Growth differentiation factor 9 (GDF9) forms an incoherent feed-forward loop modulating follicle-stimulating hormone...

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus and regulates follicle-stimulating hormone β-subunit (FSHβ) gene expression in pituitary gonadotropes in a frequency-sensitive manner. The mechanisms underlying its preferential and paradoxical induction of FSHβ by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a GnRH-suppressed autocrine inducer of FSHβ gene expression. GDF9 gene transcription and expression were preferentially decreased by high frequency GnRH pulses. GnRH regulation of GDF9 was concentration-dependent and involved ERK and PKA. GDF9 knockdown or immunoneutralization reduced FSHβ mRNA expression. Conversely, exogenous GDF9 induced FSHβ expression in immortalized gonadotropes and in mouse primary pituitary cells. GDF9 exposure increased FSH secretion in rat primary pituitary cells. GDF9 induced Smad2/3 phosphorylation, which was impeded by ALK5 knockdown and by activin receptor-like kinase (ALK) receptor inhibitor SB-505124, which also suppressed FSHβ expression. Smad2/3 knockdown indicated that FSHβ induction by GDF9 involved Smad2 and Smad3. FSHβ mRNA induction by GDF9 and GnRH was synergistic. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHβ gene. To test this, we determined the effects of GDF9 knockdown on FSHβ induction at different GnRH pulse frequencies using a parallel perifusion system. Reduction of GDF9 shifted the characteristic pattern of GnRH pulse frequency sensitivity. These results identify GDF9 as contributing to an incoherent feed-forward loop, comprising both intracellular and secreted components, that regulates FSHβ expression in response to activation of cell surface GnRH receptors.