Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells

Abstract

In the ovary, connexin-coupled gap junctions in granulosa cells play crucial roles in follicular and oocyte development as well as in corpus luteum formation. Our previous work has shown that theca cell-derived bone morphogenetic protein (BMP)4 and BMP7 decrease gap junction intercellular communication (GJIC) activity via the down-regulation of connexin43 (Cx43) expression in immortalized human granulosa cells. However, the effects of oocyte-derived growth factors on Cx43 expression remain to be elucidated. The present study was designed to investigate the effects of oocyte-derived growth differentiation factor (GDF)9 and BMP15 on the expression of Cx43 in a human granulosa cell line, SVOG. We also examined the effect relative to GJIC activity and investigated the potential mechanisms of action. In SVOG cells, treatment with BMP15 but not GDF9 significantly decreased Cx43 mRNA and protein levels and GJIC activity. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by co-treatment with a BMP type I receptor inhibitor, dorsomorphin. Furthermore, knockdown of the central component of the transforming growth factor-β superfamily signaling pathway, Smad4, using small interfering RNA reversed the suppressive effects of BMP15 on Cx43 expression and GJIC activity. The suppressive effects of BMP15 on Cx43 expression were further confirmed in primary human granulosa-lutein cells obtained from infertile patients undergoing an in vitro fertilization procedure. These findings suggest that oocyte-derived BMP15 decreases GJIC activity between human granulosa cells by down-regulating Cx43 expression, most likely via a Smad-dependent signaling pathway.

Keywords: BMP15; GDF9; connexin43; human granulosa cell; smad.

Figure 1

BMP15 but not GDF9 down-regulates Cx43 mRNA expression in SVOG cells. (A and C) Cells were treated with different concentrations (1, 10 or 100 ng/ml) of GDF9 or BMP15 for 24 h; Cx43 mRNA (A) and protein (C) levels were examined by reverse transcription and real-time quantitative PCR (RT-qPCR) and western blot analysis. (B and D) Cells were treated with 50 ng/ml GDF9 or BMP15 for 3, 6, 12 or 24 h; Cx43 mRNA (B) and protein (D) levels were examined by RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). Ctrl, control. B15, BMP15; G9, GDF9.

Figure 2

The BMP15-induced down-regulation of Cx43 in SVOG cells is abolished by treatment with the BMP type I receptor inhibitor dorsomorphin. (A and B) To localize the distribution of Cx43 in SVOG cells, cells were transfected with 25 nM control siRNA (siCtrl) or (GJA1) Cx43 siRNA (siCx43) for 48 h. The knockdown efficiency of each siRNA was examined by western blot analysis (A). Following the transfection of the cells with 25 nM siCtrl or siCx43 for 48 h, the cells were fixed in 4% paraformaldehyde in PBS, and examined for Cx43 immunofluorescence (red) (B). (C and D) Cells were treated with 50 ng/ml BMP15 for 24 h in the presence of vehicle control (DMSO) or 5 μM dorsomorphin. The levels of Cx43 protein were examined by western blot analysis (C) or immunofluorescence microscopy (D). (E and F) Cells were treated with 50 ng/ml BMP15 for 24 h in the presence of vehicle control (DMSO) or 5 μM SB431542. The levels of Cx43 protein were examined by western blot analysis (E) or immunofluorescence microscopy (F). Cell nuclei were stained with Hoechst 33258. Scale bar represents 50 μm. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). Ctrl, control; DM, dorsomorphin.

Figure 3

BMP15 but not GDF9 activates the Smad1/5/8 signaling pathway in SVOG cells. (A) Cells were treated with 50 ng/ml BMP15 or GDF9 for 30, 45 or 60 min; the phosphorylation levels of Smad1/5/8 were examined by western blot analysis. (B) Cells were treated with 50 ng/ml BMP15 for 60 min in the presence of vehicle control (DMSO) or 5 μM dorsomorphin; the phosphorylation levels of Smad1/5/8 were examined by western blot analysis. (C) Cells were treated with 50 ng/ml BMP15 for 60 min in the presence of vehicle control (DMSO) or 5 μM SB431542; the phosphorylation levels of Smad1/5/8 were examined by western blot analysis. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). Ctrl, control; DM, dorsomorphin; B15, BMP15; G9, GDF9.

Figure 4

Knockdown of Smad4 abolishes the BMP15-induced down-regulation of Cx43 in SVOG cells. (A) Cells were transfected for 48 h with transfection reagent (Lipofectamine RNAiMAX), 25 or 50 nM control siRNA (siCtrl) or Smad4 siRNA (siSmad4). The knockdown efficiency of Smad4 siRNA was examined by western blot analysis. (B) Cells were transfected for 48 h with 25 nM control siRNA (siCtrl) or Smad4 siRNA (siSmad4) and then treated for 24 h with 50 ng/ml BMP15 or vehicle control. The levels of Cx43 protein were examined by western blot analysis. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). Ctrl, control; B15, BMP15.

Figure 5

BMP15 decreases GJIC activity in SVOG cells. (A) Fully confluent cells were treated with 50 ng/ml BMP15 for 24 h in the presence or absence of 5 μM dorsomorphin (DM). (B) Cells were transfected for 48 h with 25 nM control siRNA (siCtrl) or Smad4 siRNA (siSmad4) and then treated for 24 h with 50 ng/ml BMP15. The GJIC activity was measured by monitoring the transfer of Lucifer yellow fluorescent dye between cells, and the images were captured utilizing a fluorescence microscope (top panel). The corresponding phase contrast micrographs are shown in the bottom panel. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). Ctrl, control; DM, dorsomorphin.

Figure 6

BMP15, but not GDF9, down-regulates Cx43 protein expression in hGL cells. hGL cells were treated with different concentrations (1, 10 or 100 ng/ml) of GDF9 or BMP15 for 24 h; Cx43 mRNA (A) and protein (B) levels were examined by RT-qPCR and western blot analysis. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). hGL, human granulosa-lutein; Ctrl, control; G9, GDF9; B15, BMP15.