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The Phospholipid Composition of Kangaroo Spermatozoa Verified by Mass Spectrometric Lipid Analysis

  • Original Article
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Lipids

Abstract

Cryopreservation of kangaroo sperm has not been successful so far, and yet there is no promising cryopreservation protocol for these cells available. However, conservation of gametes is extremely important, particularly in the context of preserving endangered species. As spermatozoa are comprised of different membrane systems, the composition of these membranes might account for difficulties in cryopreservation. Lipids, as the main components, affect the physical properties of biological membranes and play a major role in sperm maturation. Therefore, knowledge of the lipid composition is crucial for any further step toward the preservation of the species. We used MALDI-TOF, ESI-IT, tandem mass spectrometry, and thin layer chromatography to investigate the lipid composition of epididymal spermatozoa of four different kangaroo species. Spectra of these species were very similar with respect to the identified lipid species. Tremendous changes in the lipid composition during the transit of sperm from caput to cauda epididymis could be seen, specifically an increase in poly-unsaturated fatty acids, ether lipids, and plasmalogens, as well as a reduction in mono- and di-unsaturated fatty acids. Additionally, phosphatidylcholines containing docosatrienoic acid (22:3), a heretofore unknown fatty acid for sperm membranes, showed the highest abundance in kangaroo sperm.

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Abbreviations

CerPCho:

Sphingomyelin

CID:

Collision-induced dissociation

DHB:

2,5-Dihydroxybenzoic acid

DMA:

Dimethylacetamide

ESI-IT MS:

Electrospray ionization ion trap mass spectrometry

FAME:

Fatty acid methyl esters

GC:

Gas chromatography

HPTLC:

High-performance thin-layer chromatography

MALDI-TOF MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MS/MS:

Tandem mass spectrometry

n-3/-6/-9:

Omega-3/-6/-9 fatty acid

PBS:

Phosphate-buffered saline

PLA2 :

Phospholipase A2

PakCho:

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine

PlsCho:

1-O-Alkenyl-2-acyl-sn-glycero-3-phosphocholine

PtdCho:

1,2-Diacyl-sn-glycero-3-phosphocholine

PakEtn:

1-O-Alkyl-2-acyl-sn-glycero-3-phosphoethanolamine

PtdEtn:

1,2-Diacyl-sn-glycero-3-phosphoethanolamine

PtdIns:

1,2-Diacyl-sn-glycero-3-phosphoinositol

ROS:

Reactive oxygen species

TLC:

Thin-layer chromatography

UV:

Ultraviolet

References

  1. Rodger JC, Hughes RL (1973) Studies of the accessory glands of male marsupials. Aust J Zool 21(3):303. doi:10.1071/ZO9730303

    Article  Google Scholar 

  2. Rodger JC, White IG (1975) Electroejaculation of Australian marsupials and analyses of the sugars in the seminal plasma from three macropod species. J Reprod Fertil 43(2):233–239

    Article  CAS  PubMed  Google Scholar 

  3. White IG (1993) Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod Fertil Dev 5(6):639. doi:10.1071/RD9930639

    Article  CAS  PubMed  Google Scholar 

  4. Scarlett CJ, Lin M, Aitken RJ (2001) Actin polymerisation during morphogenesis of the acrosome as spermatozoa undergo epididymal maturation in the tammar wallaby (Macropus eugenii). J Anat 198(1):93–101. doi:10.1046/j.1469-7580.2001.19810093.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lessig J, Gey C, Suss R et al (2004) Analysis of the lipid composition of human and boar spermatozoa by MALDI-TOF mass spectrometry, thin layer chromatography and 31P NMR spectroscopy. Comp Biochem Physiol B Biochem Mol Biol 137(2):265–277. doi:10.1016/j.cbpc.2003.12.001

    Article  PubMed  Google Scholar 

  6. Fuchs B, Jakop U, Goritz F et al (2009) MALDI-TOF “fingerprint” phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa. Theriogenology 71(4):568–575. doi:10.1016/j.theriogenology.2008.08.023

    Article  CAS  PubMed  Google Scholar 

  7. Fuchs B, Muller K, Goritz F et al (2007) Characteristic oxidation products of choline plasmalogens are detectable in cattle and roe deer spermatozoa by MALDI-TOF mass spectrometry. Lipids 42(11):991–998. doi:10.1007/s11745-007-3108-7

    Article  CAS  PubMed  Google Scholar 

  8. Fuchs B, Suss R, Schiller J (2010) An update of MALDI-TOF mass spectrometry in lipid research. Prog Lipid Res 49(4):450–475. doi:10.1016/j.plipres.2010.07.001

