5.結論

 首先，表面張力測量證明牛精漿具有很好的表面活性，可能與BSP蛋白有關。其次，Langmuir膜法（先前被證明是公牛精子外細胞膜的相關脂質單層模型[35]）有助于表征BSP蛋白對脂質膜的親和力。BSP蛋白能夠到達被磷脂覆蓋的牛精子細胞表面，這得益于它們與磷脂酰膽堿的相互作用和自身的表面活性。我們的假設是，由于它們自身的表面活性，它們首先穿透精子的外小葉，然后由于與磷脂酰膽堿的強烈相互作用而留在那里。最后，利用Langmuir膜法對已知的BSP蛋白螯合劑如ldl和脂質體的作用進行了篩選。脂質體與低密度脂蛋白一樣有效地阻止BSP蛋白插入磷脂層。低密度脂蛋白和脂質體的比例相同：0.16–0.17 mg磷脂酰膽堿/mg BSP。我們還不知道在冷凍保存的時間尺度上脂質體的非常微弱的插入是否有一些生物學后果。需要進一步研究分析每種BSP蛋白的表面性質及其在膜上的作用。此外，可以測試Langmuir膜，以檢測BSP與膜的親和力隨溫度的變化[20]。最后，Langmuir膜法是一種有效的篩選BSP蛋白質新螯合劑的方法。該方法可適用于其他動物物種。

致謝

 作者感謝IMV Technologies（法國）的財務支持，Gérard Chatagnon負責精漿蛋白質定量，Véronique Solé負責凝膠電泳，Maureen Collobert負責英語校正，Alain Sire和Patrice Papineau負責手套箱的構思和實現，允許在受控大氣中使用朗繆爾槽。

附錄A.補充數據

 與本文相關的補充數據可在在線版本中找到，網址為//dx.doi.org/10.1016/j.colsurfb.2015.11.34

工具書類

 [1]F.Ardon,S.S.Suarez,Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1,BSP3,and BSP5,Reproduction 146(2013)111–117,//dx.doi.org/10.1530/rep-12-0468.

 [2]P.Manjunath,J.Lefebvre,P.S.Jois,J.Fan,M.W.Wright,New nomenclature for mammalian BSP genes,Biol.Reprod.80(2009)394–397,//dx.doi.org/10.1095/biolreprod.108.074088.

 [3]V.Nauc,P.Manjunath,Radioimmunoassays for bull seminal plasma proteins(BSP-A1/-A2,BSP-A3,and BSP-30-Kilodaltons),and their quantification in seminal plasma and sperm,Biol.Reprod.63(2000)1058–1066,//dx.doi.org/10.1095/biolreprod63.4.1058.

 [4]F.S.Esch,N.C.Ling,P.B?hlen,S.Y.Ying,R.Guillemin,Primary structure of PDC-109,a major protein constituent of bovine seminal plasma,Biochem.Biophys.Res.Commun.113(1983)861–867,//dx.doi.org/10.1016/0006-291x(83)91078-1.

 [5]N.G.Seidah,P.Manjunath,J.Rochemont,M.R.Sairam,M.Chrétien,Complete amino acid sequence of BSP-A3 from bovine seminal plasma.Homology to PDC-109 and to the collagen-binding domain of fibronectin,Biochem.J.243(1987)195–203.

 [6]J.J.Calvete,K.Mann,L.Sanz,M.Raida,E.T?pfer-Petersen,The primary structure of BSP-30K,a major lipid-,gelatin-,and heparin-binding glycoprotein of bovine seminal plasma,FEBS Lett.399(1996)147–152,//dx.doi.org/10.1016/s0014-5793(96)1310-5.

 [7]D.Salois,M.Ménard,Y.Paquette,P.Manjunath,Complementary deoxyribonucleic acid cloning and tissue expression of BSP-A3 and BSP-30-kDa:phosphatidylcholine and heparin-binding proteins of bovine seminal plasma,Biol.Reprod.61(1999)288–297,//dx.doi.org/10.1095/biolreprod61.1.288.

 [8]P.Manjunath,M.R.Sairam,J.Uma,Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography,Biosci.Rep.7(1987)231–238,//dx.doi.org/10.1007/bf01124794.

 [9]L.Desnoyers,P.Manjunath,Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid,J.Biol.Chem.267(1992)10149–10155.

 [10]L.Desnoyers,P.Manjunath,Interaction of a novel class of phospholipid-binding proteins of bovine seminal fluid with different affinity matrices,Arch.Biochem.Biophys.305(1993)341–349,//dx.doi.org/10.1006/abbi.1993.1431.

 [11]P.Müller,K.-R.Erlemann,K.Müller,J.J.Calvete,E.T?pfer-Petersen,K.Marienfeld,et al.,Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles,Eur.Biophys.J.27(1998)33–41,//dx.doi.org/10.1007/s002490050108.

 [12]P.Manjunath,I.Thérien,Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation,J.Reprod.Immunol.53(2002)109–119,//dx.doi.org/10.1016/s0165-0378(01)98-5.

