160 Lipidome Signatures in Early Bovine Embryo Development

Abstract

Mammalian pre-implantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding the membrane lipid profile fluctuation during this crucial period is fundamental to address cellular and molecular mechanisms governing embryogenesis. A full understanding of stage-specific lipid signatures in early bovine embryo development is, however, still lacking. The aim of the present work was to characterise stage-specific changes in lipid profiles during early bovine embryonic development. Immature oocytes were recovered from slaughterhouse-derived bovine ovaries and assigned among 5 in vitro developmental stages for lipid characterisation: immature oocytes, 2-cell embryos (32–40 h post-insemination), 8 to 16-cell embryos (72 h post-insemination), morulas (120 h post-insemination), and blastocysts (168 h post-insemination). Two different culture media were used for in vitro embryo production, SOFaaci medium supplemented with 2.5% of serum and serum-free SOF-BE1 medium. Cytoplasmic lipid droplets content and membrane phospholipids profiles for each development stage were assessed by lipid staining (Nile red; n = 5–9) and matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MS; n = 5–9), respectively. For statistical analysis, univariate and multivariate models were used to compare lipid droplets content and membrane phospholipids profiles. Cytoplasmic lipid droplets content increased from minimum in the immature oocyte stage to maximum at the morula stage, followed by a sharp drop at the blastocyst stage (58.4 ± 10.5ac, 62.5 ± 9.4ac, 85.9 ± 8.2a, 148.3 ± 7.4b, 37.4 ± 9.9c of fluorescence intensity per embryo area, respectively, for immature oocyte, 2-cells, 8 to 16-cells, morulas, and blastocysts; abcP < 0.05). More cytoplasmic lipid droplets were detected in morulas and blastocyts cultured in SOFaaci than in SOF-BE1 (morulas, 162.6 ± 11.3 v. 137.1 ± 9.2 of fluorescence intensity per embryo area, respectively, P < 0.05; blastocysts, 49.9 ± 9.9 v. 20.7 ± 9.9 of fluorescence intensity per embryo area, respectively, P < 0.05). Characteristic dynamic changes of unsaturation level, acyl chain length and class composition (phosphatidylcholines, sphingomyelins, and phosphatidylethanolamines) of phospholipid profiles were observed during early embryo development. This study provides a comprehensive analysis, involving lipid staining and mass spectrometry evaluation, of stage-specific lipid signatures during bovine in vitro embryo development. These results may be useful for assessing the role of specific lipid species during important events of embryogenesis. Research was supported by CNPq, FAPESP, and FAPERGS.