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

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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.