Dynamics of the mammalian sperm plasma membrane in the...

Dynamics of the mammalian sperm plasma membrane in the processof fertilization

Frits M. Flesch, Barend M. Gadella *Department of Biochemistry and Cell Biology, and Department of Farm Animal Health,

Graduate School of Animal Health and Institute for Biomembranes, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands

Received 27 June 2000; received in revised form 31 August 2000; accepted 5 September 2000

Abstract

Sexual reproduction requires the fusion of sperm cell and oocyte during fertilization to produce the diploid zygote. Inmammals complex changes in the plasma membrane of the sperm cell are involved in this process. Sperm cells have unusualmembranes compared to those of somatic cells. After leaving the testes, sperm cells cease plasma membrane lipid and proteinsynthesis, and vesicle mediated transport. Biophysical studies reveal that lipids and proteins are organized into lateral regionsof the sperm head surface. A delicate reorientation and modification of plasma membrane molecules take place in the femaletract when sperm cells are activated by so-called capacitation factors. These surface changes enable the sperm cell to bind tothe extra cellular matrix of the egg (zona pellucida, ZP). The ZP primes the sperm cell to initiate the acrosome reaction, whichis an exocytotic process that makes available the enzymatic machinery required for sperm penetration through the ZP. Aftercomplete penetration the sperm cell meets the plasma membrane of the egg cell (oolemma). A specific set of molecules isinvolved in a disintegrin^integrin type of anchoring of the two gametes which is completed by fusion of the two gameteplasma membranes. The fertilized egg is activated and zygote formation preludes the development of a new living organism.In this review we focus on the involvement of processes that occur at the sperm plasma membrane in the sequence of eventsthat lead to successful fertilization. For this purpose, dynamics in adhesive and fusion properties, molecular composition andarchitecture of the sperm plasma membrane, as well as membrane derived signalling are reviewed. ß 2000 Elsevier ScienceB.V. All rights reserved.

Keywords: Mammalian fertilization; Membrane dynamics; Cell^cell interaction; Sperm capacitation; Acrosome reaction; Signal trans-duction

1. Introduction

The earliest event in life is the meeting of thesperm cell with the egg. Enormous numbers of spermcells are deposited in the female genital tract, but

only one sperm cell will successfully fertilize theegg. The fusion of sperm and egg leads to the recom-bination of fathers and mothers genetical informa-tion resulting in a new individual. Sperm^egg inter-action and the subsequent fertilization are highlyregulated processes. Ongoing research shows the im-portant role of the sperm plasma membrane in mam-malian fertilization. The plasma membrane is notonly the border of the sperm cell, but it appears tobe a very dynamic structure. Here, we review the

0304-4157 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 4 1 5 7 ( 0 0 ) 0 0 0 1 8 - 6

* Corresponding author. Department of Biochemistry andCell Biology, Faculty of Veterinary Medicine, P.O. Box 80176,3508 TD, Utrecht, The Netherlands. Fax: +31-3025-35492;E-mail : [email protected]

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Biochimica et Biophysica Acta 1469 (2000) 197^235www.elsevier.com/locate/bba

sperm^egg interaction mainly focussing on the spermcell and particularly its plasma membrane.

Already during the formation of sperm cells in thetestis (spermatogenesis, for review see [1,2]), the plas-ma membrane and other speci¢c structures are pre-pared to react adequately to the female genital tractand the oocyte. Spermatogenesis in mammals startsat puberty by forming sperm cells in the testis fromstem cells (type A spermatogonia) arranged at thebasal lamina in seminiferous tubuli. Type A sperma-togonia divide several times to form spermatocytesand subsequent spermatids after meiosis. During thesubsequent divisions the cells migrate towards thelumen. Round spermatids are transformed intohighly di¡erentiated and polarized cells (Fig. 1A).