 [13]M.Ramakrishnan,V.Anbazhagan,T.V.Pratap,D.Marsh,M.J.Swamy,Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy,Biophys.J.81(2001)2215–2225,//dx.doi.org/10.1016/S0006-3495(01)75869-9.

 [14]D.A.Wah,C.Fernández-Tornero,L.Sanz,A.Romero,J.J.Calvete,Sperm coating mechanism from the 1.8?crystal structure of PDC-109-phosphorylcholine complex,Structure 10(2002)505–514,//dx.doi.org/10.1016/s0969-2126(02)751-7.

 [15]P.Manjunath,M.R.Sairam,Purification and biochemical characterization of three major acidic proteins(BSP-A1,BSP-A2 and BSP-A3)from bovine seminal plasma,Biochem.J.241(1987)685–692.

 [16]R.S.Damai,V.Anbazhagan,K.B.Rao,M.J.Swamy,Fluorescence studies on the interaction of choline-binding domain B of the major bovine seminal plasma protein,PDC-109 with phospholipid membranes,Biochim.Biophys.Acta:Proteins Proteomics.1794(2009)1725–1733,//dx.doi.org/10.1016/j.bbapap.2009.08.010.

 [17]A.Bergeron,M.-H.Crête,Y.Brindle,P.Manjunath,Low-density lipoprotein fraction from hen's egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane,Biol.Reprod.70(2004)708–717,//dx.doi.org/10.1095/biolreprod.103.022996.

 [18]A.Tannert,E.T?pfer-Petersen,A.Herrmann,K.Müller,P.Müller,The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability,Biochemistry 46(2007)11621–11629,//dx.doi.org/10.1021/bi7011299.

 [19]I.Thérien,G.Bleau,P.Manjunath,Phosphatidylcholine-binding proteins of bovine seminal plasma modulate capacitation of spermatozoa by heparin,Biol.Reprod.52(1995)1372–1379,//dx.doi.org/10.1095/biolreprod52.6.1372.

 [20]D.Lassiseraye,L.Courtemanche,A.Bergeron,P.Manjunath,M.Lafleur,Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes:lipid specificity and effect of the temperature,Biochim.Biophys.Acta:Biomembr.1778(2008)502–513,//dx.doi.org/10.1016/j.bbamem.2007.10.025.

 [21]I.Thérien,R.Moreau,P.Manjunath,Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm,Biol.Reprod.59(1998)768–776,//dx.doi.org/10.1095/biolreprod59.4.768.

 [22]I.Thérien,R.Moreau,P.Manjunath,Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm,Biol.Reprod.61(1999)590–598,//dx.doi.org/10.1095/biolreprod61.3.590.

 [23]A.Bergeron,P.Manjunath,New insights towards understanding the mechanisms of sperm protection by egg yolk and milk,Mol.Reprod.Dev.73(2006)1338–1344,//dx.doi.org/10.1002/mrd.20565.

 [24]P.Müller,A.Greube,E.T?pfer-Petersen,A.Herrmann,Influence of the bovine seminal plasma protein PDC-109 on cholesterol in the presence of phospholipids,Eur.Biophys.J.31(2002)438–447,//dx.doi.org/10.1007/s00249-002-0234-2.

 [25]T.S.Witte,S.Sch?fer-Somi,Involvement of cholesterol,calcium and progesterone in the induction of capacitation and acrosome reaction of mammalian spermatozoa,Anim.Reprod.Sci.102(2007)181–193,//dx.doi.org/10.1016/j.anireprosci.2007.07.007.

 [26]P.Manjunath,V.Nauc,A.Bergeron,M.Ménard,Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk,Biol.Reprod.67(2002)1250–1258,//dx.doi.org/10.1095/biolreprod67.4.1250.

 [27]M.-F.Lusignan,A.Bergeron,M.Lafleur,P.Manjunath,The major proteins of bovine seminal plasma interact with caseins and whey proteins of milk extender,Biol.Reprod.85(2011)457–464,//dx.doi.org/10.1095/biolreprod.110.089961.

 [28]L.Amirat,D.Tainturier,L.Jeanneau,C.Thorin,O.Gerard,J.L.Courtens,et al.,Bull semen in vitro fertility after cryopreservation using egg yolk LDL:a comparison with Optidyl®,a commercial egg yolk extender,Theriogenology 61(2004)895–907.

 [29]J.A.Foulkes,D.L.Stewart,Fertility of dairy-cattle after artificial-insemination with semen frozen in a lipoprotein diluent,J.Reprod.Fertil.51(1977)175–177.

 [30]J.A.Foulkes,Separation of lipoproteins from egg-yolk and their effect on motility and integrity of bovine spermatozoa,J.Reprod.Fertil.49(1977)277–284.

 [31]M.Moussa,V.Martinet,A.Trimeche,D.Tainturier,M.Anton,Low density lipoproteins extracted from hen egg yolk by an easy method:cryoprotective effect on frozen-thawed bull semen,Theriogenology 57(2002)1695–1706.

 [32]M.M.Pace,E.F.Graham,Components in egg yolk which protect bovine spermatozoa during freezing,J.Anim.Sci.39(1974)1144–1149.

