Dissection of a novel molecular pathway involved in seasonal timing in a melatonin-target tissue using an experimental and systems-level approach.

大多数野生动物物种和许多人类驯养的物种都适应于生活在季节性环境中，并在食物供应和温度方面经历显着的年度变化。季节性动物必须在一年中的适当时间开始繁殖并储存脂肪。为了实现这一点，他们操作一个季节性的时钟，控制许多激素节奏的时间。调节这个季节性计时器的关键激素称为褪黑激素，它是在大脑的松果体中产生的。褪黑激素在夜间分泌，分泌模式随季节变化，在漫长的冬夜产生较长的持续时间。众所周知，这些季节性持续时间的变化驱动季节性激素节律，并为大脑提供外部光周期变化的内部表示，作用于生理和行为。褪黑激素作用于一个专门的结构，称为结节部（PT），位于脑垂体中，靠近大脑底部的下丘脑。PT被认为通过分泌一种作用于垂体远端组织中催乳素分泌细胞的局部因子来调节催乳素分泌的季节性节律。它还在局部产生一种激素，称为促甲状腺激素（TSH），我们现在怀疑它作用于下丘脑中称为伸长细胞的细胞上的TSH受体。在这里，它调节控制甲状腺激素活性的关键酶的活性。通过这种方式，PT可以作用于垂体和下丘脑。我们发现PT中的一组基因在PT暴露于短日照长时间褪黑激素信号时变得活跃，并且也直接对褪黑激素产生反应。这些基因作用于对产热至关重要的途径，并控制储存脂肪储备的合成和使用。我们的工作旨在确定这些“代谢”基因如何在这种季节性时间结构中用于控制年度激素节律。为了监测输出，我们专注于激素催乳素，在那里我们可以通过与PT细胞培养来测量活性，以及TSH的产生，我们可以通过分析或基因表达的测量来测量。我们的目标是找出褪黑激素信号如何作用于PT驱动导致这两种激素途径激活的遗传途径。最后，我们发现，先前已知参与许多组织（包括激素分泌细胞）发育的另一组基因也在PT中激活，以响应日照变化。我们怀疑这些“发育”基因与上述代谢途径基因有关。我们的研究将使用几种不同的技术，大部分工作我们将使用羊。原因是绵羊PT易于进行解剖学研究，并且可以在实验室中培养，使我们能够测试哪些代谢途径基因可能参与激素调节。首先，我们将详细描述PT中代谢和发育基因的活性变化，以及它们如何随季节变化，以及动物何时暴露于日照长度和褪黑激素的突然变化。然后，我们将继续研究PT中的蛋白质如何相互作用，以及如何与DNA相互作用。这将最终使我们能够描述褪黑激素靶细胞内的“电路图”，并描述褪黑激素如何激活或抑制基因。我们将使用培养系统，通过测量TSH活性（直接测量）和对催乳素分泌细胞的作用（间接测量），观察褪黑激素信号的变化是否会导致激素输出的改变。我们还将使用实验室啮齿动物（仓鼠和大鼠），因为在这里我们可以更容易地管理药物作用于“代谢”途径基因，看看我们是否看到激素分泌的变化。使用仓鼠的最后一个优点是，我们将能够检查不同类型的季节性饲养者的绵羊的结果。

Most species of wild animal and many of man's domesticated species are adapted to live in seasonal environments and experience significant annual changes in food supply and temperature. It is essential that seasonal animals time the onset of breeding and lay down and store fat at the appropriate time of year. In order to achieve this, they operate a seasonal clock which controls timing of many hormone rhythms. A key hormone regulating this seasonal timer is called Melatonin, which is produced within the brain in the pineal gland. Melatonin is secreted at night and the pattern of secretion changes seasonally, with longer-duration profiles produced on the long winter nights. It is known that these changes in seasonal duration drive seasonal hormone rhythms and provide the brain with an internal representation of external photoperiod change, acting on physiology and behaviour. Melatonin acts on a specialised structure called the pars tuberalis (PT) located in the pituitary gland, in a region close to the hypothalamus in the base of the brain. The PT is thought to regulate seasonal rhythms of prolactin secretion by secreting a local factor which acts on prolactin-secreting cells in the distal pituitary tissue. It also produces a hormone locally, called thyroid stimulating hormone (TSH), which we now suspect acts on TSH receptors on cells called tanycytes in the immediate hypothalamus. Here it regulates activity of key enzymes controlling thyroid hormone activity. By this means, the PT may act both on the pituitary and also the hypothalamus. We have discovered a group of genes in the PT which become active when the PT is exposed to long-duration melatonin signals on short daylengths and are also directly responsive to melatonin. These genes act on pathways which are crucial for thermogenesis, and controlling the synthesis and use of stored fat reserves. Our work aims to establish how these 'metabolism' genes may be used in this seasonal timing structure to control annual hormone rhythms. In order to monitor output, we focus on the hormone prolactin, where we can measure activity by culturing with PT cells, and on TSH production, which we can measure by assay or measures of gene expression. The goal is to work out how the melatonin signal acting on the PT drives genetic pathways which result in activation of these two hormone pathways. Finally, we have discovered that another group of genes previously known to be involved in development of many tissues including hormone secreting cells are also activated in the PT in response to daylength change. We suspect that these 'developmental' genes are linked to the metabolic pathway genes above. Our study will use several different techniques, and for much of the work we will use sheep. The reason is that the sheep PT is easy to undertake anatomical studies and can be cultured in the laboratory, allowing us to test which metabolic pathway genes may be involved in hormone regulation. First, we will describe in detail changes in activity of the metabolic and developmental genes in the PT and how they change, both with season, and when animals are exposed to abrupt changes in daylength and melatonin. We will then go on to study how proteins in the PT interact with one another, and also with DNA. This will ultimately allow us to describe a 'circuit diagram' within a melatonin-target cell and describe how genes may be activated or suppressed by melatonin. We will use the culture system to see whether changes in the melatonin signal result in altered hormone output, by measuring TSH activity (direct measure) and action on prolactin-secreting cells (in-direct measure). We will also use laboratory rodents (hamsters and rats) as here we can more easily administer drugs which act on the 'metabolic' pathway genes and see whether we see changes in hormone secretion. A final advantage to using hamsters is that we will be able to check results from sheep in a different type of seasonal breeder.