The £agellum is the ¢rst structure to be developedduring spermatogenesis which coincides with the re-cruitment of mitochondria from the cytoplasm toform a helical pattern around the mid-piece of the£agellum (Fig. 1B). More or less simultaneously theacrosome (a large secretory vesicle in the spermhead) is formed: the perinuclear Golgi apparatusproduces small condensing vacuoles that containdense material (proacrosomal granules), which willform the acrosomal vesicle. The acrosomal vesiclespreads out over the nucleus, while the Golgi appa-ratus contributes more and more material to thedeveloping acrosome. The plasma membrane ap-proaches the nucleus due to cytoplasm redistribu-tion, with the acrosome in between both structures,

Fig. 1. Sperm cells are polarized cells with a head, £agellum and midpiece (A, schematic surface drawing). The sperm head can besubdivided in four regions: apical, pre-equatorial, equatorial and post-equatorial regions. The acrosome (large secretory vesicle, 3) issituated apical to the nucleus (B). After binding of the sperm cell to the oocyte with its apical plasma membrane, the plasma mem-brane fuses with the underlying outer acrosomal membrane at multiple sites (C). The acrosomal content (hydrolytic enzymes) will besecreted, which enables the sperm cell to digest the egg extracellular matrix (ZP). After the acrosome reaction has been completed, theinner acrosomal membrane forms a continuum with the remaining plasma membrane (D). This hairpin structure is involved in the pri-mary binding of the sperm cell to the oolemma. Note that the representations (B), (C) and (D) are cross-sections through a £attenedcell. 1: plasma membrane; 2: outer acrosomal membrane; 3: acrosomal content; 4: inner acrosomal membrane; 5: nuclear envelope;6: nucleus containing highly condensed DNA; 7: posterior ring; 8: midpiece; 9: mitochondrion; 10: annular ring; 11: £agellum; 12:mixed vesicle (i.e. plasma membrane fused with outer acrosomal membrane); 13: acrosomal secretion; 14: hairpin structure.


F.M. Flesch, B.M. Gadella / Biochimica et Biophysica Acta 1469 (2000) 197^235198

inducing polarity in the developing spermatid. TheDNA of the nucleus starts to condense by changesin histones and other speci¢c basic proteins that as-sociate with the DNA. Concomitantly, the cell elon-gates as the cytoplasm is stretched out along the£agellum. The acrosome stops growing and the Golgiapparatus migrates to the caudal site of the sperma-tid. The cell volume of spermatids is reduced to ap-proximately 25% of the original volume, due towater loss, cytoplasm loss just before sperm releaseand the separation of a cytoplasmic package (resid-ual body). Residual bodies are formed at the timesperm releases from the epithelium. These residualbodies contain packed RNA and organelles such asthe Golgi apparatus, endoplasmic reticulum (ER),lysosomes and peroxisomes.

Due to the loss of most cell organelles and DNAtranscription, spermatozoa lack protein expressionand vesicular transport. This implies that the plasmamembrane is a stable and metabolically inert struc-ture, since protein, phospholipid, cholesterol andother components of the plasma membrane cannotbe newly synthesized. However, the plasma mem-brane of released sperm cells is not yet fully matured(for review see [1]). During the transit of the spermcell through the epididymis, the plasma membranechanges for example by the release, modi¢cationand adsorption of proteins and lipids. The role ofthese surface alterations is not fully understood,although some adsorbed proteins are involved inspermôocyte binding. In most mammalian species,sperm cells are fully matured when they reach theend of the cauda of the epididymis [1].

The mature sperm cell has three highly specializedregions (Fig. 1A): (i) the sperm head, involved inspermôocyte interaction; (ii) the midpiece with mi-tochondria, involved in energy production; (iii) the£agellum, involved in motility. The sperm head plas-ma membrane is separated from the midpiece plasmamembrane by the posterior ring and this latter do-main is separated from the £agellum plasma mem-brane by the annular ring (Fig. 1B: structural ele-ments 7 and 10, respectively). The sperm headcontains, besides a very low amount of cytosol, thenucleus and the acrosome (Fig. 1B). The acrosome isa large vesicle containing hydrolytic enzymes, neces-sary for the penetration of the oocyte extracellularmatrix (zona pellucida, ZP) [3]. Furthermore, the

acrosome-overlying plasma membrane is separatedfrom the post-acrosomal membrane by the equatorialsegment. These borders can be observed by electronmicroscopy [4]. Freeze fracture studies of the mam-malian sperm plasma membrane indicated that thedi¡erent domains all contain di¡erent concentrationsand distributions of intramembranous particles,which represent transmembrane proteins (for reviewsee [1]). This lateral polarized distribution could alsobe observed using speci¢c lectins and antibodies [5].

