A growth differentiation factor 9 (GDF9) motif regulates follicle-stimulating hormone b-subunit (FSHb) gene expression: involvement of activin receptor-like kinase receptors 4/5 (ALK4/5) in the induction of FSHb by GDF9

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus. GnRH regulates follicle-stimulating hormone b-subunit (FSHb) gene expression in pituitary gonadotropes in a frequency-sensitive manner that is central to reproductive physiology. The mechanisms underlying the preferential and paradoxical induction of FSHb by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a novel autocrine inducer of FSHb gene expression. GDF9 gene expression was preferentially suppressed by high frequency GnRH pulses due to reduced transcription. Exogenous GDF9 induced FSHb mRNA expression and knockdown or immunoneutralization of GDF9 reduced FSHb gene expression. Treatment with GDF9 stimulated Smad2/3 phosphorylation. The activin receptor-like kinase (ALK) receptor inhibitor SB-505124 antagonized GDF9-induced Smad2/3 phosphorylation and FSHb mRNA induction. Smad2 and Smad3 knockdown studies indicated that the induction of FSHb by GDF9 involves both Smad2 and Smad3. GDF9 and GnRH synergistically induced FSHb mRNA expression and high frequency GnRH pulses suppressed GDF9. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHb gene. To test this, we determined the effects of GDF9 knockdown on FSHb 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, comprised of both intracellular and secreted elementscomponents, that regulates FSHb expression in response to activation of the cell surface GnRH receptor. LbT2 microarray datasets as well as RNA-Seq data (GSE42120) were interrogated to determine the levels of expression of ALK4/5/7. LβT2 cells were transfected with either scrambled siRNA or Gas siRNA for 48 h. RNA samples were snap-frozen in dry ice prior to whole-genome expression profiling analysis using MouseWG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA). A total of 6 concentrated conditioned media samples were independently prepared: 3 replicates from control siRNA-treated cells, and 3 replicates from Gas siRNA-treated cells. This submission represents the microarray component of study.