101 LIPID FINGERPRINTING OF OOCYTES AND PRE-IMPLANTATION MOUSE EMBRYOS BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY

Abstract

Lipids and metabolites can be directly detected from biological samples by desorption electrospray ionization mass spectrometry (DESI-MS). The introduction of morphologically friendly solvent systems allows nondestructive analysis of samples and because of its extractive nature, ion intensities obtained are adequate for profiling microscopic samples. This work reports the individual chemical profiling of mouse oocytes and diverse pre-implantation developmental stages of embryos by DESI-MS. Mice were superovulated with 5 IU pregnant mare serum gonadotropin (PMSG) IP followed 46 h later by 5 IU hCG IP and mated or not for embryo and oocyte recovery, respectively. Mice were killed and their oviducts removed and flushed at different intervals post-hCG to collect oocytes and embryos at different stages of development. Two-cell embryos were also cultured in vitro to obtain additional samples. For MS analysis, samples (n = 63) were taken from the flushing or culture medium, washed briefly in distilled water and placed on a glass slide for same-day analysis. Mass spectra were acquired in the negative ion mode at the mass/charge range of m/z 150 to 1000 using 1:1 (v/v) acetonitrile/DMF as a solvent system in an LTQ linear ion trap mass spectrometer (ThermoFisher Scientific, Pittsburgh, PA). Mass spectra from oocytes (n = 11) as well as zygotes (n = 9), 2-cell (n = 11), 4-cell (n = 1), 8- to 16-cell (n = 19) embryos and blastocysts (n = 12) showed the presence of deprotonated free fatty acids such as palmitic (m/z 255.2), linoleic (m/z 281.2), arachidonic (m/z 303.3) and docosahexanoenic (m/z 327.3), as well as prominent fatty acid dimers in the region of m/z 500 to 650, such as m/z 563.5 (oleic acid dimer) and glycerophospholipids in the m/z 700 to 1000 range. These complex lipids were represented by chlorinated phosphocholines such as PC 34:1, m/z 794.7; phosphatidylinositols such as PI 38:4, m/z 885.8; phosphatidylethanolamines such as PE 38:3, m/z 768.7; and phosphatidylserines such as PS 38:4, m/z 810.7. After analysis, samples observed on the glass slide displayed evident dehydration caused by the action of the organic solvent. Chemical profiling was more difficult for oocytes and blastocysts compared with other developmental stages, probably because of the presence of the intact zona pellucida in oocytes, whereas blastocysts were more prone to lysis during the rapid water wash, leading to a decrease in signal intensity. Optimization of conditions, comparison of MS spectra from a greater number of samples of different developmental stages by multivariate statistics and the influence of in vitro vs in vivo conditions are currently being investigated. Although small molecules have been shown to play a role in the direction of cell fate, the mechanisms involved are poorly understood. Perspectives for the application of this novel DESI-MS technique include the ability to study chemical signatures of hierarchical pluripotency observed during early embryonic development including embryonic stem cell differentiation.

Support from the Purdue University Center for Cancer Research Small Grants Program is gratefully acknowledged.