Figure 4: GnRH down-regulates AMH receptivity in vitro in LβT2 cells and in vivo in pituitary of female rats at pnd 18.

(a) GnRH inhibits AMH signaling. LβT2 cells were stimulated for 24 h with 2.5 μg/ml AMH, 10 nM GnRHa or with a combination of both hormones. P-Smad1/5/8 protein level was evaluated by immunoblotting and normalized with total Smad1. *P ≤ 0.05 compared with AMH-stimulated cells. (b) Combined effects of GnRH and AMH on Fshb expression. Cells were incubated with 2.5 μg/ml AMH, 10 nM GnRHa or with both hormones for 24 h. Fshb mRNA levels were analyzed by real-time qPCR and expressed as percentage of control levels. *P ≤ 0.05 ; **P ≤ 0.01 compared with control cells. (c) GnRH down-regulates Amhr2 expression. Cells were incubated with 10 nM GnRHa, 10 ng/ml activin A or 20 ng/ml BMP2 for 4 and 24 h. Amhr2 mRNA levels were determined by real-time qPCR and expressed as percentage of control cells. Results are the mean ± SEM of 8 independent experiments. *P ≤ 0.05 compared with control cells. (d) GnRH decreases AMHR2 protein level. Cells were treated for 24 h with 10 nM GnRHa and AMHR2 protein level was determined by immunoblotting after normalization with vinculin. Results are the mean ± SEM of 4 independent experiments. *P ≤ 0.05 compared with control cells. (e) GnRH down-regulates Amhr2 expression in pituitary of female rats at pnd 18. Male and female rats were injected subcutaneously at pnd 17 with 100 μl of saline solution containing or not 0.1 μg of GnRHa. Anterior pituitary Amhr2 expression was determined 24 h after injection by Taqman real time qPCR. Each value is a mean ± SEM of 8 to 14 rats. *P ≤ 0.05 compared to control rats; a, P ≤ 0.01 compared to female control rats.