SUPPLEMENTARY MATERIAL: S1 Fig. ESI spectra of lysophosphatidylcholine (LPC) fractions from boar, bull, stallion, lion and human samples.
S2 Fig. ESI spectra of sphingomyelin (SM) fractions from boar, bull, stallion, lion and human samples.
Lipid extracts were separated on a normal phase high performance thin-layer chromatography (HPTLC) plate with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. Plates were air-dried and stained with primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) (50 mg/l dissolved in acetone/water 80:20, by vol.). Lipids were made visible under UV light and marked with a pencil. SM fractions were directly analyzed by coupling a TLC plate reader to an ESI mass spectrometer. Mass spectra were recorded in the positive ion mode. For further details on ESI-IT MS see main text. For peak assignment, please see S2 Table.
https://doi.org/10.1371/journal.pone.0264675.s002
S3 Fig. ESI spectra of phosphatidylcholine (PC) fractions from boar, bull, stallion, lion and human samples.
Lipid extracts were separated on a normal phase high performance thin-layer chromatography (HPTLC) plate with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. Plates were air-dried and stained with primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) (50 mg/l dissolved in acetone/water 80:20, by vol.). Lipids were made visible under UV light and marked with a pencil. PC fractions were directly analyzed by coupling a TLC plate reader to an ESI mass spectrometer. Mass spectra were recorded in the positive ion mode. For further details on ESI-IT MS see main text. For peak assignment, please see S3 Table.
https://doi.org/10.1371/journal.pone.0264675.s003
S4 Fig. ESI spectra of phosphatidylinositol (PI) fractions from boar, bull, stallion and human lipid samples.
Lipid extracts were separated on a normal phase high performance thin-layer chromatography (HPTLC) plate with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. Plates were air-dried and stained with primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) (50 mg/l dissolved in acetone/water 80:20, by vol.). Lipids were made visible under UV light and marked with a pencil. PI fractions were directly analyzed by coupling a TLC plate reader to an ESI mass spectrometer. Mass spectra were recorded in the negative ion mode. For further details on ESI-IT MS see main text. For peak assignment, please see S4 Table.
https://doi.org/10.1371/journal.pone.0264675.s004
S5 Fig. ESI spectra of phosphatidylethanolamine (PE) fractions from boar, bull and stallion samples.
Lipid extracts were separated on a normal phase high performance thin-layer chromatography (HPTLC) plate with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. Plates were air-dried and stained with primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) (50 mg/l dissolved in acetone/water 80:20, by vol.). Lipids were made visible under UV light and marked with a pencil. PE fractions were directly analyzed by coupling a TLC plate reader to an ESI mass spectrometer. Mass spectra were recorded in the negative ion mode. For further details on ESI-IT MS see main text. For peak assignment, please see S5 Table.
https://doi.org/10.1371/journal.pone.0264675.s005
S6 Fig. ESI spectra of phosphatidylethanolamine (PE) fractions from lion and human samples.
Lipid extracts were separated on a normal phase high performance thin-layer chromatography (HPTLC) plate with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. Plates were air-dried and stained with primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) (50 mg/l dissolved in acetone/water 80:20, by vol.). Lipids were made visible under UV light and marked with a pencil. PE fractions were directly analyzed by coupling a TLC plate reader to an ESI mass spectrometer. Mass spectra were recorded in the negative ion mode. For further details on ESI-IT MS see main text. For peak assignment, please see S5 Table.
https://doi.org/10.1371/journal.pone.0264675.s006
S7 Fig. Hydrolysis of selected seminal fluid samples over time.
The plots of hydrolysis measurements from boar and stallion seminal fluid were fitted by a linear curve (f(x) = a + b×x) and the plots from bull, lion and human were fitted by an exponential growth to a maximum (f(x) = a×e-b×x). Due to these different courses of the hydrolysis reaction between the species, the absolute hydrolysis at a given time point (10 min) was used to compare the mean values of the investigated individuals between the species (see Table 2 of the main text).
https://doi.org/10.1371/journal.pone.0264675.s007
S8 Fig. Effect of artificial LPC on boar sperm.
Beltsville Thawing Solution (BTS, Minitüb GmbH)-diluted boar semen (20 × 106 sperm/ml) was mixed with 20 μM lysophosphatidylcholine (LPC 16:0, Avanti Polar Lipids®, No 855675C). After incubation at 38°C for 30 min, the ratios of total motility (blank boxes) and sperm with an intact acrosome (striped boxes) were analyzed. The lipid extract of washed sperm of this experiment was analyzed by MALDI-TOF MS and the ratio of LPC to total GPC was calculated (for details see Material and Methods of the main text). Incubation with 20 μM LPC led to 2.4 ± 3.6% inserted LPC in sperm cell membranes. Significant differences in total motility and the percentage of sperm with an intact acrosome between the incubation with 20 μM LPC and controls are marked by asterisks (P = 0.006 and 0.003, respectively, Wilcoxon signed-rank test, n = 11).
https://doi.org/10.1371/journal.pone.0264675.s008
S9 Fig. Original TLC pictures.
Lipid extracts were separated on normal phase high performance thin-layer chromatography (HPTLC) plates with chloroform/ethanol/water/triethylamine (30:35:7:35, by vol.) as the mobile phase. Plates were air-dried and stained with primuline (Direct Yellow 59, Sigma-Aldrich, Taufkirchen, Germany) (50 mg/l dissolved in acetone/water 80:20, by vol.). BP–blood plasma, SF–seminal fluid, st.–lipid standard mixture made of LPC16:0, SM16:0, PC16:0/18:1, PA 16:0/18:1, PI 16:1/18:1, PE 16:0/18:1, PG 16:0/18:1 (bottom up).
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S1 Table. Assignment of signals detected in ESI spectra from lysophosphatidylcholine (LPC) spots.
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S2 Table. Assignment of signals detected in ESI spectra from sphingomyelin (SM) spots.
n.a.—not assigned.
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S3 Table. Assignment of signals detected in ESI spectra from phosphatidylcholine (PC) spots.
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S4 Table. Assignment of signals detected in ESI spectra from phosphatidylinositol (PI) spots.
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S5 Table. Assignment of signals detected in ESI spectra from phosphatidylethanolamine (PE) spots.
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