Chemistry and Physics of Lipids 177 (2014) 41–50

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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip

Metabolic incorporation of unsaturated fatty acids into boar spermatozoa lipids and de novo formation of diacylglycerols Valentin Svetlichnyy a,b , Peter Müller c , Thomas G. Pomorski d , Martin Schulze a , Jürgen Schiller e,∗∗ , Karin Müller b,∗ a

Institute for Reproduction of Farm Animals Schönow e.V., Bernauer Allee 10, D-16321 Bernau, Germany Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, D-10315 Berlin, Germany c Humboldt-University Berlin, Department of Biology, Invalidenstr. 42, D-10115 Berlin, Germany d University of Copenhagen, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark e University of Leipzig, Department of Medical Physics and Biophysics, Härtelstr. 16-18, D-04107 Leipzig, Germany b

a r t i c l e

i n f o

Article history: Received 10 June 2013 Received in revised form 11 October 2013 Accepted 8 November 2013 Available online 16 November 2013 Keywords: Glycerophospholipid Diacylglycerol Boar spermatozoa Fatty acid

a b s t r a c t Lipids play an important role in the maturation, viability and function of sperm cells. In this study, we examined the neutral and polar lipid composition of boar spermatozoa by thin-layer chromatography/mass spectrometry. Main representatives of the neutral lipid classes were diacylglycerols containing saturated (myristoyl, palmitoyl and stearoyl) fatty acyl residues. Glycerophosphatidylcholine and glycerophosphatidylethanolamine with alk(en)yl ether residues in the sn-1 position and unsaturated long chained fatty acyl residues in sn-2 position were identified as the most prominent polar lipids. The only glycoglycerolipid was sulfogalactosylglycerolipid carrying 16:0-alkyl- and 16:0-acyl chains. Using stable isotope-labelling, the metabolic incorporation of exogenously supplied fatty acids was analysed. Boar spermatozoa incorporated hexadecenoic (16:1), octadecenoic (18:1), octadecadienoic (18:2) and octadecatrienoic (18:3) acids primarily in the diacylglycerols and glycerophosphatidylcholines. In contrast, incorporation of eicosapentaenoic acid (20:5) was not detected. The analysis of molecular species composition subsequent to the incorporation of exogenous [14 C]-octadecadienoic acid suggests two pathways for incorporation of exogenous fatty acids into glycerophosphatidylcholine: (1) de novo synthesis of glycerophosphatidylcholine via the CDP-choline pathway and (2) reacylation of lysophosphatidylcholine via an acyltransferase. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The mammalian sperm cell (Spermatozoon) is a highly differentiated motile male gamete which fuses with the female egg cell (Oocyte) in the process of sexual reproduction. Spermatozoa are transcriptionally inactive but have respiratory and glycolytic

Abbreviations: AA, aminoacridine; BTS, Beltsville Thawing Solution; DAG, diacylglycerol; DHB, dihydroxybenzoic acid; FFA, free fatty acids; GC, gas chromatography; GPA, glycerophosphatidic acid; GPC, glycerophosphatidylcholine; GPE, glycerophosphatidylethanolamine; GPI, glycerophosphatidylinositol; GPL, glycerophospholipid; GPS, glycerophosphatidylserine; LPC, lyso-glycerophosphatidylcholine (monoacylglycerophosphatidylcholine); MAG, monoacylglycerol; MS, mass spectrometry; PLA2 , phospholipase A2 ; PLC, phospholipase C; SGG, sulfogalactosylglycerolipid; TLC, thin-layer chromatography. ∗ Corresponding author. Tel.: +49 30 5168613; fax: +49 30 5126104. ∗∗ Corresponding author at: Universität Leipzig, Medizinische Fakultät, Institut für Medizinische Physik und Biophysik, Härtelstrasse 16-18, D-04107 Leipzig, Germany. Tel.: +49 341 9715733; fax: +49 341 9715709. E-mail addresses: [email protected] (J. Schiller), [email protected] (K. Müller). 0009-3084/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemphyslip.2013.11.001

abilities, primarily in relation to sustaining motility (Bedford and Hoskins, 1990). The membrane properties, based on their lipid compositions, are of crucial importance for mammalian spermatozoa function, in particular for the fusion processes which occur during fertilisation. Lipid composition of spermatozoa shows some peculiarities: firstly, neutral lipids (particularly diacylglycerols (DAG)) are present in an unusually high amount in spermatozoa compared to somatic cells (Zanetti et al., 2010; Nikolopoulou et al., 1985). Secondly, phospholipids, the most abundant spermatozoa lipids, show specific structural properties: the ester-linked fatty acids are normally accompanied by the presence of glycerophospholipids (GPL) with ether-linked alkyl and/or alkenyl residues. The ether-glycerophospholipids of mammalian spermatozoa normally possess choline or ethanolamine as the headgroup, while other GPL with different headgroups are by far less abundant (Evans et al., 1980). Thirdly, the appearance of sulfogalactosylglycerolipid (SGG), also known as the seminolipid, is highly characteristic (Kongmanas et al., 2010). The modulation of the lipid composition of mammalian spermatozoa is a key factor of membrane stabilisation/