 [33]M.Anton,V.Martinet,M.Dalgalarrondo,V.Beaumal,E.David-Briand,H.Rabesona,Chemical and structural characterisation of low-density lipoproteins purified from hen egg yolk,Food Chem.83(2003)175–183,//dx.doi.org/10.1016/s0308-8146(03)60-8.

 [34]M.-F.Lusignan,P.Manjunath,M.Lafleur,Thermodynamics of the interaction between bovine binder of sperm BSP1 and low-density lipoprotein from hen's egg yolk,Thermochim.Acta 516(2011)88–90,//dx.doi.org/10.1016/j.tca.2011.01.003.

 [35]J.Le Guillou,M.H.Ropers,C.Gaillard,E.David-Briand,S.Desherces,E.Schmitt,et al.,Organization of lipids in the artificial outer membrane of bull spermatozoa reconstructed at the air–water interface,Colloids Surfaces B:Biointerfaces 108(2013)246–254,//dx.doi.org/10.1016/j.colsurfb.2013.02.040.

 [36]A.Seelig,Local anesthetics and pressure:a comparison of dibucaine binding to lipid monolayers and bilayers,Biochim.Biophys.Acta 899(1987)196–204,//dx.doi.org/10.1016/0005-2736(87)90400-7.

 [37]P.Manjunath,L.Chandonnet,E.Leblond,L.Desnoyers,Major proteins of bovine seminal vesicles bind to spermatozoa,Biol.Reprod.50(1994)27–37,//dx.doi.org/10.1095/biolreprod50.1.27.

 [38]A.Berthold,H.Schubert,N.Brandes,L.Kroh,R.Miller,Behaviour of BSA and of BSA-derivatives at the air/water interface,Colloids Surfaces A:Physicochem.Eng.Aspects 301(2007)16–22,//dx.doi.org/10.1016/j.colsurfa.2006.11.054.

 [39]V.S.Alahverdjieva,D.O.Grigoriev,J.K.Ferri,V.B.Fainerman,E.V.Aksenenko,M.E.Leser,et al.,Adsorption behaviour of hen egg-white lysozyme at the air/water interface,Colloids Surfaces A:Physicochem.Eng.Aspects 323(2008)167–174,//dx.doi.org/10.1016/j.colsurfa.2007.12.031.

 [40]H.M.Mansour,G.Zografi,Relationships between equilibrium spreading pressure and phase equilibria of phospholipid bilayers and monolayers at the air–water interface,Langmuir 23(2007)3809–3819,//dx.doi.org/10.1021/la063053o.

 [41]L.E.Palacios,T.Wang,Egg-yolk lipid fractionation and lecithin characterization,JAOCS,J.Am.Oil Chem.Soc.82(2005)571–578,//dx.doi.org/10.1007/s11746-005-1111-4.

 [42]C.J.Thomas,V.Anbazhagan,M.Ramakrishnan,N.Sultan,I.Surolia,M.J.Swamy,Mechanism of membrane binding by the bovine seminal plasma protein,PDC-109:a surface plasmon resonance study,Biophys.J.84(2003)3037–3044,//dx.doi.org/10.1016/s0006-3495(03)70029-0.

 [43]U.Dahmen-Levison,G.Brezesinski,H.M?hwald,Specific adsorption of PLA2 at monolayers,Thin Solid Films 327–329(1998)616–620,//dx.doi.org/10.1016/s0040-6090(98)725-1.

 [44]V.Anbazhagan,R.S.Damai,A.Paul,M.J.Swamy,Interaction of the major protein from bovine seminal plasma,PDC-109 with phospholipid membranes and soluble ligands investigated by fluorescence approaches,Biochim.Biophys.Acta:Proteins Proteomics.1784(2008)891–899,//dx.doi.org/10.1016/j.bbapap.2008.03.002.

 [45]V.Anbazhagan,M.J.Swamy,Thermodynamics of phosphorylcholine and lysophosphatidylcholine binding to the major protein of bovine seminal plasma,PDC-109,FEBS Lett.579(2005)2933–2938,//dx.doi.org/10.1016/j.febslet.2005.04.046.

 [46]P.Manjunath,New insights into the understanding of the mechanism of sperm protection by extender components,Anim.Reprod.9(2012)809–815.

 [47]M.Gasset,L.Magdaleno,J.J.Calvete,Biophysical study of the perturbation of model membrane structure caused by seminal plasma protein PDC-109,Arch.Biochem.Biophys.374(2000)241–247,10.1006/abbi.1999.1593rS000398619991593X[pii].

 [48]A.Greube,K.Müller,E.T?pfer-Petersen,A.Herrmann,P.Müller,Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes,Biochemistry 40(2001)8326–8334,//dx.doi.org/10.1021/bi010552+.

 [49]I.Thérien,S.Soubeyrand,P.Manjunath,Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein,Biol.Reprod.57(1997)1080–1088,//dx.doi.org/10.1095/biolreprod57.5.1080.

 [50]M.Lafleur,L.Courtemanche,G.Karlsson,K.Edwards,J.L.Schwartz,P.Manjunath,Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes,Biochem.Biophys.Res.Commun.399(2010)406–411,//dx.doi.org/10.1016/j.bbrc.2010.07.088.