Spermôocyte interaction can be subdivided into asequence of events (Fig. 2) (for review see [3]). Ejacu-lated sperm cells need to be activated in the femalegenital tract in a process called capacitation (Fig.2A). Sperm cells become hypermotile during capaci-tation and only capacitated sperm cells bind to theZP in a species speci¢c manner (Fig. 2B). Binding ofthe sperm cell to the oocyte subsequently inducesvarious signals in the sperm cell. The concerted sig-nals trigger a Ca2� in£ux and the plasma membranefuses at multiple sites with the outer acrosomal mem-brane (acrosome reaction, Figs. 1C and 2C) [6]. Theacrosomal content, mainly hydrolytic enzymes, startsto disperse and digest the ZP (Fig. 2D). During theacrosome reaction the apical plasma membrane andthe outer acrosomal membrane form `mixed' vesiclesthat disperse (Fig. 1C). Throughout this process theinner acrosomal membrane becomes a part of theouter barrier of the cell and will form a continuousmembrane structure with the plasma membranewhich looks like a hairpin structure (Fig. 1D) [7].Consequently, the inner acrosomal membrane is ex-posed to and binds to the ZP (secondary ZP bind-ing). Hypermotile sperm cells that have reacted prop-erly upon binding to the ZP, penetrate the ZP andenter the space between the ZP and the egg plasmamembrane (perivitelline space) (Fig. 2D). Here thesperm cell binds to the egg plasma membrane (oo-lemma) with its tip. After binding of the sperm cell tothe oolemma with its tip, the sperm head binds lat-erally with its equatorial region in which the hairpinmembrane structure is involved (Fig. 2E). After lat-eral binding, the sperm plasma membrane and theoolemma fuse and, subsequently, the sperm cell isincorporated in the oocyte (Fig. 2F).

Only very recently we have gained insight into theheterogeneity of the plasma membrane. The diversi-¢cation of the cell surface probably relates to phys-


F.M. Flesch, B.M. Gadella / Biochimica et Biophysica Acta 1469 (2000) 197^235 199

iological specializations of the plasma membrane.This has been demonstrated for many epithelial cellsthat have an apical plasma membrane which is incontact with a lumen and a basolateral plasma mem-brane in contact with supporting cells and the bloodcirculation [8]. These two plasma membrane com-partments have di¡erent physiological functionsand also di¡er in molecular composition. Mixing ofmembrane components between the two plasmamembrane compartments is prevented by tight junc-tions [9]. It also has become clear that the composi-tion and lateral organization of the plasma mem-brane regulate the a¤nity for adhesion factors, thepermeability for hydrophilic solutes, cell signallingand cell fusion events.

This review will place another cell type, namely themammalian sperm cell, in the spotlight of membraneresearchers. This single cell has a variety of plasmamembrane specializations, and each of these has aunique role in the cascade of events that will leadto the ultimate goal of the sperm cell : the fusionwith the plasma membrane of the oocyte. Duringthe activation of the sperm cell (i.e. capacitation) inthe female genital tract, dramatic reorganizationstake place in the sperm plasma membrane in orderto achieve the ability to fertilize the oocyte. The cur-rent knowledge on the dynamics and functions of thesperm plasma membrane organization in relation tothe physiology of fertilization will be reviewed. Be-sides the physiological importance of this topic spe-