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destabilisation processes during the transit of spermatozoa through the male and female genital tract. The secretion of the accessory sex glands, the so-called seminal plasma, as well as the secretion of the fallopian tubes are both characterised by the presence of polyunsaturated fatty acids, as well as their transport proteins (Kalic et al., 1997; Am-In et al., 2011; Iritani et al., 1969). The spermatozoa are mixed with seminal plasma upon ejaculation and later on with fallopian tube secretion to come into contact with, amongst other compounds, free fatty acids. The ability of mammalian spermatozoa to incorporate free fatty acids into their lipids was already observed by Neill and Masters (1971, 1972, 1973), Terner and Korsh (1962) and Hamilton and Olson (1976). However, it was assumed that the lipid metabolism of spermatozoa, in comparison with somatic cells, plays a less important role since the lipid pools remain constant with only little turnover. The study by Vazquez and Roldan (1997a) showed that boar spermatozoa are capable of an active lipid biosynthesis. A de novo biosynthesis pathway was observed using the radioactively labelled GPL precursors (fatty acids, glycerol and choline) (Vazquez and Roldan, 1997a,b; Roldan and Harrison, 1992; Roldan and Murase, 1994). The incorporation of fatty acids took place first into diacylglycerophosphate (DGP), then into 1,2 diacylglycerol (1,2DAG) and, finally, into glycerophosphatidylcholine (Vazquez and Roldan, 1997a). The work by Roldan and co-workers (Vazquez and Roldan, 1997a,b; Roldan and Harrison, 1990, 1992, 1993; Roldan and Fragio, 1994) radiochemically examined the different lipid classes of mammalian spermatozoa under conditions favouring the acrosome reaction. Lipases, however, are also activated as a consequence of the acrosomal reaction which can lead to the cleavage of the headgroup as well as the related fatty acyl residues (Roldan, 1998). The fact that spermatozoa still have the capability of lipid biosynthesis indicates that this process plays an important role in the reproduction process. In this study, the intact neutral and polar lipids of boar spermatozoa were examined in detail by means of soft-ionisation mass spectrometry (MS). Using these techniques combined with stable isotope-labelling, the metabolic incorporation of selected fatty acids (those with in vivo relevance as well as some others for comparative purposes) into boar spermatozoa lipids was investigated. 2. Materials and methods 2.1. Reagents [1-14 C]-Octadecadienoic acid (specific activity 55 mCi/mmol) was obtained as solution in ethanol (Hartmann Analytic GmbH, Braunschweig, Germany); [U-13 C]-octadecadienoic acid (99% pure) was purchased from Campro Scientific GmbH (Berlin, Germany). Standard (12 C) glycerophospholipids and neutral lipids were purchased from Avanti Polar Lipids (Alabaster, USA) and used as supplied. Neutral lipids used as standards were kindly provided by Dr. Hoelzl (University Bonn, Germany). Unless otherwise stated, all other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (Taufkirchen, Germany). 2.2. Preparation, storage and labelling of spermatozoa Spermatozoa-rich ejaculate fractions were collected from boars (Sus scrofa domestica, race Piétrain or Duroc) in accordance with the rules on the care and use of domestic animals at boar stations in Brandenburg (Germany). The ejaculates were diluted to a final concentration of 2.2 × 108 spermatozoa/ml with Beltsville Thawing Solution (BTS, Minitüb® , Tiefenbach, Germany) composed of 10 mM KCl, 20.4 mM trisodium citrate, 15 mM NaHCO3 , 3.36 mM EDTA, 205 mM glucose without antibiotics, pre-warmed to 36 ◦ C

(Pursel and Johnson, 1975). The prepared spermatozoa were then gradually cooled and stored at 17 ◦ C. Spermatozoa concentration was determined with the NucleoCounter® SP-100TM (ChemoMetec A/S, Denmark). Spermatozoa motility was analysed using a CASA system (SpermVisionTM , Minitüb, Tiefenbach). For metabolic labelling, spermatozoa (0.25 to 1 × 109 ) were incubated (24 h at 17 ◦ C) with a mix of 9 ␮M [1-14 C]-octadecadienoic acid and 30 ␮M non-radiolabelled octadecadienoic acid bound to lipid-free BSA (17 ␮M) in 45 ml or 90 ml of BTS. During the labelling period, at least 75% of spermatozoa remained viable as estimated using rhodamine 123 (Rh123) and propidium iodide (PI) for assessment of their mitochondrial activity and viability according to Auger et al. (1993). A PAS flow cytometer (Partec GmbH, Münster, Germany) equipped with an argon laser emitting 100 mW at 488 nm was used. Per analysis, 15.000 events were sampled. Data was processed with the FloMax software (version 2.0; Quantum Analysis GmbH). 2.3. Lipid extraction Spermatozoa were harvested by centrifugation (10 min, 800 × g), washed with phosphate buffered saline (PBS, 20 mM Na2 HPO4 × 12 H2 O, 11 mM NaH2 PO4 × H2 O, 115 mM NaCl, sterilefiltered and adjusted to pH 7.0 with NaOH). Lipids were extracted according to Bligh and Dyer (1959), concentrated by evaporation of the solvent under a stream of nitrogen or argon, and re-dissolved in chloroform/methanol (2:1, by volume). To eliminate proteins, the lipid extract was subjected to Folch partitioning (Folch et al., 1957) using chloroform/methanol/0.9% NaCl (2:1:0.75, by volume). 2.4. Thin layer chromatography For the analysis of neutral lipids, thin layer chromatography plates (TLC, Merck or J.T. Baker) were used with a solvent system consisting of n-hexane/diethyl ether/glacial acetic acid (80:15:1, by volume). For the analysis of polar lipids, extracts were applied on TLC plates and were developed in chloroform/methanol/water (65:25:4, by volume). For two-dimensional TLC, plates were first developed (at 30 ◦ C) in chloroform/methanol/25% ammonia solution in water (90:54:7, by volume), followed by chloroform/methanol/acetone/glacial acetic acid/water (50:10:20:10:5, by volume) in the second dimension. Lipids and standards were visualised with common lipid-derivatisation agents such as iodine, ␣-naphthol-H2 SO4 , ninhydrin (0.25% ninhydrin, weight/volume in acetone) or primuline (5 mg primuline in 100 ml aceton/water, 80:20, by volume). The 14 C-containing radiolabelled spots were visualised on a 14 C-sensitive screen and quantified on a Fuji Imaging System imager and AIDA software version 3.24 (FLA3000, Raytest, Straubenhardt, Germany). For further analysis, lipid spots from primuline stained TLC plates were scraped off and extracted as described for spermatozoa (Lessig et al., 2004). 2.5. Gas chromatographic analysis of the fatty acid/fatty acyl composition Fatty acid methyl esters were prepared from free fatty acids (FFA), glycerophosphatidylcholine (GPC) or DAG (30 min at 80 ◦ C in 1 M methanolic HCl, with pentadecanoic acid (15:0) as internal standard) and determined by gas chromatography (GC) with a flame ionisation detector (Agilent HP 6890 Plus GC). A SP-2380 column (30 m column length, 750 ␮m internal diameter, 0.2 ␮m film thickness, Supelco, Munich, Germany) was used at a temperature gradient of 160 ◦ C (2 min), then heated to 200 ◦ C (5 min) at 20 ◦ C/min, followed by additional heating to 245 ◦ C (12 min) at 20 ◦ C/min and finally by cooling to 160 ◦ C at 20 ◦ C/min. Helium was used as the carrier gas at a flow-rate of 11 ml/min. Chemstation software (version 4.0.2; Agilent) was used to process the data and