    Article  CAS  PubMed  Google Scholar 

  9. Pulfer M, Murphy RC (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 22(5):332–364. doi:10.1002/mas.10061

    Article  CAS  PubMed  Google Scholar 

  10. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917. doi:10.1139/o59-099

    Article  CAS  PubMed  Google Scholar 

  11. White T, Bursten S, Federighi D et al (1998) High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography. Anal Biochem 258(1):109–117. doi:10.1006/abio.1997.2545

    Article  CAS  PubMed  Google Scholar 

  12. Schiller J, Arnhold J, Benard S et al (1999) Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: a methodological approach. Anal Biochem 267(1):46–56. doi:10.1006/abio.1998.3001

    Article  CAS  PubMed  Google Scholar 

  13. Popkova Y, Schiller J (2017) Addition of CsCl reduces ion suppression effects in the matrix-assisted laser desorption/ionization mass spectra of triacylglycerol/phosphatidylcholine mixtures and adipose tissue extracts. Rapid Commun Mass Spectrom 31(5):411–418. doi:10.1002/rcm.7806

    Article  CAS  PubMed  Google Scholar 

  14. Milne S, Ivanova P, Forrester J et al (2006) Lipidomics: an analysis of cellular lipids by ESI-MS. Methods 39(2):92–103. doi:10.1016/j.ymeth.2006.05.014

    Article  CAS  PubMed  Google Scholar 

  15. Dannenberger D, Nuernberg K, Nuernberg G et al (2012) Different dietary protein and PUFA interventions alter the fatty acid concentrations, but not the meat quality, of porcine muscle. Nutrients 4(9):1237–1246. doi:10.3390/nu4091237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Angulo J, Hiller B, Olivera M et al (2012) Dietary fatty acid intervention of lactating cows simultaneously affects lipid profiles of meat and milk. J Sci Food Agric 92(15):2968–2974. doi:10.1002/jsfa.5709

    Article  CAS  PubMed  Google Scholar 

  17. Schiller J, Müller K, Süß R et al (2003) Analysis of the lipid composition of bull spermatozoa by MALDI-TOF mass spectrometry—a cautionary note. Chem Phys Lipids 126(1):85–94. doi:10.1016/S0009-3084(03)00097-5

    Article  CAS  PubMed  Google Scholar 

  18. Schneiter R, Brugger B, Sandhoff R et al (1999) Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. J Cell Biol 146(4):741–754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Comizzoli P (2015) Biobanking efforts and new advances in male fertility preservation for rare and endangered species. Asian J Androl 17(4):640–645. doi:10.4103/1008-682X.153849

    Article  PubMed  PubMed Central  Google Scholar 

  20. Walters EM, Men H, Agca Y et al (2005) Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility. Cryobiology 50(2):193–205. doi:10.1016/j.cryobiol.2005.01.004

    Article  CAS  PubMed  Google Scholar 

  21. McClean RV, Holt WV, Johnston SD (2007) Ultrastructural observations of cryoinjury in kangaroo spermatozoa. Cryobiology 54(3):271–280. doi:10.1016/j.cryobiol.2007.03.004

    Article  PubMed  Google Scholar 

  22. Garcia BM, Ferrusola CO, Aparicio IM et al (2012) Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Theriogenology 77(7):1280–1289. doi:10.1016/j.theriogenology.2011.10.033

    Article  Google Scholar 

  23. McClean R, MacCallum C, Blyde D et al (2006) Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa. CryoLetters 27(4):253–260

    CAS  PubMed  Google Scholar 

  24. Lin M, Rodger JC (1999) Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula). J Anat 194(2):223–232. doi:10.1046/j.1469-7580.1999.19420223.x

    Article  PubMed  PubMed Central  Google Scholar 

  25. McClean R, Zee YP, Holt WV et al (2008) Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol. Cryobiology 57(3):304–307. doi:10.1016/j.cryobiol.2008.08.007

    Article  CAS  PubMed  Google Scholar 

  26. Miller RR, Sheffer CJ, Cornett CL et al (2004) Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success. Cryobiology 49(2):137–148. doi:10.1016/j.cryobiol.2004.06.002

    Article  CAS  PubMed  Google Scholar 

  27. Darin-Bennett A, Poulos A, White IG (1974) The phospholipids and phospholipid-bound fatty acids and aldehydes of dog and fowl spermatozoa. Reproduction 41(2):471–474. doi:10.1530/jrf.0.0410471

    Article  CAS  Google Scholar 

  28. Ravie O, Lake PE (1985) The phospholipid-bound fatty acids of fowl and turkey spermatozoa. Anim Reprod Sci 9(2):189–192. doi:10.1016/0378-4320(85)90023-5

