Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells

Abstract

Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated 'gene silencing' of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly (P<0.05) changed the expression of Cyclin D2, CDK4, Bcl-2, and Cyp19a1. These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.

Fig. 1

Analyses of the expression of Smad4 mRNA and protein by real-time PCR, Western blotting, and immunofluorescence at post-transfection (a) Expression of Smad4 mRNA after transfection for 48 h; (b) Expression of Smad4 protein after transfection for 48 h; (c) Silencing effect of Smad4 protein expression measured by Western blotting; (d) Silencing effect of Smad4 protein expression measured by immunofluorescence staining. After treatment, cells were fixed and stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI; blue). The data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences from the blank group (P<0.05)

Fig. 1

Analyses of the expression of Smad4 mRNA and protein by real-time PCR, Western blotting, and immunofluorescence at post-transfection (a) Expression of Smad4 mRNA after transfection for 48 h; (b) Expression of Smad4 protein after transfection for 48 h; (c) Silencing effect of Smad4 protein expression measured by Western blotting; (d) Silencing effect of Smad4 protein expression measured by immunofluorescence staining. After treatment, cells were fixed and stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI; blue). The data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences from the blank group (P<0.05)

Fig. 1

Analyses of the expression of Smad4 mRNA and protein by real-time PCR, Western blotting, and immunofluorescence at post-transfection (a) Expression of Smad4 mRNA after transfection for 48 h; (b) Expression of Smad4 protein after transfection for 48 h; (c) Silencing effect of Smad4 protein expression measured by Western blotting; (d) Silencing effect of Smad4 protein expression measured by immunofluorescence staining. After treatment, cells were fixed and stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI; blue). The data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences from the blank group (P<0.05)

Fig. 1

Analyses of the expression of Smad4 mRNA and protein by real-time PCR, Western blotting, and immunofluorescence at post-transfection (a) Expression of Smad4 mRNA after transfection for 48 h; (b) Expression of Smad4 protein after transfection for 48 h; (c) Silencing effect of Smad4 protein expression measured by Western blotting; (d) Silencing effect of Smad4 protein expression measured by immunofluorescence staining. After treatment, cells were fixed and stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI; blue). The data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences from the blank group (P<0.05)

Fig. 2

Effect of interrupted BMP/Smad signaling on cell cycle marker gene mRNA levels Data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences (P<0.05) among the groups

Fig. 3

Effect of interrupted BMP/Smad signaling on apoptosis related gene mRNA levels Data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences (P<0.05) among the groups

Fig. 4

Effect of interrupted BMP/Smad signaling on steroidogenic enzyme gene mRNA levels Data are presented as mean±SEM of three independent experiments. ^* Statistically significant differences (P<0.05) among the groups