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43

Cholesterol +H+ -H2O 369.3

front

*

375.0

A * 551.0

* 424.2 375.0

DAG (28:0)+Na+

*

DAG (30:0)+Na+ 535.4

*

B

DAG (32:0)+Na+

* 563.5 551.0

424.2

DAG (34:0)+Na+ 591.5

619.5

375.0

*

535.4

Cho / FFA 1,3-DAG 1,2-DAG

* 551.0 563.5

*

C

424.2 591.5

start 400

450

500

550

619.5

600

m/z

Fig. 1. Analysis of the neutral lipids of boar spermatozoa. A total lipid extract from boar spermatozoa was separated by one-dimensional TLC using n-hexane/diethyl ether/glacial acetic acid (80:15:1, by volume). The individual lipid species were visualised by primuline staining, scraped off, extracted and subjected to positive ion MALDITOF mass spectrometry, resulting in the spectra of the 1,2-DAG (A), the 1,3-DAG (B) and the cholesterol fraction (C) as shown. All peaks are labelled by the m/z ratio and the assignments are given in the figure. Both fatty acyl residues were combined to form a single one. Cho, cholesterol; FFA, free fatty acids; 1,2-/1,3-DAG, 1,2 and 1,3-diacylglycerols; * matrix cluster ions of the used DHB matrix.

calculate the mol% fraction of the individual fatty acids released from the lipids. Gas chromatography-mass spectrometry was performed using an Agilent HP 6890 plus GC with mass selective detector 5973inert (Agilent Technologies, Böblingen, Germany), equipped with an HP-5MS column (30 m column length, 0.25 mm internal diameter, 0.25 ␮m film thickness, Agilent) using a temperature gradient of 140 ◦ C (2 min), followed by heating to 250 ◦ C (4 min) at 10 ◦ C/min and then followed by cooling to 140 ◦ C at 20 ◦ C/min.

of 1 ␮l × min−1 using a chip-based nanospray ion source (HPLC Chip/MS 1200 with infusion chip; Agilent). Mass spectra were acquired on a quadrupole time-of-flight (Q-TOF) mass spectrometer (Q-TOF 6530; Agilent) in positive mode with a fragmentation voltage of 270 V. For structural analysis, molecular ions were selected in the quadrupole and then fragmented in the collision cell with nitrogen gas and a collision energy of 33 V for GPC und 20 V for DAG. The data were processed with the Mass Hunter Workstation software (version B.02.00; Agilent).

2.6. MALDI-TOF mass spectrometry

3. Results and discussion

For the acquisition of the positive or negative ion mass spectra, 77 mg/ml 2,5-dihydroxybenzoic acid (DHB; in methanol) or 10 mg/ml 9-aminoacridine (9-AA; in isopropanol/acetonitrile, 60:40, by volume), respectively, were used. As the quality of the spectra recorded in the presence of 9-AA depends significantly on the applied solvent system, the applied lipids were diluted with isopropanol/acetonitrile (60:40, by volume) (Fuchs et al., 2009). All samples were pre-mixed with the matrix prior to deposition onto the MALDI-TOF MS target. All MALDI-TOF mass spectra were acquired on a Bruker Autoflex mass spectrometer (Bruker Daltonics, Bremen, Germany). The system utilises a pulsed nitrogen laser, emitting at 337 nm. The extraction voltage was 20 kV and gated matrix suppression was applied to prevent the saturation of the detector by matrix ions. For each mass spectrum, 128 single laser shots were averaged. The laser fluence was kept about 10% above threshold to obtain optimum signal-to-noise (S/N) ratios. In order to enhance the spectral resolution, all spectra were acquired in the reflector mode using delayed extraction conditions. The data were processed with the FlexAnalysis software (version 2.2; Bruker Daltonics).

3.1. Lipid profile of boar spermatozoa

2.7. Q-TOF mass spectrometry Lipids were dissolved in chloroform–methanol–ammonium acetate (300:665:35, by volume) and directly infused at a flow rate

