Mechanisms underlying FSHβ gene sensitivity to GnRH pulse frequency [031615data]. Mus musculus

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.

生殖功能的正常维持依赖于下丘脑促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）的脉冲式释放，该激素可调控垂体促性腺激素卵泡刺激素（follicle-stimulating hormone, FSH）与黄体生成素（luteinizing hormone, LH）的表达。Fshb的表达对GnRH脉冲频率呈现倒U型响应。当GnRH脉冲频率提升至约每2小时1次以上时，尽管平均GnRH浓度升高，Fshb的诱导水平反而逐渐降低。为阐明Fshb基因调控的潜在机制，我们构建了3种具有生物学合理性且拓扑结构各异的数学模型。这三个模型分别对应：1）Fshb抑制性与刺激性因子的平行激活（如抑制素α、VGF）；2）具有不应期的信号组分激活（如G蛋白）；3）Fshb诱导所需因子的失活（如GDF9）。

利用这三种模型进行的模拟，均复现了不同GnRH脉冲频率下标准灌流实验中测得的Fshb表达水平。值得注意的是，针对平均浓度、脉冲时长与频率的模拟显示，模型1所呈现的表观频率依赖性Fshb表达模式，实则源于平均GnRH浓度的变化。与之相反，模型2与模型3则展现出“真正的”频率感知特性。

为明确GnRH信号的哪些组分可诱导Fshb表达，我们开发了大规模并行实验系统。对约40组实验中的4000余份样本进行分析后发现：尽管早期基因Egr1与Fos仅响应GnRH浓度的变化，但Fshb的诱导可在维持平均浓度不变的前提下，对GnRH脉冲频率的改变产生敏感响应。上述结果为理解不同调控因子在调节Fshb基因应答过程中的作用提供了理论框架。

总体实验设计：将750,000个细胞接种于经组织培养处理的盖玻片上，于添加10%胎牛血清（fetal bovine serum, FBS）的DMEM培养基中培养两天。脉冲实验前，将细胞置于添加2%活性炭处理FBS与20mM HEPES的DMEM培养基中孵育过夜。将盖玻片置于惰性盖玻片架中，通过在维持于37℃水浴槽内的培养腔中依次切换GnRH、冲洗液与静置液，实现不同的脉冲给药模式。水浴槽内的培养腔溶液为添加2%活性炭处理FBS与20mM HEPES的DMEM培养基。针对每一种条件/时间点，均收集至少3份生物学重复样本。细胞的脉冲周期（T）分别设置为30分钟、1小时、2小时或4小时，脉冲时长分别为2.5分钟、5分钟或10分钟，脉冲GnRH浓度分别为0.5nM、1nM或2nM。同时设置“无脉冲对照”样本。ET为起始点（即第1次脉冲）至样本收集时刻的经过时间。