Fig. 2. Sequence of mammalian fertilization. (A) Freshly ejaculated sperm cells are activated in the female genital tract during a pro-cess called capacitation. (B) Capacitated sperm cells are hypermotile and are able to bind to the egg extracellular matrix (ZP). (C)Binding of sperm cells to the ZP triggers the acrosome reaction and acrosomal enzymes are secreted. (D) Hydrolytic enzymes secretedfrom the acrosome degrade the ZP and subsequent sperm cells penetrate the ZP, enter the perivitelline space and bind to the oolemmawith the apical tip. (E) Subsequent to apical tip binding, oolemma binding changes to the hairpin structure of the acrosome reactedsperm cell. (F) After hairpin structure binding to the oolemma, the sperm cell fuses with the oocyte and the sperm cell is subsequentlyincorporated in the oocyte. 1: perivitelline space; 2: ZP; 3: oolemma (egg plasma membrane).



cial attention will be paid to the potential of thesperm cell and its plasma membrane as a biologicalmodel system for future studies on the dynamicalaspects in the regulation of membrane heterogeneityand its consequences for cell physiology.

2. The lipids of the sperm cell and their role insperm^oocyte interaction

As mentioned in Section 1 the mature sperm celllacks a set of organelles important for the synthesisof lipids (e.g. the ER and the Golgi complex) and thebreakdown of lipids (e.g. lysosomes and peroxi-somes). In the mature sperm cell its surface mem-brane is not in contact with intracellular membranesbecause vesicle mediated membrane transport isblocked. The only exception to this is when the ap-ical plasma membrane fuses with the underlying out-er acrosomal membrane, which only takes place dur-ing the highly regulated acrosome reaction (Fig. 1).The unusual composition and organization of lipidsin the sperm plasma membrane are probably re£ec-tions of these speci¢c sperm properties.

2.1. Lipid composition

The lipid composition of the sperm plasma mem-brane of several mammalian species has been eluci-dated. Although there is considerable variation be-tween di¡erent mammalian species, in general theplasma membrane contains approximately 70% phos-pholipids, 25% neutral lipids and 5% glycolipids (onmolar base) [10].

2.1.1. PhospholipidsPhospholipids can be divided into phosphoglycer-

olipids and sphingomyelin (SM). The phosphoglycer-olipids vary in molecular structure because of di¡er-ent polar head groups at the sn-3 position of theglycerol backbone (phospholipid classes). Each phos-pholipid class comprises a number of molecular spe-cies due to di¡erent aliphatic acyl-, alkyl or alk-/-enylchains attached to either the sn-1 or sn-2 positions ofthe glycerol backbone (phospholipid species). Apartfrom some variations among di¡erent mammalianspecies [11], the phospholipid class composition ofsperm cells is generally comparable with that of so-

matic cell types. For example, human sperm cellscontain 50% phosphocholine glycerides (PC), 30%phosphoethanolamine glycerides (PE), 12.5% SM,3% phosphatidylserine (PS), 2.5% cardiolipin (CL)and about 2% of phosphatidylinositol (PI) [10]. Con-trarily, the molecular species composition of PC andPE, and perhaps also of the other phospholipidclasses, is quite unique for sperm cells. The sn-2 po-sition of the glycerol backbone of these phospho-lipids is predominantly esteri¢ed with long chainpolyunsaturated fatty acids (almost exclusivelydocosahexaenoic acid, 22:6 and docosapentaenoicacid, 22:5). Furthermore, at the sn-1 position theycontain predominantly saturated aliphatic chainswith a carbon chain length of 16 atoms, of whichapproximately 55% is attached as a vinylether (plas-menylcholine or plasmenylethanolamine) and 25%as a saturated alkyl-group (phosphatidylcholineor phosphatidylethanolamine, respectively) [12,13].Only 20% of the PC and PE contain the normalsn-1 ester linkage of a fatty acid. During capacitationthe PC levels may increase due to methylation of PE[14].

2.1.2. Neutral lipidsMajor variations in neutral lipid composition of

sperm membranes can be found between di¡erentspecies, individual males but also among di¡erentejaculates. The major variable factor is the amountof cholesterol in the sperm plasma membrane. Hu-man sperm cells for instance contain rather highamounts of cholesterol (40 mol percent of total lip-ids) whereas boar sperm cells contain much less cho-lesterol (22 mol percent). The cellular sterol contentseems to be related with the length of capacitation[3]. In fact, it has been demonstrated that cholesterolis depleted from the plasma membrane of sperm cellsupon capacitation (see Section 2.3.1). Besides choles-terol, low amounts of desmosterol, cholesterol sul-phate and cholesteryl esters can be found [10].