Fractionation of total lipid extracts of boar spermatozoa by thin layer chromatography and subsequent MALDI-TOF MS analysis identified neutral and polar lipids. Neutral lipids comprised almost exclusively cholesterol, diacylglycerols (DAG) and free fatty acids (FFA) (Fig. 1). The evaluation of the masses of the DAG quasi-molecular ions (sodium adducts) showed that the 1,2- and 1,3-DAG-isomers are characterised by saturated fatty acyl residues (Fig. 1B and C). The observed masses unequivocally indicate that the diacylglycerols in the preserved boar spermatozoa are represented by DAG 28:0, 30:0, 32:0 and 34:0. Although we did not perform additional MS/MS experiments, the observed m/z ratios can presumably be attributed to the Na+ adducts of DAG (14:0/14:0), (14:0/16:0), (16:0/16:0) and (16:0/18:0). It is a particular advantage of the MALDI-TOF MS analysis that DAG exclusively appear as sodiated ions; thus, there is no interference between differences in the fatty acyl composition and different adducts (e.g., H+ and Na+ ) (Fuchs et al., 2010). Cholesterol is also obvious (m/z 369.3), while the mass range of the FFA is not shown because DHB is no suitable matrix for FFA detection. DAG species with unsaturated or longchain, polyunsaturated fatty acyl residues could not be detected. The fatty acyl compositions of all identified DAG were additionally verified by means of GC (see Supplementary data, Fig. S3). Among

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Fig. 2. Analysis of the polar lipids of boar spermatozoa. A total lipid extract from boar spermatozoa was separated by one-dimensional TLC using chloroform/methanol/water (65:25:4, by volume). The individual lipid species were visualised by primuline staining, scraped off, extracted and identified by MALDI-TOF mass spectrometry. Glycerophosphatidylcholine (GPC) and glycerophosphatidylethanolamine (GPE) species were further characterised by positive ion Q-TOF mass spectrometry. The assignments of the most pronounced peaks are given in the text. Please note that the Q-TOF exhibits a much higher mass accuracy than the TOF mass analyser (about 25 ppm versus < 1 ppm). GPE, glycerophosphatidylethanolamine; SGG, sulfogalactosylglycerolipids; GPC, glycerophosphatidylcholine; GPS, glycerophosphatidylserine; GPA, glycerophosphatidic acid; GPI, glycerophosphatidylinositol; PSL, phosphosphingolipids; lyso-GPL, lyso-derivatives of glycerophospholipids.

the glycerophospholipids (GPL), glycerophosphatidylcholine (GPC) and glycerophosphatidylethanolamine (GPE) were the most prominent polar lipids (Fig. 2, left), while glycerophosphatidylserine (GPS), glycerophosphatidylinositol (GPI), phosphosphingolipids and sulfoglycoglycerolipids (SGG) were present only as minor constituents. The reader should note that assignments are based on the high mass accuracy of the Q-TOF instrument (less than 1 ppm) while no MS/MS has been performed. Therefore, potential isomers (for instance, lipids with the unsaturated residue in the sn-1 position) are not resolved. Q-TOF und MALDI-TOF MS analysis revealed that diacyl- as well as ether-species are present in both the GPC and the GPE fractions of boar spermatozoa (Fig. 2). The evaluation of the obtained peaks indicates that both GPL classes (GPE und GPC) are characterised by unsaturated long chain residues; the assignment of the most intense peaks is provided directly in Fig. 2. This agrees favourably with the data by Evans et al. (1980) and Lessig et al. (2004). On the basis of the calculated masses of the detected peaks, GPL with long chain multiple unsaturated residues (fatty acids, fat aldehydes) could be identified. The observed m/z values unequivocally indicate that all GPL, in the presence of ammonium acetate, are detectable mainly as [M+H]+ and to a lesser extent as [M+Na]+ adducts. Both can be easily differentiated (even if there is a complex distribution of the apolar residues) by using the high mass

accuracy of the Q-TOF mass spectrometer. As evident from Fig. 2, the majority of the GPE species in the preserved boar spermatozoa are represented by GPE 18:0-alkenyl/18:2-acyl (m/z 728.5557), GPE 18:0-alkenyl/22:6-acyl (m/z 750.5415) both detected as the H+ adducts and diacyl-18:0/20:4 GPE (m/z 768.5515, m/z 790.5336 and m/z 812.5169 corresponding to the H+ , Na+ and -H+ +2Na+ adducts, respectively). GPC, compared with GPE, is represented by alkenyl-acyl, alkyl-acyl- as well as -diacyl subclasses. As evident from Fig. 2, in the preserved boar spermatozoa, GPC are represented particularly by the phosphatidylcholines 14:0/14:0 (m/z 678.5076), 14:0/16:0 (m/z 706.5405), 14:0/18:3 (m/z 728.5208), 16:0/16:0 (m/z 734.5700), 16:0/18:1 (m/z 760.5872), 18:2/18:2 (m/z 782.5701), 18:1/18:2 (m/z 784.5858), 16:0/22:6 (m/z 806.5693), 16:0/22:5 (m/z 808.5848) and 16:0/22:3 (m/z 812.5971). All these peaks may be assigned to the H+ adducts due to the high mass accuracy of the measurements. Ether-GPC species are mainly represented by GPC 16:0 alkenyl/22:6-acyl (m/z 790.5785), GPC 16:0-alkyl/22:6-acyl (m/z 792.5918) and GPC 16:0-alkyl/22:5-acyl (m/z 794.6067), that are all detected as the H+ adducts. Further GPL from boar spermatozoa are GPS and GPI as well as sphingomyelin as the most abundant sphingolipid. However, no ether species could be detected in the GPS and GPI fractions at all. Thus, GPS and GPI are exclusively represented by diacyl species (data not shown). In boar spermatozoa, all the glycoglycerolipids are exclusively present as

