Identification and characterization of non-phosphorylcholine-binding and phosphorylcholine-binding proteins of canine seminal plasma

Marzena Mogielnicka-Brzozowska 1 , Mariola Słowińska 2 , Leyland Fraser 1 , Paweł Wysocki 1 , Rafał Strzeżek 1 , Karolina Wasilewska 1  and Władysław Kordan 1
  • 1 Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-719 , Olsztyn, Poland
  • 2 Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Tuwima 10, 10-748 , Olsztyn, Poland


Seminal plasma (SP) proteins participate in the process of fertilization by binding to the sperm membrane, particularly to the phosphorylcholine-containing lipids. This study aimed to identify and characterize non-phosphorylcholine-binding and phosphorylcholine-binding proteins (nPch- BPs and PchBPs, respectively) of canine SP. The nPchBPs and PchBPs were isolated from canine SP by affinity chromatography. Electrophoretic studies revealed that the nPchBPs and PchBPs occurred in their native state as high-molecular-weight aggregates. Immunofluorescent staining showed preferential binding of nPchBPs to the sperm acrosome membrane, whereas PchBPs coating was uniformly distributed on the sperm post-acrosomal membrane, mid-piece and tail regions. Analysis with mass spectrometry confirmed that canine prostate specific esterase (CPSE) is a component of the nPchBPs and PchBPs, which is implicated in key mechanisms of protein-coating on the sperm plasma membrane surface. In addition, proteins of known binding properties such as prostaglandin-H2 D-isomerase and lipocalin-like 1 protein, identified in canine SP, might have a specific role in the fertilization-associated processes.

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  • Adlerova L., Bartoskova A., Faldyna M. (2008). Lactoferrin: Areview. Vet. Med., 53: 457-468.

  • Aquino - Cortez A.,da Silva L.D.M,de Araujo A.A.,da Silva Bezerrade Menezes E.,de Alencar Araripe Noronha Moura A. (2016). Proteins of the canine seminal plasma. Cienc. Rural, Santa Maria, 46: 901-908.

  • Calvete J.J., Raida M., Gentzel M., Urbanke C., Sanz L., Töpfer - Petersen E. (1997). Isolation and characterization of heparin and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine p B1. FEBS Lett., 407: 201-206.

  • De Souza F.F., Martins M.I.,dos Santos Fernandes C.E., Ribolla P.E., Lopes M.D. (2006). Heparin-binding proteins of canine seminal plasma. Theriogenology, 66: 1606-1609.

  • Desnoyers L., Manjunath P. (1992). Interaction ofanovel class of phospholipid-binding proteins of bovine seminal fluid with different affinity matrices. Arch. Biochem. Biophys., 305: 341-349.

  • Flower D.R., North A.C.T., Sansom C.E. (2000). The lipocalin protein family: structural and sequence overview. Biochim. Biophys., 1482: 9-24.

  • Fraser L., Dziekonska A., Strzezek R., Strzezek J. (2007). Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics. Theriogenology, 67: 994-1003.

  • Gwathmey T.M., Ignotz G.G., Mueller J.L., Manjunath P., Suarez S.S. (2006). Bovine SPproteins PDC-109, BSP-A3, and BSP-30-k Da share functional roles in storing sperm in the oviduct. Biol. Reprod., 75: 501-507.

  • Isaacs W., Coffey D. (1984). The predominant protein of canine SPis an enzyme. J. Biol. Chem., 259: 11520-11526.

  • Ivanova N., Gradinarska D., Daskalova D. (2015). Reproductive biotechnology in animal husbandry - current status and future prospects. Biotechnol. Anim. Husb., 31: 467-480.

  • Kiso W.K., Selvaraj V., Nagashima J., Asano A., Brown J.L., Schmitt D.L., Leszyk J., Travis A.J., Pukazhenthi B.S. (2013). Lactotransferrin in Asian elephant (Elephas maximus) seminal plasma correlates with semen quality. PLOS ONE, 8: e71033.

  • Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.

  • Leblond E., Desnoyers L., Manjunath P. (1993). Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma. Mol. Reprod. Dev., 34: 443-449.

  • Linde- Forsberg C. (1991). Achieving canine pregnancy using frozen and chilled extended semen. Vet. Clin. North. Am. Small Anim. Pract., 21: 467-485.

  • Lowry O.H., Rosenbrough N.J., Farr A.L., Randall R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275.