2.1.3. GlycolipidsGlycolipids are normally formed by the addition

of a glycosidic head group to ceramide. The onlyexception in vertebrates is the glycolipid seminolipidwhich is found only in mammals, in sperm cells andSchwann cells (for review see [2]). About 98% of theseminolipid has the structure 1-O-hexadecyl, 2-O-



hexadecanoyl, 3-3P-sulfogalactosyl glycerol. Besidesseminolipid, only trace amounts of other glycolipidscan be found in mammalian sperm cells [15]. Afterejaculation seminolipid can be desulfated by secretedarylsulfatases that originate from the accessory sexglands [16]. Both seminolipid and its desulfatedcounterpart are believed to participate in certain fer-tilization processes (see Sections 2.2.1, 2.3.3, 2.5 and4.2).

2.2. Lipid organization

2.2.1. Lateral membrane topologyThe structure of the sperm cell and the functional

division of its surface into lateral domains and sub-domains are summarized in Fig. 1. In the 1970s ¢rstclues were found for a lateral heterogeneity of thetopology of surface molecules in the plasma mem-brane of the sperm head. Lectins bound heterogene-ously to the surface of the sperm head (see Section3.1.3). Furthermore, freeze fracture replicas revealedthat integral membrane proteins were polarly distrib-uted among di¡erent regions of the plasma mem-brane of the sperm head as well as other parts ofthe sperm cell (for review see [1]). The delicate sur-face organization of the sperm cell alters upon ca-pacitation. This is partly due to decoating of theremoval of glycocalyx components [17] and adsorp-tion of new components from the female genital £u-ids and partly a result of enzymatic modi¢cations ofglycocalyx components [18,19]. In addition, a lateralreorganization of transmembrane proteins takesplace within the sperm head plasma membrane [20].

All these changes have been observed in the spermhead plasma membrane, which makes the interpreta-tion of the lateral heterogeneity di¤cult: (i) the headplasma membrane is separated from the mid-pieceand tail plasma membrane by linear arrangementsof transmembrane proteins forming the so-calledposterior ring [1]. However, such structural barriersare not present in the sperm head plasma membraneand therefore lateral separation of sperm head plas-ma membrane components must be accomplished byother means [20^22]. (ii) One other possible explan-ation for the polar distribution of the integral mem-brane proteins could be that they are attached to thepolarized sperm cytoskeleton (Fig. 3). However, thelateral organization of membrane proteins alters dra-

matically upon capacitation whereas changes in thecytoskeleton have not been observed [1,23]. A morelikely explanation for the lateral heterogeneity in thismembrane is provided by the di¡erential electrostaticinteractions of the membrane components with thelateral polarized glycocalyx (Fig. 3). A strong indica-tion for the validity of this model is that capacitationinduces remodelling of the glycocalyx which explainsthe dynamic alterations in lateral topology of thetransmembrane proteins. Moreover, the glycocalyxmay induce lateral polarity changes of lipids in thesperm plasma membrane which in turn may inducetopological rearrangements of freely di¡usible trans-membrane proteins. Evidence has been provided thatthe lipids in the sperm head plasma membrane areindeed also organized into lateral membrane do-mains. The lateral polarity of lipids in the spermplasma membrane was ¢rst detected using probesthat complex unesteri¢ed sterols (¢lipin [24]) oranionic phospholipids (polymyxin [21]). The lipid^probe particles were visualized on freeze fracture rep-licas and, although the underlying phenomenon isstill not understood, the particles were polarly organ-ized in the sperm head plasma membrane. Filipincomplexes were distributed over the entire head plas-ma membrane surface, although a lower density ofparticles was detected in the post-equatorial subdo-main in fresh sperm cells. After capacitation thepost-equatorial region was devoid of ¢lipin com-plexes and a slight increase in complexed ¢lipinwas found in the apical head plasma membrane[21]. Weak polymyxin B labelling was found onfreshly ejaculated sperm cells whereas, after capaci-tation, a strong increase of labelling was detectable inthe apical ridge region of the sperm head surface [25].Most likely polymyxin B labels PS, an anionic phos-pholipid that is localized in the inner lipid lea£et ofthe sperm cell but that becomes partly exposed at theplasma membrane exterior during capacitation [26].This phenomenon appears to be restricted to the ap-ical area of the sperm head (see Section 2.2.2 and[27]).