V. Svetlichnyy et al. / Chemistry and Physics of Lipids 177 (2014) 41–50

sulfogalactosylglycerolipid (SGG) and this compound exclusively contains 16:0-alkyl- and 16:0-acyl residues. Hence, plasmanyl-SGG (32:0) is the compound that is most sensitively detectable by negative ion MS due to the sulphate residue as a strong electrolyte (see Supplementary data, Fig. S1). This has already been shown in numerous studies (Evans et al., 1980; Lessig et al., 2004; Ishizuka et al., 1973). According to the TLC data, very small amounts of lysolipids are also detectable. A more detailed analysis of these compounds was beyond the scope of this paper. An extensive hydrolysis of phosphatidylcholines to the lyso compounds (LPC) and free fatty acids leads to the vesiculation of the spermatozoa plasma membranes and the outer acrosomal membranes; and finally to the acrosome reaction (Abou-haila and Tulsiani, 2009; Flesch and Gadella, 2000; Roldan and Shi, 2007; Regazzi and Tomes, 2007). This hydrolysis can be caused either by phospholipase (PLA2 ) activity on acyl lipids or the reaction of reactive oxygen species (ROS) with unsaturated fatty acyl residues and particularly the plasmalogen lipids where the vinyl linkages are particularly oxidation-sensitive (Roldan and Shi, 2007; Lessig et al., 2007). Under in vitro conditions, the presence of such hydrolysis products (lysophospholipids and FFA) can predominantly be detected in the supernatant of the storage medium (Zanetti et al., 2010) due to the detergent properties of these compounds. As physiologically intact spermatozoa (those not having undergone the acrosome reaction) were exclusively used in this study, the amounts of lysolipids and FFA are only very small and thus were not further examined. We have shown (vide supra) that there are pronounced differences in the fatty acyl compositions between DAG and GPL and emphasised also the fact that sperm-derived DAG contain only medium-chained, saturated fatty acyl residues. It is known that mammalian spermatozoa possess amongst other enzymes, glycerophosphatidylcholine-specific phospholipases (GPC-PLC) and, thus, DAG can be generated directly by the PLC-mediated hydrolysis of GPC (Roldan and Shi, 2007). Under physiological conditions, DAG are early markers of spermatozoa capacitation due to the hydrolysis of GPL catalysed by the GPI- or GPC-PLC enzymes (O’Toole et al., 1996; Roldan, 1998; Roldan and Shi, 2007). However, under the in vitro conditions of our experiment, no decline in the viability of the spermatozoa (triggered by undergoing the acrosome reaction) could be monitored. We did not observe any DAG with a fatty acyl profile resembling that of the phospholipids (mainly long chained and unsaturated residues). Therefore, a major contribution of PLC is not very likely and other enzymes must be involved in this process: it is known that PLA2 plays an essential role in the release of fatty acids and lysophospholipids involved in spermatozoa membrane fusion during acrosomal exocytosis (Roldan and Fragio, 1994). Moreover, this enzyme is part of the cellular repair system which predominantly affects the long chained unsaturated fatty acyl residues of GPL and could provide the background for reacylation of lysolipids and subsequent DAG formation (Fuchs et al., 2012). To elucidate this phenomenon, we examined the metabolic incorporation of unsaturated fatty acids (that were not detected in the DAG fraction of native spermatozoa) to obtain further insights as to whether the incorporation of such fatty acids into the spermatozoa lipids is possible. 3.2. Incorporation of [1-14 C]-octadecadienoic acid into the lipids of boar spermatozoa In order to investigate the metabolic incorporation of unsaturated fatty acids into the polar and nonpolar lipids, spermatozoa were incubated with [1-14 C]-octadecadienoic acid. Metabolic incorporation of [1-14 C]-octadecadienoic acid took place in the neutral and polar lipids (cf. Supplementary data, Fig. S3). Radiolabelled neutral lipids were identified as 1,2-DAG. This result agrees

45

Fig. 3. Incorporation of [1-14 C]-octadecadienoic acid into lipids of boar spermatozoa. Boar spermatozoa were incubated for 24 h at 17 ◦ C with [1-14 C]-octadecadienoic acid. Lipids were extracted, separated by two-dimensional thin layer chromatography (alkaline run: chloroform/methanol/25% ammonium hydroxide solution (90:54:7, by volume), acidic run: chloroform/methanol/acetone/glacial acetic acid/water (50:10:20:10:5, by volume, at 30 ◦ C) and then visualised by iodine staining (A) or phosphorimager scanning (B, overlayed with iodine staining and marked here in red). The location of individual species was verified by MALDITOF mass spectrometry. Unidentified lipids are not marked. FFA, free fatty acids; GPE, glycerophosphatidylethanolamine; SGG, sulfogalactosylglycerolipids; GPC, glycerophosphatidylcholine; GPS, glycerophosphatidylserine; GPA, glycerophosphatidic acid; GPI, glycerophosphatidylinositol. (For interpretation of the references to color in this legend, the reader is referred to the web version of the article.)

perfectly with previous radiochemical experiments that were performed with palmitic and arachidonic acid (Vazquez and Roldan, 1997). It could be shown that these radiolabelled fatty acids are primarily incorporated into glycerophosphatidic acid (GPA) and GPC. Upon the evaluation of the autoradiogram of the 2D thin-layer chromatogram, an inhomogeneous distribution of the radioactive signal was clearly discernable within the GPC spots (Fig. 3B). If [1-14 C]-octadecadienoic acid would have been incorporated randomly into all GPC classes, a homogenous distribution of the signal would have been expected. In the plasma membranes of boar spermatozoa, about 20% of the GPC is present as diacyl-GPC and approximately 80% as ether-GPC (Evans et al., 1980). On the basis of the distribution of the radioactive signals within the total GPC spot, it can be assumed that a metabolic incorporation of the free fatty

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DAG (28:0)+Na+ DAG (30:0)+Na+ 535.5 563.5

