Impaired production of BMP-15 and GDF-9 mature proteins derived from proproteins WITH mutations in the proregion

Abstract

Mutations in the bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) genes have been identified in women with primary ovarian insufficiency (POI) and mothers of dizygotic twins. Here, we show that biological activities of the conditioned media from human embryonic kidney 293F cells transfected with two representative BMP-15 and GDF-9 mutants identified in the affected women have significantly reduced biological activities compared with the corresponding wild-type. Moreover, this difference is due to decreased production of the mature proteins, attributed most likely to impaired posttranslational processing of the proprotein. As genetic studies of the BMP-15 and/or GDF-9 genes in ewes established that a reduction of these proteins is associated with an increased ovulation rate, it is conceivable that women affected with these mutations may have an increased probability of bearing dizygotic twins during active reproductive ages before diagnosis with POI at later ages due to an earlier exhaustion of ovarian reserve.

Fig. 1

Effect of wild-type and mutant rhBMP-15 (A) and rhGDF-9 (B) on the Smad signaling pathways. COV-434 (A) and P19 (B) cells were cultured for 1 h with conditioned medium collected from 293F cells cultured for 4 days after the transfection with the indicated plasmids. Cell lysates were then subjected to Western immunoblotting analysis using indicated Smad antibodies. Representative data obtained from three independent experiments are presented in the upper panels. The band intensity of scanned images was statistically analyzed and the cumulative data from three independent experiments are shown with mean ± SEM mean in the lower panels. Each experiment was performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.

Fig. 2

Effect of wild-type and mutant rhBMP-15 (A) and rhGDF-9 (B) on rat granulosa cell mitosis. Granulosa cells were cultured for 24 h with conditioned medium collected from 293F cells cultured for 4 days after transfection with the indicated plasmids, followed by additional 24 h culture in the presence of 10 µM BrdU. The BrdU incorporated into the cells was quantified by a BrdU ELISA assay. All results are shown as mean ± SEM of data from at least three separate experiments, each performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.

Fig. 3

Quantitative RT-PCR analysis for transfected 293F cells. Total cellular RNAs were extracted from 293F cells transfected with the indicated plasmids. For the quantification of BMP-15 (A) and GDF-9 (B) mRNA levels, real-time PCR was performed, and the expression levels of target genes were standardized by RPL19 level in each sample. All results are shown as mean ± SEM of data from at least three separate experiments, each performed with triplicate samples.

Fig. 4

Production of BMP-15 by 293F cells transfected with wild-type and mutant plasmids. The 293F cells transfected with the indicated BMP-15 plasmids were cultured for the indicated hours, and cell lysates were subjected to SDS/PAGE immunoblotting analysis using anti-FLAG antibody (Ab) and anti-β-actin Ab. Note that all expression plasmids have a Flag epitope at the C-terminus. CTRL: control cells transfected with the empty vector. Results are shown as representative of those obtained from three independent experiments in panel A. The band intensity of scanned images was statistically analyzed, and the cumulative data from three independent experiments are presented with mean ± SEM in panel B. Each experiment was performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.

Fig. 5

Production of BMP-15 by 293F cells transfected with wild-type and mutant plasmids. The 293F cells transfected with the indicated plasmids were cultured for the indicated hours, and conditioned media were treated with acetone to precipitate proteins followed by the SDS/PAGE immunoblotting analysis using anti-FLAG Ab. CTRL: control cells transfected with the empty vector. Results are shown as representative of those obtained from three independent experiments in panel A. The band intensity of scanned images was statistically analyzed (panel B). All results are shown as mean ± SEM of data from at least three separate experiments, each performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.

Fig. 6

Production of GDF-9 by 293F cells transfected with wild-type and mutant plasmids. The 293F cells transfected with the indicated plasmids were cultured for the indicated hours, and cell lysates were subjected to SDS/PAGE immunoblotting analysis using anti-FLAG Ab and anti-β-actin Ab. CTRL: control cells transfected with the empty vector. Results are shown as representative of those obtained from three independent experiments in panel A. The band intensity of scanned images was statistically analyzed, and the cumulative data from three independent experiments are presented with mean ± SEM in panel B. Each experiment was performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.

Fig. 7

Production of GDF-9 by 293F cells transfected with wild-type and mutant plasmids. The 293F cells transfected with the indicated plasmids were cultured for the indicated hours, and conditioned media were treated with acetone to precipitate proteins followed by the SDS/PAGE immunoblotting analysis using anti-FLAG Ab. CTRL: control cells transfected with the empty vector. Results are shown as representative of those obtained from three independent experiments in panel A. The band intensity of scanned images was statistically analyzed (panel B). All results are shown as mean ± SEM of data from at least three separate experiments, each performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.

Fig. 8

Ratios in the expression levels of the mature proteins to the corresponding proproteins produced by 293F cells transfected with wild-type and mutant BMP-15 (A) and GDF-9 (B). The 293F cells transfected with the indicated plasmids were cultured for 96 h. Cell lysates and conditioned media were then subjected to immunoblotting analysis using anti-FLAG Ab. The ratios of the mature proteins to the corresponding proproteins were determined using densitometric analysis of the SDS/PAGE immunoblotting results. All results are shown as mean ± SEM of data from at least three separate experiments, each performed with triplicate samples. *, P <0.05; **, P < 0.01 between the indicated groups.