Analysis and characterisation of bovine oocyte and embryo biomarkers by matrix-assisted desorption ionisation mass spectrometry imaging.

In the field of 'single cell analysis', many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na](+) (m/z 725) and phosphatidylcholine [PC (32:0)+Na](+) (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development.

Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI).

Lipid pathways play important biological roles in mammalian embryology, directing early developmental pathways to differentiation. Phospholipids and triglycerides, among others, are the main composing lipids of zona pellucida in several embryo species. Lipid analysis in embryos by mass spectrometry usually requires sample preparation and/or matrix application. This novel approach using silica plate laser desorption/ionization mass spectrometry imaging (SP-LDI-MSI) allows direct single-cell imaging and embryo region discrimination with no matrix coating. Its application is herein described for two- and eight-cell embryos. Lipid biomarkers for blastomere and intact zona pellucida are reported and corroborated by both fragmentation reactions (MS/MS) and images. Results obtained in this work are understood to be of great use for further developments on in vitro bovine fertilization. Since much of the processes can be monitored by characteristic biomarkers, it is now possible to precisely identify cell division errors during early embryo stages, as well as evaluate pre-implantation conditions.