DAG (32:0)+Na+

DAG (34:0)+Na+

A

591.6

* 551.0

619.6 639.5

DAG (18:2/18:2)+Na+

535.5

B

563.5 * 551.0

591.6

619.6

535.5

DAG ([U-13C]-18:2/[U-13C]-18:2)+Na+

675.7

C

563.5 * 551.0 540

591.6 560

580

619.6 600

620

640

660 Δ 36

680 m/z

u

Fig. 4. Incorporation of octadecadienoic acid into diacylglycerol of boar spermatozoa. Boar spermatozoa were incubated for 24 h at 17 ◦ C with [12 C]-octadecadienoic acid or with uniformly labelled [U-13 C]-octadecadienoic acid. Lipids were extracted, separated by one-dimensional TLC using n-hexane/diethyl ether/glacial acetic acid (80:15:1, by volume). The individual lipid species were visualised by primuline staining, scraped off, extracted and identified by MALDI-TOF mass spectrometry. Compounds related to peaks (m/z = 535.5, m/z = 563.5, m/z = 591.6 and m/z = 619.6) are present in all samples and can be easily assigned to DAG with different acyl chain lengths (DAG-28:0 up to DAG-34:0) (A–C). Peaks at m/z 639.5 and 675.5 are indicative of the supplementation conditions and are not present in the control (A). Please note that DAG are exclusively detected as sodiated (never as protonated) ions under conditions of MALDI-TOF MS. Supplementation of the spermatozoa with octadecadienoic acid results in the de novo generation of DAG with two octadecadienoyl residues. DAG, diacylglycerol; * matrix cluster ions of the used DHB matrix.

acid took place exclusively into the diacyl-GPC. This incorporation could also be verified by subsequent MS analyses. The incorporation of the radioactively labelled fatty acid could be also observed for GPA, a precursor of the de novo biosynthesis of GPC. In both, GPE and GPI, however, the intensity of the radioactive signal was significantly weaker which confirms the preferred incorporation of the fatty acid into GPC. The incorporation of the radiochemical into SGG and GPS could not be observed. Thus, metabolic incorporation of the FFA into these lipid classes can be excluded. Furthermore, a so far unknown compound migrated close above the GPE spot. 3.3. Incorporation of octadecadienoic acid into the neutral lipids of boar spermatozoa and de novo formation of diacylglycerols A detailed characterisation of selected neutral lipid species was performed by labelling experiments using [12 C]- (i.e. “native” octadecadienoic acid) and [U-13 C]-octadecadienoic acid. Subsequent to supplementation with the indicated fatty acid, lipids were extracted from spermatozoa, separated by TLC and the obtained DAG fractions were characterised by MALDI-TOF MS. The corresponding positive ion mass spectra are shown in Fig. 4. The peaks at m/z = 535.5, m/z = 563.5, m/z = 591.6 and m/z = 619.6 are present in all samples and can be easily assigned to DAG with

different acyl chain lengths (DAG-28:0 up to DAG-34:0) (Fig. 4A–C). The analysis of the samples supplemented with octadecadienoic acid, however, shows that a metabolic incorporation of this dedicated fatty acid into the DAG fraction is obvious and, thus, a de novo generation of diacylglycerols (Fig. 4B and C) must have taken place. A characteristic peak with m/z = 639.5 (Fig. 4B) was only detected in the case of the spermatozoa supplemented with [12 C]-octadecadienoic acid. This peak corresponds to the sodium adduct of DAG-36:4, whereby accompanying post source decay (PSD) experiments revealed the exclusive presence of [12 C]octadecadienoyl residues. Unfortunately, the quality of the PSD spectra is poor and the deprotonated fatty acids would be easier to detect by negative ion PSD MALDI MS. However, we were so far not able to generate convincing negative ion spectra of DAG. Therefore, an additional experiment was performed. The spermatozoa were additionally supplemented with [U13 C]-octadecadienoic acid and a characteristic peak at m/z = 675.5 (Fig. 4C) is detectable with high intensity. Both peaks (m/z 639.5 as well as 675.5) are indicative of the supplementation conditions and are not present in the control sample at all. The mass difference between m/z = 675.5 and m/z = 639.5 is 36 mass units (u). This corresponds exactly to the double of the mass difference between “native” [12 C]-octadecadienoic acid and a uniformly labelled

V. Svetlichnyy et al. / Chemistry and Physics of Lipids 177 (2014) 41–50

47

Fig. 5. Incorporation of octadecadienoic acid into glycerophosphatidylcholine of boar spermatozoa. Boar spermatozoa were incubated for 24 h at 17 ◦ C with [12 C]octadecadienoic acid or with uniformly labelled [U-13 C]-octadecadienoic acid. Lipids were extracted, separated by one-dimensional TLC using chloroform/methanol/water (65:25:4, by volume). The individual lipid species were visualised by primuline staining, scraped off, extracted and identified by Q-TOF mass spectrometry. Two new peaks are present in sample C (m/z 776.6281 as well as 818.6890; see bold print). Supplementation of the boar spermatozoa with octadecadienoic acid results in the generation of GPC (16:0/[U-13 C]-18:2) and GPC ([U-13 C]-18:2/[U-13 C]-18:2) but not the de novo formation of GPC. Identification of peaks is described in more detail in the text. GPC, glycerophosphatidylcholine.

[U-13 C]-octadecadienoic acid. Thus, in the spermatozoa samples B and C, the de novo generation of DAG containing two octadecadienoyl residues is obvious. The fatty acyl composition of DAG was additionally investigated by gas-chromatography and an increase

of the linoleic acid content in the DAG fraction could be clearly monitored (cf. Supplementary data, Fig. S3). The MALDI-TOF mass spectra also unequivocally indicate that the supplied octadecadienoic acid is directly metabolised into DAG, while processes such

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V. Svetlichnyy et al. / Chemistry and Physics of Lipids 177 (2014) 41–50

