Novel Aspect Detection of characteristic chemical signatures of individual embryos by DESI-MS represents a sensitive and informative analytical technology for embryology research. Introduction Analysis of small molecules present in individual embryos is of interest for embryology and cellular pluripotency research. Analytical approaches usually involve the pooling of tens to hundreds of individuals due to their microscopic size (100-200µm of diameter). Nonetheless, by using DESI-MS with the compatible solvent systems, enough ion signal from free fatty acids, phospholipids and other small molecules can be obtained from single preimplantation embryos. The objective of this work is to report the differences observed by chemometric analysis of the DESI-MS chemical signatures from 2-cell embryos and blastocysts, as well as the changes observed in embryos cultured in vitro. We have also observed the intake of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), an organic zwitterionic buffer commonly used in embryo manipulation media. Methods Mouse embryos at the two-cell stage and blastocysts were collected in vivo using FHM (HEPES-buffered K modified simplex optimized medium) for oviduct and uterus flushing. Blastocysts cultured in vitro from the 2-cell stage were also used. All embryos were maintained in FHM medium during collection and manipulation until they were washed three times in H2O/MeOH 1:1 (v/v) and placed in glass slides. DESI mass spectra were acquired during ca. 1 min in the negative ion mode at the m/z range of 150 to 1000 using 1:1 (v/v) acetonitrile/DMF as solvent. Principal component analysis (PCA) was performed by MatLab software (version 7.0) on selected informative m/z values and relative ion counts from accumulated mass spectra. Preliminary Data Free fatty acids (FA) were mostly represented by palmitic, linoleic, arachidonic and docosahexanoenic acids. FA dimers were observed in the region of m/z 500-650. Phospholipids were detected in all the groups and were mostly represented by the phosphatidylcholine (34:1; m/z 794.5). Nonetheless, phosphatidylinositols (such as PI 38:4, m/z 885.6), phosphatidylethanolamines (such as PE 38:3, m/z 768.3) and phosphatidylserines (such as PS 38:4, m/z 810.7) were also detected. The mass spectra of 2-cell embryos (N=18) displayed a larger contribution of fatty acids and fatty acid dimers compared to the blastocysts. Mass spectra obtained from blastocysts produced in vivo (N=15) vs. cultured in vitro were also distinctive, mainly because blastocysts cultured in vitro gave mass spectra with higher intensity of fatty acids ions and dimers and few or no ions of phospholipids. On the other hand, blastocysts in vivo showed, besides the FA and FA dimers, a variety of PC, PS and PI ions, which are involved in membrane structure and signaling functions. Interestingly, there were larger individual variations in spectra of in vivo-derived embryos compared to the group exposed to in vitro conditions. The PCA also indicated that the ion at m/z 237 was significant and present in most samples. The fragmentation pattern of m/z 237 generated m/z 207 (neutral loss of CH2O), m/z 193 (neutral loss of C2H4O) and m/z 107 (C3H3SO3-) and m/z 80 (SO3-), which were indicative of deprotonated HEPES buffer, which was accumulated during embryo manipulation in the FHM medium. This attribution was confirmed with the HEPES standard fragmentation and when this ion influence was removed from the PCA analysis, chemical signatures were preserved. Therefore, DESI-MS allowed the identification of characteristic chemical signatures of single embryos in different developmental stages, as well as the chemical characterization of molecules related to the differences observed.
DESI-MS from Individual Mouse Pre-implantation Embryos Allow Identification of Characteristic Chemical Signatures Related to Distinct Developmental Phases
PIRRO, VALENTINA;
2012-01-01
Abstract
Novel Aspect Detection of characteristic chemical signatures of individual embryos by DESI-MS represents a sensitive and informative analytical technology for embryology research. Introduction Analysis of small molecules present in individual embryos is of interest for embryology and cellular pluripotency research. Analytical approaches usually involve the pooling of tens to hundreds of individuals due to their microscopic size (100-200µm of diameter). Nonetheless, by using DESI-MS with the compatible solvent systems, enough ion signal from free fatty acids, phospholipids and other small molecules can be obtained from single preimplantation embryos. The objective of this work is to report the differences observed by chemometric analysis of the DESI-MS chemical signatures from 2-cell embryos and blastocysts, as well as the changes observed in embryos cultured in vitro. We have also observed the intake of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), an organic zwitterionic buffer commonly used in embryo manipulation media. Methods Mouse embryos at the two-cell stage and blastocysts were collected in vivo using FHM (HEPES-buffered K modified simplex optimized medium) for oviduct and uterus flushing. Blastocysts cultured in vitro from the 2-cell stage were also used. All embryos were maintained in FHM medium during collection and manipulation until they were washed three times in H2O/MeOH 1:1 (v/v) and placed in glass slides. DESI mass spectra were acquired during ca. 1 min in the negative ion mode at the m/z range of 150 to 1000 using 1:1 (v/v) acetonitrile/DMF as solvent. Principal component analysis (PCA) was performed by MatLab software (version 7.0) on selected informative m/z values and relative ion counts from accumulated mass spectra. Preliminary Data Free fatty acids (FA) were mostly represented by palmitic, linoleic, arachidonic and docosahexanoenic acids. FA dimers were observed in the region of m/z 500-650. Phospholipids were detected in all the groups and were mostly represented by the phosphatidylcholine (34:1; m/z 794.5). Nonetheless, phosphatidylinositols (such as PI 38:4, m/z 885.6), phosphatidylethanolamines (such as PE 38:3, m/z 768.3) and phosphatidylserines (such as PS 38:4, m/z 810.7) were also detected. The mass spectra of 2-cell embryos (N=18) displayed a larger contribution of fatty acids and fatty acid dimers compared to the blastocysts. Mass spectra obtained from blastocysts produced in vivo (N=15) vs. cultured in vitro were also distinctive, mainly because blastocysts cultured in vitro gave mass spectra with higher intensity of fatty acids ions and dimers and few or no ions of phospholipids. On the other hand, blastocysts in vivo showed, besides the FA and FA dimers, a variety of PC, PS and PI ions, which are involved in membrane structure and signaling functions. Interestingly, there were larger individual variations in spectra of in vivo-derived embryos compared to the group exposed to in vitro conditions. The PCA also indicated that the ion at m/z 237 was significant and present in most samples. The fragmentation pattern of m/z 237 generated m/z 207 (neutral loss of CH2O), m/z 193 (neutral loss of C2H4O) and m/z 107 (C3H3SO3-) and m/z 80 (SO3-), which were indicative of deprotonated HEPES buffer, which was accumulated during embryo manipulation in the FHM medium. This attribution was confirmed with the HEPES standard fragmentation and when this ion influence was removed from the PCA analysis, chemical signatures were preserved. Therefore, DESI-MS allowed the identification of characteristic chemical signatures of single embryos in different developmental stages, as well as the chemical characterization of molecules related to the differences observed.
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