Lipid droplets in oocytes: shedding new light on why fats are good or bad for development.

肥胖在发达国家是一个日益严重的健康问题，它对生殖有许多影响。此外，肥胖母亲的孩子健康状况明显较差，更有可能肥胖。肥胖妇女在怀孕和维持妊娠方面有更大的问题。人们发现，与肥胖和高脂肪饮食有关的许多问题都存在于卵细胞中。当卵母细胞暴露于脂肪中时，它们会在细胞质中积聚过量的由脂质构成的小液滴。当卵母细胞中有太多的脂滴时，它们的发育就会很差，即使它们作为胚胎移植到不肥胖的养母体内也是如此。脂肪的破坏作用见于细胞内的结构，如线粒体和内质网。然而，人们仍然不知道脂肪是如何破坏这些结构的。目前还不清楚卵母细胞是如何调节它们的脂滴的，因为虽然过多的脂肪对卵母细胞有害，但脂肪代谢也是必不可少的。卵母细胞利用储存在脂滴中的脂肪在线粒体中产生能量，这一过程被称为氧化。如果线粒体中的脂肪氧化过程被阻断，卵母细胞或胚胎的发育就会很差。因此，我们知道卵母细胞需要一些脂肪，但不是太多。我们不知道的是为什么会出现这种情况，以及到底多少才算太多。这一领域的研究受到研究脂质技术的限制，这些脂质使用染料，涉及固定并因此破坏卵母细胞。因此，在本项目中，我们将采用一种新的成像方法来测量小鼠卵母细胞中的脂滴。这种方法被称为相干反斯托克斯拉曼散射显微镜（CARS显微镜）。在保持小鼠卵母细胞和胚胎存活的情况下，我们可以利用红外线观察到小鼠卵母细胞和胚胎中的脂滴。CARS探测到由脂肪酸的碳氢链中振动的化学键散射的光。它是专门的，精确的，不需要任何化学品或染料。我们将使用CARS显微镜来量化喂食高脂肪饮食的小鼠卵母细胞的脂质含量，或者保存在不同脂肪酸培养中的卵母细胞。我们将精确地量化多少脂质对发育是最适宜的，并精确地显示在不同脂肪中孵育的效果是如何模仿高脂肪饮食的效果的。然后，我们将结合CARS显微镜与其他活细胞成像技术。这将使我们能够验证卵母细胞需要使用碳水化合物和脂肪的特定平衡来产生能量的想法。我们还将研究过多的饱和脂肪会导致卵母细胞失去对Ca2+水平的控制，这就是为什么这些脂肪是有毒的。最后，我们将建立一种研究卵母细胞脂质代谢的新方法，用CARS显微镜观察在氢被氘原子取代的脂肪酸中培养的卵母细胞。这给了碳氘键一个不同的振动频率，这意味着它们有一个独特的CARS信号。这将允许我们实时观察脂肪酸被吸收并在活卵母细胞中使用。我们的工作将确定与良好发育一致的脂质含量范围，准确解释为什么卵母细胞和胚胎需要平衡使用脂质和碳水化合物，并引入一种量化脂滴中脂肪摄取和代谢的新方法。这项工作将大大促进我们对肥胖为什么会损害人类和动物的卵母细胞和胚胎的理解，并为如何逆转这种损害开辟新的研究。这一新发现将为旨在扭转不良饮食和肥胖不良影响的研究提供基础，从而支持几代人终生改善健康状况。

Obesity is a growing health problem in developed countries and it has numerous effects on reproduction. Moreover the children of obese mothers are significantly less healthy and more likely to be obese. Obese woman have greater problems in conceiving and in maintaining pregnancy. It has been found that many of the problems associated with obesity and a high fat diet are seen in the egg cells, called oocytes. When oocytes are exposed to fats they accumulate an excess of small droplets made from lipid that can been seen in their cytoplasm. When there are too many of these lipid droplets in oocytes they show very poor development, even when they are transferred as embryos to foster mothers who are not obese. The damaging effects of fats are seen in structures inside the cell such as the mitochondria and the endoplasmic reticulum. However, it is still not known how fats damage these structures. It is also unclear exactly how oocytes regulate their lipid droplets, because whilst an excess of fat is bad for oocytes, fat metabolism is also essential. The oocyte uses fats stored in the lipid droplets to make energy in their mitochondria in a process called oxidation. If this process of fat oxidation in mitochondria is blocked, the oocyte or embryo shows very poor development. Hence, we know that oocytes needs some fat, but not too much. What we don't know is why this is the case, and exactly how much is too much. Studies in this field are limited by techniques for studying lipids which use dyes and involve fixing and hence destroying oocytes. So in this project we shall use a new imaging method to measure lipid droplets in mouse oocytes. This method is called Coherent Antistokes Raman Scattering microscopy (CARS microscopy). We can visualise lipid droplets in mouse oocytes and embryos using infrared light, while keeping them alive. CARS detects light scattered by the vibrating chemical bonds in the carbon hydrogen chains of the fatty acids. It is specific, precise, and does not require any chemicals or dyes. We will use CARS microscopy to quantify the lipid content of oocytes from mice fed a high fat diet, or from oocytes that have been kept in culture with different fatty acids. We shall quantify exactly how much lipid is optimal for development, and show precisely how well the effects of incubation in different fats mimics the effects of the high fat diet. We shall then combine CARS microscopy with other live cell imaging techniques. This will allows us to test the idea that to be viable the oocytes need to use a specific balance of carbohydrates as well as fats to produce their energy. We shall also investigate the idea that too much saturated fat causes the oocyte to lose control of its Ca2+ levels and that this is why such fats are toxic. Finally we shall establish a novel method to study lipid metabolism in oocytes by using CARS microscopy on oocytes incubated in fatty acids where the hydrogens are replaced by deuterium atoms. This gives a different frequency of vibration of the carbon deuterium bonds and this means they have a distinctive CARS signal. This will then allow is to see fatty acids being taken in and used in living oocytes in real time. Our work will establish the range of lipid content consistent with good development, exactly why oocytes and embryos need a balance use of lipids and carbohydrates, and introduce a new way of quantifying uptake and metabolism of fats in lipid droplets. This work will significantly advance our understanding of why obesity damages oocytes and embryo in humans and animals, and open up new investigations into how to reverse some of this damage. This new knowledge will underpin investigations aimed at reversing the ill effects of poor diet and obesity supporting life-long improvements in health across the generations.