Effects of ODC interference on the levels of ODC mRNA and protein in the ovarian granulosa cells of geese
si-ODC-331, si-ODC-511 and si-ODC-894 were transfected into GCs for 24 h, 48 h and 72 h, respectively. The interference efficiency of ODC was the highest when transfected with si-ODC-894 plasmid for 48 h, which was much lower than that of the Blank and NC groups (P < 0.01) (Fig. 1A). Then, western blot analysis was utilized to further verify the level of ODC protein after the transfection of si-ODC-894 at different times. Figure 1B shows that the protein expression decreased significantly when ODC was interfered with for 48 h (P < 0.05), which was aligned with the results acquired by qRT-PCR. Therefore, si-ODC-894 transfected for 48 h was selected as the subsequent experimental condition.
Effects of ODC overexpression on the levels of ODC mRNA and protein in goose ovarian granulosa cells
As shown in Fig. 2A, when the ODC overexpression plasmid was transfected into GCs for 24 h, 48 h and 72 h, ODC expression was considerably upregulated by 19.9 times, 19.7 times, and 13.1 times, respectively (P < 0.01). Figure 2B shows that after 24 h of overexpression, the protein level of ODC was significantly upregulated (P < 0.05), which was consistent with the results obtained by qRT-PCR. Thus, the transfection of GCs with the ODC overexpression plasmid for 24 h was selected as the subsequent experimental condition.
Effects of ODC on polyamine contents and polyamine metabolism-related gene expression in GCs
Upon ODC interference, the putrescine content in GCs was significantly decreased (P < 0.05) (Fig. 3A). When ODC was overexpressed, the putrescine content in GCs was greatly increased (P < 0.05) (Fig. 3B). Both the concentrations of spermidine and spermine were not significantly different under ODC interference or overexpression conditions (P > 0.05).
Then, we studied the effect of ODC interference and overexpression on the gene expression of polyamine-metabolizing enzymes in GCs. The change in the expression of SPMS and SAMDC was not significant after interference with ODC (P > 0.05), while OAZ1, OAZ2, AZIN1, SPDS, APAO, SSAT and SMO gene expression was significantly upregulated (P < 0.05), and SPDS expression was remarkably decreased (P < 0.05) (Fig. 3C). OAZ2, SPMS and APAO expression was not significantly changed when ODC was overexpressed in GCs (P > 0.05), and the expression of OAZ1, SPDS, AZIN1, SSAT and SMO was significantly increased, while SAMDC expression was greatly reduced (P < 0.05) (Fig. 3D).
Effect of ODC on the gene expression of reproductive hormone receptors in GCs and P4 and E2 levels in culture medium
After interfering with ODC, the expression of ER, FSHR and LHR genes in GCs was greatly decreased, while the AR gene was markedly increased (P < 0.05) (Fig. 4A). The P4 level in the culture medium was remarkably increased (P < 0.05) (Fig. 4C), and the E2 content was tremendously decreased (P < 0.01) (Fig. 4D). As shown in Fig. 4B, when ODC was overexpressed, the expression of the ER gene in GCs was markedly downregulated (P < 0.05). LHR, FSHR, AR and PR were not remarkably impacted (P > 0.05). The P4 level was significantly decreased (P < 0.05) (Fig. 4E), while the E2 level did not markedly change (P > 0.05) (Fig. 4F).
The viability of GCs was analyzed after ODC interference and overexpression
After ODC expression interference in goose ovarian GCs, the number of decreased and dead GCs was observed under the microscope and compared with that of the NC group (Fig. 5A). MTT results showed that granulosa cell activity was remarkably reduced (P < 0.05) (Fig. 5A). As shown in Fig. 5C, after ODC was overexpressed, the number of GCs was significantly increased, and the cell morphology was fuller than that of the NC group. MTT results indicated that the activity of GCs was greatly increased after ODC overexpression (P < 0.01) (Fig. 5D). Then, we explored the effects of ODC on proliferation-related proteins. Figure 5E and 5F show that the protein level of CCND1 decreased significantly when ODC was downregulated (P < 0.01), and the protein level of CCND1 was considerably increased when ODC was upregulated (P < 0.01).
Effect of ODC interference and overexpression on the apoptosis of GCs
qRT-PCR was applied to detect the expression of cell apoptosis-related genes after ODC interference and overexpression. As shown in Fig. 6C, under ODC expression interference, the relative expression of BAX, CASPASE 8 and CASPASE 9 mRNA was greatly upregulated (P < 0.05). When ODC was overexpressed (Fig. 6D), the expression of the proapoptotic gene BAX was remarkably reduced (P < 0.05), and that of BCL-2, CASPASE 3, CASPASE 8 and CASPASE 9 was not significantly changed (P > 0.05).
Western blotting was applied to assess the influence of ODC interference and overexpression on the protein levels of apoptosis-related genes in GCs. BAX and BCL-2 increased significantly after ODC interference (P < 0.05) (Fig. 6E). BAX was markedly reduced after ODC overexpression (P < 0.05), whereas BCL-2 showed no notable change (P > 0.05) (Fig. 6F).
Furthermore, flow cytometry was utilized to test the influence of ODC interference and overexpression on the apoptosis of GCs. The apoptosis rate of GCs was greatly upregulated after interfering with ODC (P < 0.05) (Fig. 6A), and the apoptosis rate of GCs was significantly downregulated when ODC was overexpressed (P < 0.05) (Fig. 6B).