In our laboratory we have investigated the topol-ogy of seminolipid in the plasma membrane of ¢xedand living sperm cells using indirect immuno£uores-cent labelling techniques and vesicle mediated load-ing of a £uorescent acylated seminolipid analog, re-spectively [28,29]. Both techniques revealed that



Fig. 3. Current models for lateral membrane polarity dynamics upon sperm capacitation. (A) Lateral phase separation of gel phase(excluding freely di¡usible membrane proteins) and liquid crystalline phase (aggregations of transmembrane proteins). Melting of gelphase preferring lipids results in lateral mixing of membrane lipids and free membrane proteins. (B) The extracellular matrix (ECM)of the sperm cell is very heterogeneous and is organized into lateral domains. Therefore, the ECM may create membrane domains viaelectrostatic interactions with glycolipids or with membrane proteins. Disruption of the ECM will allow mixing of lipids and proteinsbetween the two lateral domains. (C) A similar scenario as described for (B) may be valid for the polarly organized cytoskeleton ofthe sperm cell. (D) Processes (A)^(C) may induce dimerization of transmembrane proteins (or dissociation of transmembrane proteinaggregates). Dimerization is probably further facilitated by increased membrane £uidity due to the bicarbonate induced e¥ux of mem-brane cholesterol and the scrambling of bilayer phospholipid asymmetry.



seminolipid is highly concentrated in the apical ridgeregion of the sperm head plasma membrane in con-trol cells, whereas, in capacitated cells almost theentire seminolipid fraction migrates towards theequatorial region of the sperm head plasma mem-brane (Fig. 4). As is valid for most other glycolipidsthis lipid is con¢ned to the outer lipid lea£et of thesperm plasma membrane [16] and is most likely incontact with the glycocalyx components (e.g. by link-

age to its seminolipid immobilizing protein (SLIP),see Section 2.3.4). Reorganizations in the lateral po-larity of lipids in the sperm head may therefore verywell originate from altered seminolipid glycocalyxinteractions [28].

It has been suggested that the lipid segregation andreorientation phenomena in the sperm plasma mem-brane could also be explained by lipid phase transi-tions (freezing of lipids from the £uid liquid crystal-line phase to the frozen gel phase, see Fig. 3) [30].However, these lipid phase transitions were only ob-served after cooling and they emerged within thesubdomains [21,31]. The small circular areas in whichthe lipids were in the gel phase had a surface ofmaximally only 0.04 Wm2, which is very small incomparison with the average size of the subdomains(12 Wm2). The absence of the small gel phase struc-tures in the head plasma membrane of capacitatingsperm cells indicate that lipid phase separations donot occur under these conditions and that anotherphenomenon must drive the (re-)organizations of lat-eral lipid subdomains in the sperm plasma mem-brane. Although lateral di¡usion properties of lipidsand free membrane proteins vary considerably be-tween the di¡erent subdomains of the sperm headplasma membrane [32], this is not due to lipid phaseseparation phenomena, but probably a re£ection ofthe di¡erent subdomain lipid compositions due toregionalization of the glycocalyx (see Section 2.2.3).

Conclusively, we believe that the polarized organi-zation of the glycocalyx is important for the laterallipid heterogeneity in the sperm head plasma mem-brane. However, the capacitation dependent reorgan-ization in lipid polarity most likely re£ects changes inthe glycocalyx as well as a collapse of the phospho-lipid asymmetry (see Section 2.2.2). Recently it hasbeen demonstrated that lateral membrane specializa-tions called membrane rafts can function as a focalpoint for cellular signalling [33]. It is well possiblethat the dynamic lateral organization of the spermplasma membrane enables the cell to respond appro-priately to the variable stimuli it will encounterthroughout its voyage to the oocyte.

