Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt(+/⁻) mice

Abstract

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt(+/⁻) mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt⁻/⁻ males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt(+/⁻) males sire offspring. The higher than 50% expression level of SGG in Cgt(+/⁻) animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt(+/⁻) mice were sufficient for normal spermatogenesis.

Fig. 1.

A: Structure of C16:0/C16:0 SGG (1-O-hexadecyl-2-O-hexadecanoyl-3-O-β-D-(3′-sulfo)-galactopyranosyl-sn-glycerol). B: Deuterium-labeled internal standard. C: C24:1 sulfatide (also known as cerebroside sulfate and sulfogalactosylceramide, SGC), the other main mammalian sulfolipid. MS/MS fragmentation positions that generate ions used for MRM analyses are indicated. SGG, sulfogalactosylglycerolipid.

Fig. 2.

Purity of pig testis SGG standards. Top panel: HPTLC of partially purified SGG (lanes a and c) and highly purified SGG (lanes b and d). Plates were treated with Coomassie Blue (lanes a and b) and Azure A (lanes c and d). The samples were prepared from a pig testis as described in “Materials and Methods.” Bottom panel: Sulfate (m/z 97) parent ion electrospray tandem spectra of the same partially purified SGG (A) and highly purified SGG (B) samples.

Fig. 3.

Purity of mouse testis lipid extracts. Top panel: HPTLC of total mouse testis lipids (lanes a and c) and partially purified SGG (lanes b and d). Plates were treated with Coomassie Blue (a and b) and Azure A (c and d). Bottom panel: Sulfate (m/z 97) parent ion electrospray tandem spectra of mouse total testis lipids (A) and partially purified SGG (B).

Fig. 4.

MRM chromatograms of C16:0/C16:0 SGG in the total mouse testis lipid extract and the deuterated SGG (²H₃-SGG) internal standard. Parent to fragment ion transitions included two for both SGG (A and B) and ²H₃-SGG (C and D) (m/z 97 for both SGG and ²H₃-SGG, m/z 539.3 for SGG, and m/z 542.3 for ²H₃-SGG). The peak heights in arbitrary units for the signals in traces A, B, C, and D were 1526, 3094, 1348 and 1530, respectively. For quantitation, the average of the values obtained for the two transitions was used.

Fig. 5.

Levels of SGG and total sulfolipids in testes and sperm of Cgt^+/− mice and their wild-type littermates. SGG was quantified by LC-MS/MS-MRM, and total sulfolipids were quantified by the Azure A assay. Three Cgt^+/− mice and three wild-type littermates were used for SGG quantification. For total sulfolipid quantification, two additional Cgt^+/− and wild-type mice were included. *Significant difference of the values from Cgt^+/− mice compared with the wild-type values (P < 0.05).