Different glycolipids in sperm from different freshwater fishes – A high-performance thin-layer chromatography/electrospray ionization mass spectrometry study

Rationale: Glycolipids play important roles in many physiological processes – despite their commonly low abundance. This study summarizes selected data on the (glyco) lipid composition of sperm from different fish species. Methods: Lipid extraction of fish sperm was performed according to the procedure by Bligh and Dyer. The lipid composition of the organic extracts was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap (ESI-IT)MS coupled to high-performance thin-layer chromatography (HPTLC). Results: It was shown that sperm from carp, northern pike, rainbow trout and burbot contain high amounts of neutral and acidic glycosphingolipids as well as sulfoglycolipids. These particular lipids are presumably involved in reproduction requirements. Conclusions:


| INTRODUCTION
Even though sperm cells have the same and only mission in every sexually reproducing species, namely to fertilize a female oozyte, sperm cells are the most heterogeneous cells regarding size, shape and molecular composition (reviewed in Ramón et al 1 ). The molecular composition is particularly heterogeneous when it comes to lipids. [2][3][4][5][6] In sperm most lipids are integrated parts of the different membrane systems, namely the sperm plasma membrane, the outer and the inner acrosomal membrane, the nuclear envelope and mitochondrial membranes. 7 Due to distinct fertilization modalities among different species, the lipid composition of the sperm membrane has to be adjusted according to the respective requirements. The lipid composition changes continuously during the life-time of a spermatozoon 8 and helps the sperm membrane to become more fluid and to overcome the different fertilization obstacles. 9 In mammals, sperm membranes are mainly composed of phospholipids, particularly of phosphatidylcholines (PC) and phosphatidylethanolamines (PE), which can be easily differentiated by mass spectrometry. In addition to these lipids, there is also a special glycolipid called seminolipid which is exclusively present in the membranes of mature mammalian sperm. 10 Seminolipid is a sulfoglycolipid with an ether-bound alkyl residue at the sn-2 position (also called an ether-glycerolipid) and, just like all other glycolipids, it is an important component of the outer cell membrane layer. Due to its negatively charged sulfate group, it provides the sperm cell membrane with an overall negative charge.
Seminolipid has been described as being important for the adhesion of mammalian sperm to the respective egg cell. 11 It should be noted that sperm-egg interaction during the fertilization process occurs in different taxa-specific environmental conditions (e.g. the environment of the female reproductive tract in species with internal fertilization and external water environment in species with external fertilization). These conditions are so diverse that it is really difficult to understand the biological meanings of the known differences in sperm lipid composition. In this respect fishes with external fertilization are the group which is of interest for comparative studies oriented on understanding the basic principles of sperm physiology in relation to the fertilization process. While quite a lot of work in this field has already been done, the present study will focus on poorly described aspects of sperm lipidology in externally fertilizing fishesthe presence of glycolipids.
In the testes of salmon and trout, sulfogalactosylglycosylceramide has been detected as the most abundant glycolipid. 12 However, there are so far no data available on whether this lipid is also present in the sperm of other fish species. In a recent study we showed that a neutral glycosphingolipid, namely Galβ-Cer(d18:1/16:0), represents one of the most abundant lipids in sterlet spermatozoa. 13 During earlier investigations of the phospholipid profiles of sperm from the common carp (Cyprinus carpio), the northern pike (Esox lucius) and the burbot (Lota lota), 14 dedicated lipid fractions, that could not be assigned to any common phospholipid class, were also detected. These spots presumably represent different glycolipids. Therefore, the present investigation aimed at elucidating the unknown lipid fractions from sperm of several freshwater fishes with external fertilization by coupling highperformance thin-layer chromatography (HPTLC) to electrospray ionization ion trap mass spectrometry (ESI-ITMS). This rapid method is very convenient in the field of lipid analysis, because (1) the different lipid classes of a separated lipid mixture are visible at first glance after staining of the TLC plate and (2) ion suppression effects which are the major problem with soft ionization mass spectrometric methods can be avoided.

| Fish rearing conditions and sperm collection
The study was performed on sperm from the common carp (Cyprinus carpio), the northern pike (Esox lucius), the burbot (Lota For each fish species, sperm samples from five males were collected by abdominal massage and stripped into dry collecting vials.
One mL of each sperm sample was concentrated by centrifugation at 5000 g for 15 min at 4 C. The obtained cell pellet was frozen at −80 C until the time of lipid extraction.

| Chemicals
All solvents were obtained in the highest commercially available purity from Sigma-Aldrich (Taufkirchen, Germany). Lipids for the HPTLC lipid standard mix were purchased from Avanti Polar Lipids (Alabaster, AL, USA). All chemicals were used as supplied.

| Lipid extraction
Lipids were extracted according to the procedure by Bligh and Dyer 15 with slight modifications. Briefly, 100 μL of concentrated sperm samples were mixed vigorously with 4 mL methanol/chloroform (1:1, v/v) in glass vials for cell lysis and good homogeneity. Then 2 mL of distilled water was added, and the batches were mixed vigorously and centrifuged for 10 min at 1400 g to achieve good phase separation.
The lower organic (CHCl 3 ) layer was withdrawn using a glass syringe and lipid extraction was repeated once more with an additional volume of 2 mL of chloroform. The organic phases were combined, and aliquots were evaporated to dryness.

| Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)
Dried lipid extracts of fish sperm (see section 2.3) were mixed 1:1 (v/v) with 0.5 M 2,5-dihydroxybenzoic acid (DHB) in methanol as matrix 16 for positive polarity and 10 mg/mL 9-aminoacridine (9-AA) in isopropanol/acetonitrile (60/40, v/v) for negative polarity 17 and vortexed for good homogeneity. A sample of 0.75 μL was transferred onto an aluminum-coated MALDI target (Bruker Daltonics GmbH, Bremen, Germany). MALDI-TOF spectra were recorded on a Bruker Autoflex Speed time-of-flight mass spectrometer, which utilizes a 2 kHz solid-state laser emitting at 355 nm. Spectra were recorded in reflector mode by using the predefined laser firing algorithm "random walk". The extraction voltage was 20 kV. Gated matrix suppression was applied to prevent saturation of the detector by matrix ions. For each mass spectrum 1000 single laser shots were averaged. Laser-induced sample alterations were kept to a minimum by setting the laser energy only slightly above the threshold level.

| HPTLC and ESI-MS
Lipid extracts were analyzed as described earlier. 6

| RESULTS AND DISCUSSION
The investigation of the lipid composition of sperm from different freshwater fish species with external fertilization revealed some lipids that have not been described for fish sperm, so far. In addition to established phospholipids such as PC or PE, 14 Figure 4B) represent -NaHSO 4 and -SO 3 , respectively. However, there is no loss of one hexose modified with sulfate. This indicates that the sulfate is present at the first hexose unit (Figures 4C and 4D). The loss of 404 u might stem from the cleavage of two hexose moieties plus -SO 3 .
Therefore, the ions at m/z 986.5 and 940.5 were assigned to II 3 SO 3 -LacCer(d18:1/16:0). Thus, the ions at m/z 1068.6 (+) and 1022.6 (−) could be assigned to II 3 SO 3 -LacCer(d18:1/22:1). The same sulfoglycolipid has also been found in rainbow trout sperm (data not shown). It is very surprising that a marked loss of the sulfate residue is only observed with low intensity. Former investigations using MALDI-TOFMS to characterize sulfated carbohydrates of the glycosaminoglycan type, for instance, showed that the spectra are dominated by a typical loss of 102 u (-SO 3 Na + H) and that it is very difficult to suppress the generation of this production. 25 The reasons that the sulfate loss only plays a minor role in the present ESI-ITMS investigation needs to be elucidatedalthough ionization by ESI is gentler than by MALDI.
Burbot sperm extracts are characterized, in addition to well-known phospholipids, 14   It is obvious from the data given in this manuscript that there are major differences in the glycolipid composition of sperm from different fish species. Despite these differences, very limited attention has been paid to sperm glycolipids and their role remains largely unknown. The glycocalyx is involved in cell adhesion. In the context of sperm, it encodes information specific for its species of origin. This represents an example of reproductive incompatibility 28 and might be important to prevent the generation of offspring from incompatible species. Glycolipids are modifications of the F I G U R E 4 ESI-IT mass spectra of an unknown lipid fraction from northern pike sperm after HPTLC separation. A, Singly charged ions were detected in the positive as well as in the negative ion mode with a mass difference of 46, representing the exchange of two protons by Na + . Additional information is available in Table S1 (supporting information). B, Ions at m/z 986 and 940 were fragmented by tandem mass spectrometry (CID) in the respective ion mode. Collecting all the available data, the observed ions could be assigned to a sulfoglycosphingolipid (C and D). The Y1 and Y0 product ions represent the loss of a hexose (B1) and the loss of a hexose plus a sulfated hexose moiety (B2), respectively. The linkage of the hexose moieties is only exemplarily given and remains to be elucidated molecular architecture of the cell surface. The hexoses bound to those lipids may be either glucose or galactose residues of different numbers which result in alterations in the hydrophobicity.
Gangliosides have already been found in the sperm heads of rainbow trout together with related epitope structures near the egg micropyle, of the position where the sperm enter the female oozyte. 29 In sea urchin sperm gangliosides and sulfatides were found to be highly expressed in low-density detergent-insoluble moieties of the sperm membrane 30  F I G U R E 5 ESI-IT mass spectra from three unknown lipid fractions from burbot sperm after HPTLC separation. A, Singly and doubly charged ions were detected in the negative ion mode, whereas only doubly charged ions were detected in the positive ion mode. Additional information is available in Table S1 (supporting information). B, Ions at m/z 1387 and 1401 were fragmented by tandem mass spectrometry in the negative ion mode. For explanation of the MS/MS spectra, see main text procedures for preserving fish biodiversity and aquaculture). The authors are particularly indebted to the Merck Company for their continuous support. Open access funding enabled and organized by Projekt DEAL.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/rcm.8875.