MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes

Figures & data

Table 1. Phospholipids (PL) and triacylglycerols (TAG) identified via MALDI(+)-MS in the supplements, in the oocytes, and in the denuded oocytes.

m/z	Lipid ion (carbons:unsaturation)	Reference
700.6	[PC (30:3) + H]^+	1
703.5	[SM (16:0) + H]^+	2, 3, 1
706.6	[PC (30:0) + H]^+	1
720.6	[PCe (32:0) + H]^+, [PE (34:0) + H]^+	4, 1
725.5	[SM (16:0) + Na]^+	1-4
728.6	[PC (32:3)+ H]^+, [PEp (36:2) + H]^+, [PEe (36:3)+ H]^+	4, 1
732.6	[PC (32:1) + H]^+	4,1
734.6	[PC (32:0) + H]^+	1
748.6	[PCe (34:0) + H]^+	1
754.6	[PC (32:1) + Na]^+, [PC (34:4) + H]^+	1,4
756.6	[PC (32:0) + Na]^+, [PC (34:3) + H]^+	1-6
758.6	[PC (34:2) + H]^+	1,2,4,5
760.6	[PC (34:1) + H]^+	1,2,5
780.6	[PC (36:5) + H]^+, [PC (34:2) + Na]^+	1,2,4,5
782.6	[PC (36:4) + H]^+, [PC (34:1) + Na]^+	1,2,4,5
784.6	[PC (36:3) + H] ^+, [PC (34:0) + Na]^+	1,2
798.6	[PC (34:1) + K]^+	6
806.6	[PC (38:6) + H]^+, [PC(36:3) + Na]^+	1,2
808.6	[PC (38:5) + H]^+, [PC (36:2) + Na]^+	1-5
809.6	[SM (22:0) + Na]^+	4
810.6	[PC (38:4) + H]^+, [PC (36:1) + Na]^+	1-5
834.6	[PC (40:6) + H]^+	2
835.7	N/A
836.6	[PC (40:5) + H]^+	2
855.7	[PPO (50:1) + Na]^+	2
856.6	[PC (40:6) + Na]^+	2, 6
881.7	[POO (52:2) + Na]^+	2
907.7	[OOO (54:3) + Na]^+, [SOL (54:3) + Na]^+	2
929.7	[OOO (52:2) + Na]^+, [SSL (52:2) + Na]^+	6
935.7	N/A
943.7	N/A
955.8	[TAG (58:7) + Na]^+, [TAG (60:10) + H]^+	7
957.7	[TAG (59:13) + Na]^+	7
959.7	N/A
971.7	[TAG (60:13) + Na]^+, [TAG (62:16) + H]^+	7

Identification is based on earlier studies and MS/MS data. L: linoleic acid; Ln: linolenic acid; O: oleic acid; P: palmitic acid; PC: phosphatidylcholines; S: stearic acid; SM: sphingomyelin. Reference 1: Milne et al. [2006]; 2: Ferreira et al. [2010]; 3: Fuchs et al. [2009]; 4: Brugger et al. [1997]; 5: Petkovic et al. [2001]; 6: Hasayaka et al. [2008]; 7: Shrestha et al. [2014].

Figure 1. MALDI-MS lipid profiles of serum used as supplements for oocytes IVM, before solubilization to culture medium. Spectra were obtained in the positive mode using 2,5-Dihydroxybenzoic acid (DHB) as matrix. Analyses were performed directly on fetal bovine serum - FBS (A) and synthetic serum substitute – SSS (B).

Figure 2. MALDI-MS lipid profiles of pooled cumulus cells from immature and mature oocytes supplemented with sera during 24 h of IVM. (A) iCC: immature cumulus cells; (B) FBS-CC: cumulus cells obtained from FBS-supplemented oocytes after IVM; (C) SSS-CC: cumulus cells obtained from SSS-supplemented oocytes after IVM. FBS: fetal bovine serum; SSS: synthetic serum substitute; IVM: in vitro maturation. Spectra were obtained in the positive mode using 2,5-Dihydroxybenzoic acid (DHB) as matrix.

Figure 3. Three-dimensional PLS-DA score plot of samples from immature cumulus cells (iCC), FBS-treated cumulus cells (FBS-CC), and SSS-treated cumulus cells (SSS-CC) during IVM. FBS: fetal bovine serum; SSS: synthetic serum substitute; IVM: in vitro maturation. A clear separation between three clusters was achieved. See Supplemental Figure S2 for related variable importance in projection (VIP) plot of the ions.

