Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts.

In a 2×2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7±2.0% vs 27.0±2.0%) cows. The total numbers of ova or embryos recovered (5.5±0.9 vs 3.7±0.8) and transferable embryos (3.8±1.0 vs 2.3±0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n=75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n=89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.

Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts.

Lipid droplets, subspecies (Bos taurus indicus vs. Bos taurus taurus), and in vitro culture are known to influence cryopreservation of bovine embryos. Limited information is available regarding differences in membrane lipids in embryo, such as phosphatidylcholines (PC) and sphingomyelins (SM). The objective of the present study was to compare the profiles of several PC and SM species and relate this information to cytoplasmic lipid levels present in Nellore (B. taurus indicus) and Simmental (B. taurus taurus) blastocysts produced in vitro (IVP) or in vivo (ET). Simmental and IVP embryos had more cytoplasmic lipid content than Nellore and ET embryos (n = 30). Blastocysts were submitted to matrix-assisted laser desorption/ionization mass spectrometry. Differences in the PC profile were addressed by principal component analysis. The lipid species with PC (32:1) and PC (34:1) had higher ion abundances in Nellore embryos, whereas PC (34:2) was higher in Simmental embryos. IVP embryos had less abundant ions of PC (32:1), PC (34:2), and PC (36:5) compared to ET embryos. Moreover, ion abundance of PC (32:0) was higher in both Nellore and Simmental IVP embryos compared to ET embryos. Therefore, mass spectrometry profiles of PC and SM species significantly differ with regard to unsaturation level and carbon chain composition in bovine blastocysts due to subspecies and in vitro culture conditions. Because PC abundances of Nellore and Simmental embryos were distinct (34:1 vs. 34:2), as were those of IVP and ET embryos (32:0 vs. 36:5), they are potential markers of postcryopreservation embryonic survival.

Lipidome signatures in early bovine embryo development.

Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development.

Optimal single-embryo mass spectrometry fingerprinting.

In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research.