Abstract
Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.
Highlights
The early stages of embryonic development following fertilization in animals are characterized by synchronous cell divisions, the onset of transcription of genes that will pattern the embryo, and local signaling events that transition a ball of equipotent cells into regions that express different tissue fates [1, 2]
To reveal the biochemical makeup, elucidate low abundance proteins, and study the kinetics of developmental and cellular events, X. laevis oocytes have been analyzed by high performance liquid chromatography (HPLC) [10, 11][12], time-of-flight (TOF) secondary ion mass spectrometry (SIMS) [13], gas chromatography (GC) mass spectrometry (MS) [14], 2D gel electrophoresis followed by electrospray ionization (ESI) MS [15], and liquid chromatography-tandem MS (LC-MS/MS) [2]
There has been extensive of the transcriptome that regulates the major developmental transitions in X. laevis at the subcellular level. These investigations have been limited to the detection of mRNAs by in situ hybridization and RT-PCR approaches and to the detection of proteins by immunohistochemistry or Western blotting techniques
Summary
The early stages of embryonic development following fertilization in animals are characterized by synchronous cell divisions, the onset of transcription of genes that will pattern the embryo, and local signaling events that transition a ball of equipotent cells into regions that express different tissue fates [1, 2] These events are common across different species of animals, but can occur for different lengths of times and at different stages of morphogenesis. 3D molecular images of freeze dried Xenopus oocytes were obtained under optimized sample preparation conditions using TOF-SIMS [13] While all of these methods for X. laevis oocytes and embryo analysis have been shown to capture important aspects of its biochemistry, most of them require elaborate sample preparation prior to analysis that might alter subcellular localization of important developmental molecules
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