The Development Analysis Of Intracellular Lipid Droplets In Pig Oocytes

Lipid droplets as an organelles for energy storage, regulating lipid dynamic balance and intracellular signal transduction. Lipid droplets plays an important role in oocyte maturation, fertilization and early embryo development. This study is quantitative analysis of lipid droplets based on the characteristics of abundant lipid droplets in pig oocyte. Lipid mainly exist as lipid droplets form in pig oocytes. In order to restore cell morphological structure and detecting the number, volume, density and spatial distribution of lipid droplets. Two methods of 3D reconstruction method and the flat optical density method, are used to quantitative lipid drops which are fluorescence staining by Nile Red dye solution. By comparing methodology, we found that the method of 3D reconstruction based on the confocal laser scanning can quantify the content of lipid droplets in the cell with higher accuracy and reproducibility. Comparative analysis the accuracy and repeatability between 3D reconstruction method and flat optical density method,the result showed that:(1) the accuracy:3D reconstruction method can reflect the variation of content of lipid droplets more accurately (coefficient of variation C.V.= 0.86), but the flat optical density method C.V.= 0.68. The accuracy of the 3D reconstruction method is higher. (2) the repeatability:The ICC (interclass correlation coefficient) of the volume of lipid drops in pig oocytes are 0.995,0.936,0.995, which is counted by the 3D reconstruction method. The ICC (interclass correlation coefficient) of the total fluorescence intensity of lipid drops in pig oocytes are 0.225,-3.413, 0.225, which is counted by the flat optical density method. While scaning the same cell in multiple angles for repeat observatiom, the 3D reconstruction method had higher repeatability repeatability (coefficient of variation C.V.= 0.07, interclass correlation coefficient ICC>0.9), but the flat optical density method C.V.= 0.37, ICC<0.4.Then 3D reconstruction method is used to quantitative lipid drops in different development stage of pig oocytes, the results showed that:(1) 3D reconstruction method had a good reduction of the morphology, structure and spatial distribution in pig oocytes lipid droplets. (2) Lipid drops are appeared in the early stages of the oocyte development. The average volume of oocytes in the different development stages as:GC period (285.91μm3), PF period (895.61μm3), PRF period (1339.96μm3), SFperiod (3348.04μm3), the content of lipid drops is increasing as the development of pig oocytes step by step.AF and MF period oocytes volume is too big and fat droplets in cells is too dense, so can’t use this method for quantitative analysis.(3)PRF period is a critical period for significantly increasing the number and volume of lipid drops in porcine oocytes.We detected the genes expression of related lipogenesis in pig oocytes by RT-PCR. Six lipid-related genes were screening out to compared with granular cell, follicular fluid, muscle, liver, and subcutaneous fat. We have finded that the lipogenic transcription in AF period pig oocytes mainly regulated by PPARy. SREBP-1 May indirectly involved in the oocytes lipid generated transcription by granulosa cells. Fatty acid synthesis in pig oocyte mainly is regulated by ACC generated cholesterol, triglycerides and phospholipids synthesis mainly rely on follicular fluid in the regulation of SCD-1. Oocytes in fatty acids related gene transcription LPL and aP2 expression quantity is very low, the oocyte itself the transcription of fatty acids is very weak, oocyte is mainly composed of fatty acids in follicular fluid, granular cells or other organizations to provide. The ability of lipid degradation and fatty acid transport is very weak in pig oocyte, lead to deposit a large amount of lipid in porcine oocytes.These results showed that the content of lipid droplets in different development stages of pig oocytes and the molecular mechanism of lipid development of pig oocytes, provide the basis data for the unique model of lipid deposition and fat metabolism in pig oocyte.