Figure 4. MALDI-MS lipid profiles of immature oocytes (IO) and oocytes supplemented with sera during IVM in the presence (COC) or absence (DO) of cumulus cells. Spectra were obtained in the positive mode using 2,5-dihydroxybenzoic acid (DHB) as matrix. Analyses were performed directly on pools of 5 mechanically denuded oocytes from 15 replicates. (A) IO; (B) FBS-COC; (C) SSS-COC; (D) FBS-DO; and (E) SSS-DO. IVM: in vitro maturation; COC: cumulus-oocyte complex; DO: denuded oocyte; FBS: fetal bovine serum; SSS: synthetic serum substitute; CC: cumulus cells.

Figure 5. Three-dimensional PLS-DA score plot of samples from FBS-treated denuded oocytes (FBS-DO) and FBS-treated cumulus-oocyte complexes (FBS-COC). A good separation between two clusters was achieved. See Supplemental Figure S4 for related variable importance in projection (VIP) plot of the ions. FBS: fetal bovine serum; SSS: synthetic serum substitute.

Figure 6. Three-dimensional PLS-DA score plot of samples from SSS-treated denuded oocytes (SSS-DO) and SSS-treated cumulus-oocyte complexes (SSS-COC). A clear separation between two clusters was achieved. See Supplemental Figure S6 for related variable importance in projection (VIP) plots reporting the ions responsible by clusters separation. FBS: fetal bovine serum; SSS: synthetic serum substitute.

Table 2. Embryonic development of bovine denuded oocytes (DO) or cumulus-oocyte-complexes (COC) matured in vitro in TCM-199 medium supplemented with fetal bovine serum (FBS) or synthetic serum substitute (SSS).

Supplement	Groups	Number of Oocytes	Cleaved Embryos (%)	Blastocysts (% of cultured)
FBS	COC	262	187 (71.4±3.5%)^a	85 (32.4±7.1%)^a
FBS	DO	266	154 (57.9±1.9%)^b	69 (25.9±0.8%)^a
SSS	COC	262	150 (57.3±8.6%)^b	52 (19.8±5.8%)^a,b
SSS	DO	252	135 (53.6±5.4%)^b	40 (15.8±0.5%)^b

Each experiment involved 3 replicates. ^a,bp < 0.05, per column; FBS: fetal bovine serum; SSS: serum synthetic substitute.

Milne, S., Ivanova, P., Forrester, J., Alex Brown, H. (2006) Lipidomics: an analysis of cellular lipids by ESI-MS. Methods 39: 92–103.

 PubMed Web of Science ®Google Scholar

Ferreira, C.R., Saraiva, S.A., Catharino, R.R., Garcia, J.S., Gozzo, F.C., Sanvido, G.B., et al. (2010) Single embryo and oocyte lipid fingerprinting by mass spectrometry. J Lipid Res 51: 1218–1227.

 PubMed Web of Science ®Google Scholar

Fuchs, B., Jakop, U., Goritz, F., Hermes, R., Hildebrandt, T., Schiller, J. et al. (2009) MALDI-TOF “fingerprint” phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa. Theriogenology 71:568–575.

 PubMed Web of Science ®Google Scholar

Brugger, B., Erben, G., Sandhoff, R., Wieland, F.T., Lehmann, W.D. (1997) Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci USA 94: 2339–2344.

 PubMed Web of Science ®Google Scholar

Petkovic, M., Schiller, J., Müller, M., Bernard, S., Reichl, S., Arnold, K., et al. (2001) Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species. Anal Biochem 289: 202–206.

 PubMed Web of Science ®Google Scholar

Hayasaka, T., Goto-Inoue, N., Sugiura, Y., Zaima, N., Nakanishi, H., Ohishi, K., et al. (2008) Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALSI-QIT-TOF)-based imaging mass spectrometry reveals a layered distribution of phospholipid molecular species in the mouse retina. Rapid Commun Mass Spectrum 22: 3415.

 PubMed Web of Science ®Google Scholar

Shrestha, B., Sripadi, P., Reschke, B.R., Henderson, H.D., Powell, M.J., Moody, S.A., et al. (2014) Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry. PLoS ONE 9(12): e115173.

 Google Scholar