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

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

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.

Technical Summary

Fatty acids oxidation is essential for mammalian oocyte maturation and development, but excess non-esterified fatty acids have toxic effects on oocytes. In this project we shall use Coherent Antistokes Raman Scattering (CARS) microscopy to study lipid droplets and fatty acid beta oxidation in mouse oocytes. Firstly, we will use CARS microscopy, with hyperspectral imaging, or simultaneous differential interference contrast microscopy, to determine the number, the distribution, and the saturated to unsaturated ratio of fatty acid chains in lipid droplets in oocytes from mice fed a high fat diet compared to control oocytes from mice on a conventional diet. We will compare these oocytes to those maturated in vitro in the presence of defined amounts of palmitic acid or oleic acid. We will determine the ability of oocytes imaged using CARS microscopy to undergo fertilization and development up to the blastocyst stage and after implantation. Secondly, we will establish the way in which excess saturated fatty acids produce toxic effects on oocytes. Specifically, we will investigate the link between excess saturated fatty acids and the loss of Ca2+ in the endoplasmic reticulum, and establish this as a major cause of the stress response and damage to mitochondria. Third, we will investigate the need for fatty acid oxidation in mitochondria by showing how a loss of beta-oxidation leads to excess pyruvate oxidation which then causes enhanced reactive oxygen species production and damage to the embryo. Finally, we will introduce a new technique to the field by using CARS imaging of oocytes incubated in deuterated fatty acids. This will be used to establish with high chemical specificity the uptake of fatty acids into and out of lipid droplets during in vitro maturation and development. This work will establish the optimal amount of lipid for oocyte viability and determine the biochemical reasons why too little, or too much saturated fatty acid, is detriment to development.

Planned Impact

The beneficiaries from this research will be:
The academic community. The outcome of this research will benefit researchers investigating the links between high fat diet, or obesity, and its effects on reproduction in animals and humans. It will also promote new methods for investigating the way embryos and cells handle excess fatty acid exposure.
The commercial sector. Food supplements are commonly used for cattle, to boost milk yields, and can interfere with reproduction. Our work will provide a framework for understanding the consequence of excess fatty acids in animal reproduction. We strongly expect that our results will encourage the use of CARS imaging in oocytes of domestic animals, where lipid droplets appear to provide the bulk of the energy supply for early development. Hence, our studies will showcase CARS microscopy and promote its widespread use, which will also strengthen the case for its commercial development as microscopy instrumentation. We expect that our work will show the extent to which DIC microscopy can be used to estimate the amount of lipid droplets in oocytes. This will encourage the developments of better standard high resolution microscopy to make assessments of oocyte and possibly embryos in IVF clinics and reproductive biology laboratories.
The public sector. Maternal obesity is an increasing concern in reproductive biology. Obesity in parents, and particularly the mother, not only affects the chances of achieving a pregnancy, but there is evidence that it affects the health of the foetus and may have transgenerational effects upon of the long term health of the offspring. The immediate problems associated with the oocyte appear to be caused by the exposure to fatty acids during growth and maturation. Our work will provide for the first time a mechanistic explanation for the effects of fatty acids upon oocytes and embryos. Furthermore, it has become apparent that the free fatty acid profile of human follicular fluid can have some predictive value of the success rate of IVF treatment. Our work will allow this correlation to be understood in direct and quantitative terms for the embryo. It will provide the basis for future proposals to study the relationship between human oocyte lipid content and the success rates in IVF treatment. It may be possible to either improve the chances of embryo development by tailoring culture media to the lipid content of oocytes, or else use the lipid content of embryos as a basis for selecting the embryos used for transfer to prospective mothers.
 
Description We planned to investigate the use of deuterium labelling of fatty acids combined with CARS microscopy as a new way of assessing lipid metabolism in mouse oocytes. We have carried out some experiments with CARS microscopy on oocytes with deuterium labelled palmitic acid. We have found that we can image the deuterium labelled lipid droplets in live mouse oocytes and that the loss of label reflects the metabolism of the fatty acids by mitochondria. In addition we have made measurements of ATP production in live oocytes. We have quantified the contribution that lipid oxidation by mitochondria makes to the overall ATP production in mouse oocytes. This work has now been published in the Journal of Cell Science as an original research paper: 'Dynamic label-free imaging of lipid droplets and their link to fatty acid and pyruvate oxidation in mouse eggs', by J Bradley, I Pope, W Langbein, P Borri and K Swann. This work has also been presented by Dr Josie Bradley at the UK conference 'Photon 2018', at Aston University, in 3-6 September 2018. It was also presented by Prof Paola Borri at SPIE Photonics West 2019 (San Francisco, USA) in Feb 2019. A review of our work was also be published soon as a result of the SPIE Photonics West Conference. The review is called 'Imaging lipids in living mammalian oocytes and early embryos by Coherent Raman Scattering microscopy' by Paola Borri, Josephine Bradley, Iestyn Pope, Wolfgang Langbein, and Karl Swann.
We have contined our studies of lipid droplets and recently found that palmitic acid can cause an increase in lipid content of oocytes as well disrupt internal membranes. These effects are reversed by co-incubation with oleic acid, and can be seen in the abscence of any clear change in mitochondrial redox state or Ca2+ homeostasis. This data is now being completed and analysed and will be prepared for submission as another research paper. The work on this paper has been delayed by the closue of our University during the last three months of the project. However, the work was completed during the period from Sept to Dec 2020 when further work as funded via the UKRI COVID-19 Grant Extension Allocation. The work has now been written up and a submission of this work is expected shortly.

During the course of this project we made two new discoveries that were not anticipated in the original proposal. One is as a result of preliminary imaging experiments we carried out on human oocytes to compare them with mouse oocytes where we noticed differences in the lipids droplets. We the checked the oocytes for ATP levels we noticed a significant difference between species. This developed into a project on its own that uncovered the signficance of this differences in ATP between mouse and human oocytes. This is to be regarded as a serendipitous offshoot of the orginal project. In addition, we also carried out some experiments tracking lipid droplet in mouse oocytes and their movements during metabolic manipulations. We subsequently found that movements of the whole cytoplasm are linked to the metabolism in the oocyte. We have now obtained short term funding from a Wellcome Trust ISSF project to establish the relationship between oocyte metabolism and cytoplasmic movements. This might lead to new ways of non invassively assessing embryo viability. This is now part of collaboration involving some of the original team members (Swann, Borri and Langbein) and a new member of the School of Mathematics in Cardiff Univeristy (Dr Thomas Woolley).
Exploitation Route The CARS technique using deuterium labelling might be used in other oocytes or other cell types as a way of assessing lipid metabolism by mitochondria.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description WELLCOME TRUST ISSF3 CROSS DISCIPLINARY AWARD
Amount £22,699 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2020 
End 06/2021