    Article  CAS  Google Scholar 

  29. Aveldaño MI, Rotstein NP, Vermouth NT (1992) Occurrence of long and very long polyenoic fatty acids of the n-9 series in rat spermatozoa. Lipids 27(9):676–680. doi:10.1007/BF02536023

    Article  PubMed  Google Scholar 

  30. Saether T, Tran TN, Rootwelt H et al (2007) Essential fatty acid deficiency induces fatty acid desaturase expression in rat epididymis, but not in testis. Reproduction 133(2):467–477. doi:10.1530/REP-06-00294

    Article  CAS  PubMed  Google Scholar 

  31. Pyttel S, Nimptsch A, Bottger J et al (2014) Changes of murine sperm phospholipid composition during epididymal maturation determined by MALDI-TOF mass spectrometry. Theriogenology 82(3):396–402. doi:10.1016/j.theriogenology.2014.04.017

    Article  CAS  PubMed  Google Scholar 

  32. Sato D, Ando Y, Tsujimoto R et al (2001) Identification of novel nonmethylene-interrupted fatty acids, 7E, 13E-20:2, 7E, 13E, 17Z-20:3, 9E, 15E, 19Z-22:3, and 4Z, 9E, 15E, 19Z-22:4, in Ophiuroidea (brittle star) lipids. Lipids 36(12):1371–1375

    Article  CAS  PubMed  Google Scholar 

  33. Palmer DN, Husbands DR, Jolly RD (1985) Phospholipid fatty acids in brains of normal sheep and sheep with ceroid-lipofuscinosis. Biochim Biophys Acta 834(2):159–163

    Article  CAS  PubMed  Google Scholar 

  34. Nervi AM, Brenner RR (1965) Identification and origin of docosatrienoic acid of rat phospholipids. Biochim Biophys Acta 106(1):205–207

    Article  CAS  PubMed  Google Scholar 

  35. Parks JE, Hammerstedt RH (1985) Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane. Biol Reprod 32(3):653–668

    Article  CAS  PubMed  Google Scholar 

  36. Banerjee N, Rosenthal MD (1986) Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts. Biochim et Biophys Acta (BBA) Lipids Lipid Metab 878(3):404–411. doi:10.1016/0005-2760(86)90249-3

    Article  CAS  Google Scholar 

  37. Brenner RR (1974) The oxidative desaturation of unsaturated fatty acids in animals. Mol Cell Biochem 3(1):41–52. doi:10.1007/BF01660076

    Article  CAS  PubMed  Google Scholar 

  38. Godwin S, Kang A, Gulino L et al (2014) Investigation of the microbial metabolism of carbon dioxide and hydrogen in the kangaroo foregut by stable isotope probing. ISME J 8(9):1855–1865. doi:10.1038/ismej.2014.25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zoeller RA, Lake AC, Nagan N et al (1999) Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether. Biochem J 338(3):769–776. doi:10.1042/bj3380769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Yeste M (2015) Recent advances in boar sperm cryopreservation: state of the art and current perspectives. Reprod Domest Anim 50(Suppl 2):71–79. doi:10.1111/rda.12569

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Dr. Gudrun Wibbelt for the necropsy of the analyzed animals and the provision of kangaroo testes and epididymides and Christiane Franz for technical assistance. This work was supported by the German Research Council (DFG Schi 476/12-2, DFG Schi 476/16-1, DFG MU 1520/4-2 and SFB 1052/Z3).

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Authors and Affiliations

  1. Institute of Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Härtelstr. 16/18, 04107, Leipzig, Germany

    Kathrin M. Engel & Jürgen Schiller

  2. Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, 10315, Berlin, Germany

    Karin Müller & Ulrike Jakop

  3. Function of Bioactive Lipids Unit, Institute of Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology, Willhelm-Stahl-Allee 2, 18196, Dummerstorf, Germany

    Dirk Dannenberger

Authors
  1. Kathrin M. Engel
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  2. Jürgen Schiller
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  5. Ulrike Jakop
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Contributions

Conception and design, Analysis and interpretation of the data, Drafting of the article, Critical revision of the article for important intellectual content, Final approval of the article: KME, UJ, KM, JS.

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Correspondence to Kathrin M. Engel.

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Engel, K.M., Schiller, J., Müller, K. et al. The Phospholipid Composition of Kangaroo Spermatozoa Verified by Mass Spectrometric Lipid Analysis. Lipids 52, 857–869 (2017). https://doi.org/10.1007/s11745-017-4283-9

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  • DOI: https://doi.org/10.1007/s11745-017-4283-9

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