DAG (28:0)+Na+ DAG (30:0)+Na+ 535.4

DAG (32:0)+Na+

563.4

*

550.9

591.5

DAG (34:0)+Na+

A

619.5

DAG (16:1/16:1)+Na+

535.4 563.4

*

587.4

551.0

B 591.5

619.5

DAG (18:1/18:1)+Na+

535.4

*

563.4

551.0

535.4

591.5

619.5

DAG (18:2/18:2)+Na+

563.4

*

551.0

535.4

C

643.4

591.5

619.5

D

639.4

DAG (18:3/18:3)+Na+

563.4

*

551.0

E

635.4

591.5

619.5

535.4

* 551.0 540

F

563.4 591.5

560

580

619.5

600

620

640

660

680 m/z

Fig. 6. Incorporation of hexadecenoic, octadecenoic, octadecadienoic und octadecatrienoic acids into diacylglycerols of boar spermatozoa. Boar spermatozoa were incubated for 24 h at 17 ◦ C with hexadecenoic (16:1), octadecenoic (18:1), octadecadienoic (18:2), octadecatrienoic (18:3) and eicosapentaenoic (20:5) acids. Lipids were extracted, separated by one-dimensional TLC using n-hexane/diethyl ether/glacial acetic acid (80:15:1, by volume). The individual lipid species were visualised by primuline staining, scraped off, extracted and identified by MALDI-TOF mass spectrometry. Compounds related to peaks (m/z = 535.4, m/z = 563.4, m/z = 591.5 and m/z = 619.5) are present in all samples and can be easily assigned to DAG with different acyl chain lengths (DAG-28:0 up to DAG-34:0) (A–E). Peaks at m/z 587.4 (B), 643.4 (C), 639.4 (D) and 635.4 (E) are indicative of the supplementation conditions and not present in the control (A). Identification of peaks is described in more detail in the text. Supplementation of the spermatozoa with 16:1-, 18:1-, 18:2-, 18:3 fatty acids, results in the de novo generation of DAG, in each case with two fatty acyl residues. No DAG 20:5/20:5 (expected at m/z = 683.5) was detected in the boar spermatozoa supplemented with eicosapentaenoic acid. DAG, diacylglycerol; * matrix cluster ions of the used DHB matrix.

as elongation, desaturation or oxidation of the used fatty acid did not take place.

absolutely regioselective. However, a more detailed evaluation of these aspects was beyond this investigation.

3.4. DAG isomers: 1,2-DAG and 1,3-DAG

3.5. Incorporation of unsaturated fatty acids into the polar lipids of boar spermatozoa

Surprisingly, the 1,2-DAG as well as the 1,3-DAG isomers were both detectable by means of TLC (Fig. 1, left). The isomerisation of 1,2-, respectively 2,3-DAG to 1,3-DAG might be an artefact since migration of fatty acyl residues is known to occur under conditions of TLC if protic solvents (for instance alcohols) are used (Kodali et al., 1990). In studies performed by Vazquez and Roldan (1997) where boar spermatozoa were incubated with [1-14 C] palmitic acid, a very early incorporation of the radiochemical label into 1,3-DAG was also seen. When boar spermatozoa, however, were labelled with [1-14 C] arachidonic acid, the authors reported an incorporation of the unsaturated fatty acid exclusively into the 1,2-DAG but not in the 1,3-DAG isomer. In our investigation, when boar spermatozoa were incubated with [1-14 C] octadecadienoic acid, likewise no incorporation of the radiochemical label into 1,3-DAG could be detected. Another potential explanation for the presence of the 1,3-DAG-isomers in spermatozoa lipids could be the direct intake of 1,3-DAG through the supplied food (Castellano et al., 2010; Strzezek et al., 2004; Mourvaki et al., 2010). This particularly applies as lipases are (in contrast to the majority of phospholipases) not

As already indicated in the context of the neutral spermatozoa lipids, a more detailed characterisation of the GPC species was also carried out using the labelling experiment with [12 C](i.e. native octadecadienoic acid) and [U-13 C]-octadecadienoic acid. Subsequent to supplementation with the indicated fatty acids, spermatozoa lipids were extracted, the lipid mix separated by TLC and the obtained GPC fraction characterised by Q-TOF MS. The corresponding positive ion mass spectra are shown in Fig. 5. The majority of peaks (m/z = 758.5702, m/z = 760.5874, m/z = 784.5861, m/z = 808.5849 and m/z = 782.5702, m/z = 812.6119) are present in all samples and can easily be assigned either to the H+ adducts of diacyl GPC (16:0/18:2, 16:0/18:1, 18:2/18:2, 16:0/22:5, 16:0/22:3) or the H+ adducts of ether-GPC 16:0 alkenyl/22:6-acyl (m/z 790.5789) and 16:0 alkyl/22:6-acyl (m/z 792.5921). Upon the comparison of the mass spectra of the native (non-supplemented) sample A and sample B (Fig. 5) supplemented with [12 C]-octadecadienoic acid), no significant differences are detectable. This is a clear indication

