120 EFFECTS OF BOVINE PLASMA LIPOPROTEINS ON ACCUMULATION OF CYTOPLASMIC LIPID DROPLETS AND MITOCHONDRIAL MORPHOLOGY IN BOVINE EMBRYOS

Abstract

The excessive accumulation of cytoplasmic lipid droplets (LD) may affect the embryo quality such as the sensitivity of embryos to chilling and freezing. Previous studies demonstrated that the presence of serum in embryo culture medium caused the abnormal accumulation of cytoplasmic LD. However, the mechanism and source of cytoplasmic LD accumulation in bovine embryos cultured in serum-containing medium is unclear. In this study, the effects of bovine plasma lipoproteins (BPL) on the accumulation of cytoplasmic LD were investigated in bovine embryos. Bovine oocytes were matured in IVMD101 serum-free medium and inseminated in BO-based medium. After insemination, embryos were cultured in IVMD101 supplemented BPL (Sigma) at various concentrations (60, 150, 300, 600 μg mL^–1) or 5% calf serum (CS) in a humidified atmosphere of 5% CO₂ in air at 38.5°C. The embryos of 2-, 8-cell, morula, and blastocyst stages were used for experiments. Embryos were fixed and stained with Sudan black B (SBB) for histochemical evaluation. Some embryos were prepared for transmission electron microscopy. The effect of media supplemented with BPL on in vitro development of bovine embryos is shown in Table 1. The proportion of embryos developing to morula and blastocyst stages decreased with increased levels of BPL (Table 1). Histochemical examinations demonstrated that embryos cultured in BPLcontaining media had numerous lipid droplets in their cytoplasm as observed in the embryos developed in CS-supplemented medium. The increase in number of LD was dose-dependent. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of morulae and blastocysts cultured in medium supplemented with BPL (300 μg mL^–1). Many elongated mitochondria were found in embryos developed in IVMD101 medium at the morula and blastocyst stages, whereas many of the mitochondria in the morulae and blastocysts developed in IVMD101 medium supplemented with BPL or CS were of an immature form such as spherical or ovoid shape. These results indicate that bovine embryos cultured in BPL-containing media abnormally accumulated LD in their cytoplasm, suggesting that the BPL directly affects the accumulation of cytoplasmic LD. Furthermore, the excessive accumulation of cytoplasmic LD in embryos may affect the mitochondrial development of bovine embryos.

Table 1. Effects of BPL on in vitro development of IVM/IVF bovine embryos

This work was parially supported by Grant-in-Aid for Scientific Research (17380164), on Priority Areas ‘Lifesurveyor’(19021006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Special Coordination Funds for Promoting Science and Technology of Japan, and the Japan Livestock Technology Association.