Development of the GnRH neuronal network and effects of prenatal androgen exposure

项目摘要 促性腺激素释放激素(GnRH)神经元形成了调节生育的最终共同中枢通路。 正常模式的GnRH释放是生育所必需的，在患有多囊卵巢的女性中经常被干扰 综合征(PCOS)。高雄激素性多囊卵巢综合征影响约8%-10%的女性。在这些女性身上，有一种坚持不懈的 高频率的黄体生成素，可能还有促性腺激素释放激素。产前雄激素(PNA)小鼠 有类似于多囊卵巢综合征女性的神经内分泌表型，包括高促黄体生成素脉冲频率，并且可以 用于研究这种增长的机制。类似于多囊卵巢综合征的病理生理学在年轻患者中被检测到 年龄，这表明这种疾病的前驱可能是发育编程的。在前面的工作中 从不同的筹资机制(NCTRI)，我们表征了GnRH神经元活性和 GABA传递到这些细胞，表明PNA在青春期之前扰乱了这两个参数，而PNA- 青春期前后引起的变化是不同的。其背后的神经生物学机制 观察结果在很大程度上是未知的。我们解释这些发现的工作模型是：1)PNA改变了 促性腺激素释放激素神经元及其传入的生物物理特性；2)至少部分改变了表观遗传程序 3)PNA在青春期前增加对GnRH神经元的兴奋性GABA突触驱动 4)在PNA小鼠青春期前，GnRH神经元启动内源性变化 以适应增加的GABA驱动，并减少放电输出；5)PNA小鼠的发育变化 导致这些GnRH神经元适应失败，因此在PNA成人中，GABA驱动增加有助于 GnRH神经元放电增加；6)神经内分泌驱动增加雄激素，这是至关重要的 以维持成人的神经内分泌PNA表型。我们将在两个目标上测试这个模型。目标1将确定 PNA小鼠GnRH神经元对GABA驱动增强的青春期前适应机制。 目的2将描述促性腺激素释放激素神经元在发育过程中的表观遗传格局，以及由此引起的变化 被巴勒斯坦民族权力机构。卵巢和雄激素替代在建立和维持表观遗传学改变中的作用 也将接受评估。初步数据表明，GnRH神经元动作电位放电、钙电流和 与对照组相比，青春期前PNA小鼠和成年PNA小鼠的钾电流都有不同的调节。至 作为电生理学研究的补充，我们已经将表观遗传学图谱修改为由几个 数百个神经元和建立的荧光细胞分选方案，产生足够数量的浓缩细胞 用于这些分析的GnRH神经元。因此，我们可以研究分子和生物物理。 观察到PNA小鼠GnRH神经元功能变化的基础。这项工作将提供 目前缺乏对促性腺激素释放激素神经元典型功能发育的机械论见解 青春期过程，相关的表观遗传学变化，以及这些参数可能如何在 高雄激素性疾病；不可能从人类研究中获得这些见解。

Project Summary Gonadotropin-releasing hormone (GnRH) neurons form the final common central pathway regulating fertility. Properly patterned GnRH release is required for fertility and is often disrupted in women with polycystic ovary syndrome (PCOS). Hyperandrogenic PCOS affects ~8-10% of women. In these women, there is a persistent high frequency of luteinizing hormone (LH), and likely GnRH, release. Prenatally androgenized (PNA) mice have neuroendocrine phenotypes similar to women with PCOS, including high LH pulse frequency, and can be used to study mechanisms of this increase. Pathophysiology similar to PCOS is being detected at younger ages, suggesting the antecedents of this disorder may be developmentally programmed. In the previous work from a different funding mechanism (NCTRI), we characterized the development of GnRH neuron activity and GABA transmission to these cells, showing that PNA disrupts both parameters before puberty, and that PNA- induced changes before and after puberty are different. The neurobiological mechanisms underlying these observations are largely unknown. Our working model to explain these findings is that 1) PNA alters the biophysical properties of GnRH neurons and their afferents; 2) altered epigenetic programing at least in part underlies these changes; 3) PNA increases excitatory GABA synaptic drive to GnRH neurons before puberty and this increase continues in adults; 4) before puberty in PNA mice, GnRH neurons initiate intrinsic changes to adapt to the increased GABA drive, and firing output is reduced; 5) developmental changes in PNA mice lead to failure of these GnRH neuron adaptations, so that in PNA adults, increased GABA drive contributes to increased GnRH neuron firing; 6) the increased neuroendocrine drive increases androgens, which are critical to maintain neuroendocrine PNA phenotypes in adults. We will test this model in two aims. Aim 1 will identify the mechanisms underlying prepubertal adaptation of GnRH neurons in PNA mice to increased GABA drive. Aim 2 will characterize the epigenetic landscape in GnRH neurons during development, and changes induced by PNA. The role of the ovary and androgen replacement in establishing and maintaining epigenetic changes will also be assessed. Preliminary data indicate that GnRH neuron action potential firing, calcium currents and potassium currents are all differentially regulated in prepubertal vs adult PNA mice compared to controls. To complement the electrophysiology studies, we have adapted epigenetic profiling to libraries made from a few hundred neurons and established fluorescent cell sorting protocols that yield sufficient numbers of enriched GnRH neurons for these analyses. We are thus positioned to examine the molecular and biophysical underpinnings of the functional changes of GnRH neurons observed in PNA mice. This work will provide mechanistic insight currently lacking on the typical functional development of GnRH neurons through the pubertal process, associated epigenetic changes, and how these parameters may be altered in hyperandrogenic disorders; is not possible to obtain these insights from human studies.