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that the native octadecadienoic acid is incorporated into GPL that are already present in the mixture. Thus no differentiation is possible. This agrees with the results of the labelling experiments with radioactive octadecadienoic acid (Fig. 3). In contrast, in trace C (Fig. 5), where the spermatozoa were supplemented with [U-13 C]-octadecadienoic acid, two minor but characteristic peaks at m/z = 818.6890 and m/z = 776.6281 are detectable. These peaks can be used as a measure of the de novo generation of GPL. The mass difference between the peaks at m/z = 818.6890 and m/z = 782.5699 is 36.1191 u. This corresponds exactly to the mass difference between two “native” [12 C]-octadecadienoyl residues in GPC 18:2/18:2 and two uniformly labelled [U-13 C]-octadecadienoyl residues. Thus, the generation of GPC containing two octadecadienoyl residues unequivocally took place in trace C (Fig. 5). The presence of labelled octadecadienoic acid was additionally confirmed by GC–MS (Supplementary data, Fig. S3). We were also able to detect DAG (18:2/18:2) in the neutral lipid fractions from spermatozoa supplemented with octadecadienoic acid (vide supra, 3.3). 1,2-DAG is a precursor for the synthesis of GPC via the cholinephosphotransferase pathway. In mammals, GPC is synthesised via phosphorylation of the choline headgroup, and activated as CDP-choline followed by condensation with 1,2-DAG (Nelson and Cox, 2008). The mass difference between the peak at m/z = 776.6281 and m/z = 758.5691 is 18.0590 u. This corresponds exactly to the mass difference between “natural” GPC (16:0/18:2) containing octadecadienoic acid (12 C-18) and GPC (16:0/18:2) containing a uniformly 13 C-labelled octadecadienoic acid (U-13 C-18). Thus, in sample C (Fig. 5) the generation of GPC containing only a single octadecadienoyl is also evident. We did not, however, detect a precursor for the synthesis of this GPC (i.e. DAG 16:0/18:2) upon the examination of the neutral lipid fraction from samples supplemented with U-13 C-18. Therefore, the de novo synthesis of this GPC via the cholinephosphotransferase pathway, as described above, can be ruled out. Thus, an acyl transfer to LPC 16:0 is the only possible explanation. The required lysolipid species can easily be generated by PLA2 -induced hydrolysis of GPC. Lysolipids (e.g. LPC) and FFA destabilise membranes (Henriksen et al., 2010). Spermatozoa are able to incorporate FFA into GPC and to reacylate LPC; both processes are important to stabilise the membrane structures. In this way a premature acrosomal exocytosis through the hydrolysis of the GPC of spermatozoa during ejaculation and in the female genital tract can be avoided. The location of all enzymes that are responsible for the synthesis of GPC as well as the acyl transfer to lysolipids in spermatozoa is, however, so far unknown. Our results suggest that upon ejaculation, enzymes (involved in lipid biosynthesis) from the secretion of the accessory sex glands, the so-called seminal plasma, may bind to the surface of spermatozoa. Washing the spermatozoa from seminal plasma with BTS led to the reduction but not the complete loss of the radioactive label (see Supplementary data, Fig. S2). In this context, further research on the capacity of seminal plasma for lipid biosynthesis is necessary. 3.6. Metabolic incorporation of endogenously present unsaturated fatty acids into DAG of boar spermatozoa Although it could be unequivocally shown that octadecadienoic acid (18:2) is incorporated into spermatozoa lipids, the question whether the metabolic incorporation of different fatty acids is dependent on their chain-length as well as their degree of saturation remains to be clarified. To clarify this aspect, boar spermatozoa were incubated for 24 h at 17 ◦ C with hexadecenoic (16:1), octadecenoic (18:1), octadecadienoic (18:2), octadecatrienoic (18:3) and eicosapentaenoic (20:5) acid, the lipids were extracted, separated by TLC and analysed by MS.

49

As already shown in Fig. 1, the native composition of the acyl residues of DAG of boar spermatozoa is (28:0, 30:0, 32:0 and 34:0). As monitored by MS, all (artificially added) fatty acids with the exception of eicosapentaenoic acid are obviously incorporated into the spermatozoa. As evident from Fig. 6, DAG 16:1/16:1 (m/z 587.4), 18:1/18:1 (m/z 643.5), 18:2/18:2 (m/z 639.5) and 18:3/18:3 (m/z 635.5) could be detected. In contrast, no DAG 20:5/20:5 (expected at m/z 683.5) was detectable in the boar spermatozoa supplemented with eicosapentaenoic acid. This indicates that eicosapentaenoic acid was not metabolically incorporated in the spermatozoa lipids and, thus, this fatty acid is not accepted by the spermatozoa for the biosynthesis of DAG. 1,2-DAG are a precursor for the synthesis of GPC via the CPT-pathway, as described above. Therefore the synthesis of GPC (20:5/20:5) can be completely ruled out. 4. Conclusions This is the first study that precisely characterises native boar spermatozoa lipid species as well as those which are only generated after the supplementation of the spermatozoa with unsaturated fatty acids under in vitro conditions of liquid sperm preservation. Unsaturated middle chained octadecadienoic acid (18:2), present in GPC but not in 1,2-DAG, was chosen as the most promising substrate. The details of incorporation were analysed: the metabolic incorporation of octadecadienoic acid took place into the boar spermatozoa lipids (GPA, 1,2-DAG, GPC). The spermatozoa lipid species were identified as DAG (18:2/18:2), GPC (16:0/18:2) and GPC (18:2/18:2). Considering two independent pathways may explain these findings: on the one hand, the de novo synthesis of GPC via the CDP-choline pathway and, on the other hand, the reacylation of LPC catalysed by lysophosphatidylcholine acyltransferase (LPCAT). Additionally, the metabolic incorporation of selected free fatty acids such as 16:1, 18:1 and 18:3 could be verified; no metabolic incorporation was observed for 20:5 indicating that docosapentaenoic acid is not a substrate of the related enzymes. We suggest that the synthesis of lipids in spermatozoa (e.g. from endogenously present fatty acids in seminal plasma and/or in fluids of the female genital tract) is important for the function of spermatozoa during their stay in the female genital tract and during the fertilisation process. Future research on the functional characterisation of enzymes (e.g. choline kinase, CTP-choline phosphate cytidyltransferase, CDP-choline phosphotransferase und lysophosphatidylcholine-acyltransferase) that are involved in the lipid biosynthesis of spermatozoa is, thus, very important and may lead to an improved understanding of (in)fertility as well as the improvement of the preservation of spermatozoa in breeding and conservation programmes. Acknowledgements This work was supported by Minitüb® (Tiefenbach, Germany), by the German Research Council (DFG: MU 1520/4-1, Schi 476/121) and by the German Academic Exchange Service (DAAD). The authors thank Peter Dörmann, Georg Hölzl (University of Bonn) for Q-TOF measurements and Anita Retzlaff, Karin Rüdiger (Institute for Reproduction of Farm Animals Schönow e.V.) for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.chemphyslip.2013.11.001.

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