Single embryo and oocyte lipid fingerprinting by mass spectrometry

Abstract

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.

Figures

Fig. 1.

Schematic of sample preparation and spectrum acquisition for MALDI-MS analysis of a single embryo or oocyte. (A) Individual samples are removed from the culture dish, washed, transferred to a spot on a MALDI target plate, dried and moistened with the matrix solution. (B) The laser is focused on the single unit and the MALDI-MS data is collected. MALDI plates typically allow for the simultaneous loading of either 96 or 384 samples in individual spots, and full spectra acquisition requires a few milliseconds of spectra accumulation.

Fig. 2.

MALDI-MS in the positive ion mode for a single and intact oocyte or embryo: (A) human (Homo sapiens) oocyte; (B) bovine (Bos taurus) oocyte; (C) bovine embryo; (D) sheep (Ovis aries) oocyte; (E) fish (Mugil spp.) oocyte; and (F) ant (Solenopsis spp.) egg.

Fig. 3.

PCA plot for the MALDI-MS data of single embryos and oocytes of (A) all samples from the five species: (a1) human (Homo sapiens) oocytes (N = 10), (a2) sheep (Ovis Aries) oocytes (N = 6), (a3) bovine (Bos taurus) oocytes (N = 6), (a4) fish (Mugil spp.) oocytes (N = 9), and (a5) insect (Solenopsis spp.) eggs (N = 6); (B) of the three mammalian species: (b1) human, (b2) sheep and (b3) bovine; and (C) of only (c3) bovine oocytes (N = 6) and (c6) bovine embryos (N = 8).

Fig. 4.

MALDI-MS in the positive ion mode for single and intact bovine embryos cultured using four different in vitro conditions regarding incubator atmosphere and culture medium supplementation, respectively: (A) 5% O2 and BSA (Group 1; N = 14); (B) 20% O2 and BSA (Group 2; N = 12); (C) 20% O2 and FCS (Group 3; N = 10); and (D) 5% O2 and FCS (Group 4; N = 14).

Fig. 5.

3D PCA using MALDI-MS lipid data from bovine embryos cultured in four different in vitro conditions regarding culture medium supplementation: (A) 5% O2 and BSA (Group 1; red dots) versus 20% O2 and BSA (Group 2; light blue dots), and (B) 20% O2 and FCS (Group 3; dark blue dots) versus 5% O2 and FCS (Group 4; brown dots), and incubator atmosphere: (C) 5% O2 and BSA (Group 1; red dots) versus 5% O2 and FCS (Group 4; brown dots); and (D) 20% O2 and BSA (Group 2; light blue dots) versus 20% O2 and FCS (Group 3; dark blue dots).

Fig. 6.

Single bovine embryo MALDI-MS/MS for the ions of: (A) m/z 810.6 [PC (38:4) + H]+ or [PC (36:1) + Na]+ and (B) m/z 725.5 [SM (16:0) + Na]+. Both spectra display characteristic NL of 59 Da (trimethylamine) and of 124 Da from the loss of the cyclophosphane ring. Also, the sodiated cyclophosphane (m/z 147.0) and the choline polar head group (m/z 184.0) ion can be observed.