butyric acid treatment were higher than the 2.3 ?M Bupivacaine treatment(P<0.05),and the ER-?,StAR mRNA expression were lower than the 2.3?M Bupivacaine treatment(P<0.05).Summary:SCFAs influence the secretion of P4 and E2 in the PGCs,which may thought the GPRs-cAMP-PKA signalling pathway. |
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