Reproduction requires pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates expression of the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Fshb expression shows an inverted U-shaped response to GnRH pulse frequency. Increasing GnRH pulse frequency beyond ~one pulse/2 hours, despite increasing the average GnRH concentration, induces progressively less Fshb. To clarify regulatory mechanisms underlying Fshb gene control, we developed three biologically inspired and topologically distinct mathematical models. The models represent: 1) parallel activation of Fshb inhibitory and stimulatory factors (e.g. inhibin α, VGF), 2) activation of a signaling component with a refractory period (e.g. G protein), and 3) inactivation of a factor needed for Fshb induction (e.g. GDF9). Simulations with all three models recapitulated the Fshb expression levels obtained in standard perifusion experiments at different GnRH pulse frequencies. Notably, simulations altering average concentration, pulse duration and frequency showed that the apparent frequency-dependent pattern of Fshb expression obtained with model 1 actually resulted from variations in average GnRH concentration. In contrast, models 2 and 3 showed “true” frequency sensing. To resolve which components of this GnRH signal induce Fshb, a massively parallel experimental system was developed. Analysis of over 4000 samples in ~40 experiments indicated that, while early genes Egr1 and Fos respond only to variations in GnRH concentration, Fshb induction is sensitive to GnRH pulse frequency changes, whilst maintaining the same average concentration. These results provide a framework for understanding the role of different regulatory factors in modulating the responses of the Fshb gene.
Overall design: 750,000 cells were seeded on each tissue culture-treated coverslip and were grown for two days in DMEM supplemented with 10% FBS. Cells were incubated in DMEM supplemented with 2% charcoal-treated FBS and 20mM HEPES overnight before the pulse experiment. Coverslips were placed in inert coverslip racks and pulse patterns were achieved by moving racks among GnRH, wash and resting solutions in chambers maintained at 37°C in a water bath. The chamber solution in the water bath was DMEM supplemented with 2% charcoal-treated FBS and 20mM HEPES. For each condition/time point, a minimum of 3 biological replicates were collected. Cells were pulsed with a cycle period (T) of either 30 min, 1 h, 2 h or 4 h, a pulse duration of either 2.5 min, 5 min, or 10 min, and a pulse amplitude of either 0.5 nM, 1 nM, or 2 nM GnRH. 'No pulse control' samples were included. ET, elapsed time between starting point (i.e. pulse 1) and time of collection.
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