5.結(jié)論

首先，表面張力測(cè)量證明牛精漿具有很好的表面活性，可能與BSP蛋白有關(guān)。其次，Langmuir膜法（先前被證明是公牛精子外細(xì)胞膜的相關(guān)脂質(zhì)單層模型[35]）有助于表征BSP蛋白對(duì)脂質(zhì)膜的親和力。BSP蛋白能夠到達(dá)被磷脂覆蓋的牛精子細(xì)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(shè)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(qiáng)烈相互作用而留在那里。最后，利用Langmuir膜法對(duì)已知的BSP蛋白螯合劑如ldl和脂質(zhì)體的作用進(jìn)行了篩選。脂質(zhì)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(zhì)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(shí)間尺度上脂質(zhì)體的非常微弱的插入是否有一些生物學(xué)后果。需要進(jìn)一步研究分析每種BSP蛋白的表面性質(zhì)及其在膜上的作用。此外，可以測(cè)試Langmuir膜，以檢測(cè)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(zhì)新螯合劑的方法。該方法可適用于其他動(dòng)物物種。

致謝

作者感謝IMV Technologies（法國）的財(cái)務(wù)支持，Gérard Chatagnon負(fù)責(zé)精漿蛋白質(zhì)定量，Véronique Solé負(fù)責(zé)凝膠電泳，Maureen Collobert負(fù)責(zé)英語校正，Alain Sire和Patrice Papineau負(fù)責(zé)手套箱的構(gòu)思和實(shí)現(xiàn)，允許在受控大氣中使用朗繆爾槽。

附錄A.補(bǔ)充數(shù)據(jù)

與本文相關(guān)的補(bǔ)充數(shù)據(jù)可在在線版本中找到，網(wǎng)址為http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://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,http://dx.doi.org/10.1016/j.bbrc.2010.07.088.