Figure 1.
FGF9 increases steroidogenesis in both mouse primary and tumor Leydig cells.
(A) Mouse primary Leydig cells were treated with different concentrations of FGF9 for 24 hr (Con = control). (B) MA-10 mouse Leydig tumor cells were treated without (control) or with 50 ng/ml FGF9 for 0 and 24 hr. At the end of each incubation, culture medium was collected, and testosterone (A) or progesterone (B) production was measured by RIA. Each bar represents the mean ± SEM of the fold difference in testosterone or progesterone production compared with the control group. Different letters above the bars indicate significant differences between treatments (p<0.05).
Figure 2.
Temporal effect of FGF9 on the expression levels of phospho-Akt, -JNK, -p38, and -ERK1/2 in mouse primary Leydig cells.
Mouse primary Leydig cells were treated without (control) or with FGF9 (50 ng/ml) for the indicated times. The expression levels of phospho-Akt (A), -JNK (B), -p38 (C), and -ERK1/2 (D) were detected by immunoblotting. Immunoblots represent the observations from one single experiment repeated in three different times. The integrated optical density of each protein was normalized to β-actin (43 kDa) in each lane. The fold difference of each of phospho-Akt, -JNK, -p38, and -ERK1/2 in the treatment group was normalized to the control group at each time point, and the mean ± SEM of three separate experiments is shown. Different letters above the bars indicate statistically significant differences between treatments (p<0.05).
Figure 3.
Temporal effect of FGF9 on the expression levels of phospho-Akt, -JNK, -p38, and -ERK1/2 in MA-10 cells.
MA-10 cells were treated without or with FGF9 (50 ng/ml) for the indicated times. The expression levels of phospho- Akt (A), -JNK (B), -p38 (C), and -ERK1/2 (D) were detected by immunoblotting. Immunoblots represent the observations from one single experiment repeated at least in three different times. The integrated optical density of each protein was normalized to β-actin (43 kDa) in each lane. The fold difference of each of phospho-Akt, -JNK, -p38, and -ERK1/2 in the treatment group was normalized to the control group at each time point, and the mean ± SEM of three separate experiments is shown. Different letters above the bars indicate statistically significant differences between treatments (p<0.05). P represents the positive control.
Figure 4.
Effect of inhibitors on FGF9-induced activation of Akt, JNK, p38, and ERK1/2 in mouse primary Leydig cells.
Mouse primary Leydig cells were pretreated with the Akt inhibitor wortmannin (100 nM) (A), JNK inhibitor SP600125 (1 µM) (B), p38 inhibitor SB202190 (1 µM) (C), or ERK inhibitor PD98059 (25 µM) (D) for 30 min and then incubated without or with FGF9 (50 ng/ml) for an additional 15 min. Immunoblots represent the observations from one single experiment repeated at least in three different times. The integrated optical density of each protein was normalized by β-actin (43 kDa) in each lane. Each bar represents the mean ± SEM fold difference of phospho-Akt, -JNK, -p38, and -ERK1/2 compared with the control group (Con) from three independent experiments. Different letters above the bars indicate statistically significant differences between treatments (p<0.05). W, wortmannin; SP, SP600125; SB, SB202190; PD, PD98059.
Figure 5.
Effect of inhibitors on FGF9-induced activation of Akt, JNK, p38, and ERK1/2 in MA-10 cells.
MA-10 cells were pretreated with the Akt inhibitor wortmannin (100 nM) for 1 hr (A), JNK inhibitor SP600125 (100 µM) for 30 min (B), p38 inhibitor SB202190 (1 µM) for 30 min (C), or ERK inhibitor PD98059 (25 µM) for 30 min (D). Then, cells were incubated without or with FGF9 (50 ng/ml) for 3 hr in the Akt inhibitor group, or for 15 min in the JNK, p38, and ERK1/2 inhibitor groups. Immunoblots represent the observations from one single experiment repeated at least in three different times. The integrated optical density of each protein was normalized by β–actin (43 kDa) in each lane. Each bar represents the mean ± SEM fold difference of phospho-Akt, -JNK, -p38, and -ERK1/2 compared with the control group (Con) from three independent experiments. Different letters above the bars indicate statistically significant differences between treatments (p<0.05). W, wortmannin; SP, SP600125; SB, SB202190; PD, PD98059.