  • Manaskova P., Jonakova V. (2008) Localization of porcine SP (PSP) proteins in the boar reproductive tract and spermatozoa. J. Reprod. Immunol., 78: 40-48.

  • Manjunath P., Therien I. (2001). Role of SPphospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. J. Reprod. Immunol., 53: 109-119.

  • Mogielnicka- Brzozowska M., Fraser L., Czarzasta J., Kordan W. (2012). Isolation and characterization of zinc-binding proteins of canine seminal plasma. Pol. J. Vet. Sci., 15: 493-498.

  • Mogielnicka- Brzozowska M., Dziekońska A., Strzeżek R., Załęcki M., Majewska A., Tołścik K., Kordan W. (2014 a). Effect of zinc-binding proteins of SPon motility and membrane integrity of canine spermatozoa stored at 5°C. Bull. Vet. Inst. Pulawy, 58: 163-168.

  • Mogielnicka- Brzozowska M., Strzeżek J., Dziekońska A., Kordan W. (2014 b). The ability of zinc-binding proteins from boar SPto bind heparin and phosphorylcholine. Anim. Sci. Pap. Rep., 32: 327-336.

  • Mogielnicka- Brzozowska M., Kowalska N., Fraser L., Kordan W. (2015). Proteomic characterization of zinc-binding proteins of canine seminal plasma. Reprod. Dom. Anim., 50: 1017-21.

  • Niżański W., Partyka A., Prochowska S. (2016). Evaluation of spermatozoa function - useful tools or just science. Reprod. Dom. Anim., 51: 37-45.

  • Nynca J., Dietrich M.A., Bilińska B., Kotula - Balak M., Kiełbasa T., Karol H., Ciereszko A. (2011). Isolation of lipocalin-type protein from rainbow trout SPand its localization in the reproductive system. Reprod. Fert. Develop., 23: 381-389.

  • O ’ Farrell P.Z., Goodman H.M., O ’ Farrell P.H. (1977). High resolution of two-dimensional electrophoresis of basic as well as acidic proteins. Cell, 12: 1133-1142.

  • Rijsselaere T.,van Soom A., Maes D.,de Kruif A. (2002). Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. Theriogenology, 57: 1669-1681.

  • Rodriguez- Martinez H., Kvist U., Ernerudh J., Sanz L., Calvete J.J. (2011). SP proteins: What role do they play? Am. J. Reprod. Immunol., 66: 11-22.

  • Schäfer- Somi S., Palme N. (2016). Seminal plasma characteristics and expression of ATP-binding Cassette Transporter A1 (ABCA1) in canine spermatozoa from ejaculates with good and bad freezability. Reprod. Domest. Anim., 51: 232-239.

  • Strzeżek R., Fraser L. (2009). Characteristics of spermatozoa of whole ejaculate and sperm-rich fraction of dog semen following exposure to media varying in osmolality. Reprod. Biol., 9: 113-126.

  • Tannert A., Kurz A., Erlemann K.R., Muller K., Herrmann A., Schiller J., Topfer - Petersen E., Manjunath P., Muller P. (2007). The bovine SPprotein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet. Eur. Biophys. J., 36: 461-475.

  • Therien I., Bleu G., Manjunath P. (1995). Phosphatidyl choline binding proteins of bovine SP modulates capacitation of spermatozoa by heparin. Biol. Reprod., 52: 1372-1379.

  • Watson P.F. (1975). Use ofa Giemsa stain to detect changes in acrosomes of frozen ram spermatozoa. Vet. Rec., 1: 12-15.

  • Zakharchenko O., Greenwood C., Lewandowska A., Hellman U., Alldridge L., Souchelnyrskyi S. (2011). Meta-data analysis asastrategy to evaluate individual and common features of proteomic changes in breast cancer. Cancer Genom. Proteom., 8: 1-14.

  • Zelli R., Orlandi R., Verstegen J., Troisi A., Elad Ngonput A., Menchetti L., Cardinali L., Polisca A. (2016). Addition of different concentrations of prostasome-like vesicles at neutral or slightly alkaline p Hdecreases canine sperm motility. Andrology, 5: 160-168.

  • Zhou Y., Shaw N., Li Y., Zhao Y., Zhang R. (2010). Structure-function analysis of human l-prostaglandin Dsynthase bound with fatty acid molecules. FASEB J., 24: 4668-4677.


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