2.2.2. Membrane bilayer topologyLike plasma membranes of somatic cells the lipids

in the sperm plasma membrane are asymmetricallydistributed over the lipid bilayer. Reliable methods

Fig. 4. Lateral distribution of £uorescent glycolipids after theirincorporation into the plasma membrane on boar sperm cellswith intact acrosomes and plasma membranes. (A) The distri-bution of SGalCer(C12-LRh) (a £uorescent analog of semino-lipid) in the plasma membrane of a freshly ejaculated spermcell. (B) Idem, but after capacitation in vitro in Tyrode's basedmedium containing 2 mM CaCl2 for a period of 2 h. The dis-tribution of the C12-LRh lipid label intensity was measured insitu on the sperm surface with an epi£uorescence microscope(rhodamine ¢lter setting) connected to a CCD camera and animage analyzer [29]. The in situ biophysical behavior of the£uorescence probe was analyzed by £uorescence lifetime imag-ing microscopy [28]. This enabled quanti¢cation in situ of the£uorescent probe at the surface of the sperm cell as indicatedhere in peak height and in warm color table. Cells were stainedwith Hoechst 33258 and a peanut agglutinin^FITC conjugate inorder to assess the intactness of the plasma membrane and theacrosome, respectively. The length of the sperm head is approx-imately 8 Wm.



have revealed that the choline phospholipids SMand, to a lesser extent, PC are mainly found in theouter lipid lea£et [27,34]. The aminophospholipidsPE and especially PS are located in the inner lipidlea£et probably due to active inward transport by anaminophospholipid translocase. Some older studiesreport equal distributions of PE and PS in the spermplasma membrane bilayer [35,36]. In those studiesprotocols were used that had been well establishedto measure lipid asymmetry in erythrocytes, such asphospholipases or TNBS-labelling at low tempera-ture [37]. These methods are, however, very detri-mental for the highly sensitive sperm plasma mem-brane organization [27]. Even in freshly ejaculatedsperm samples, a proportion of 7^15% of the cellshave a deteriorated plasma membrane and this pro-portion will only increase during sperm activation(see Section 3.1). Therefore, it has now become stan-dard to detect lipid asymmetry for sperm cells in a£ow cytometer for living cells only [27,38].

We recently performed a set of tests to see whetherlipid asymmetry was a¡ected by in vitro capacitation[26]. In order to compare the lipid asymmetry incontrol cells with capacitating sperm cells it is impor-tant to work at physiological temperature (38.5³C,pig body temperature) and to block endogenousphospholipases (see Section 2.4 and [27]). Scramblingof lipid asymmetry was observed: after 2 h a markedincrease of PC and SM translocation into the innerlea£et was observed from 14 to 30% of total PC and2 to 23% of total SM, for control and capacitatedcells, respectively. Furthermore the inward move-ment of PE and PS were considerably slowed downin capacitated cells (up to 10 times). The activationof a scramblase would explain the increased inwardtranslocation of PC and SM, but also the decrease inthe net PS and PE translocation rates. Recently wedetected that PS and PE are only exposed at theapical head plasma membrane using annexin V-FITC [39] and Ro1100-FITC [40] as probes for PSand PE exposure, respectively [26,27,41]. Probably asimilar observation has been made almost 20 yearsago by Bearer and Friend who studied the anionicphospholipid distributions with polymyxin B in con-trol (only faintly labelled) and capacitating spermcells (where intense labelling took place at the apicalplasma membrane) [25].

The scrambling of phospholipids is under control

of a bicarbonate mediated signalling pathway. Bicar-bonate directly activates a sperm speci¢c adenylatecyclase (AC) and thereby switches on protein kinaseA (PKA). Most likely PKA in its turn directly orindirectly activates the proposed scramblase resultingin the altered phospholipid asymmetry of the capaci-tating life sperm cells (see Section 3.1.1 and Fig. 5).At any rate the scrambling is dose dependent onbicarbonate levels (half maximal response at 7 mM)and can be mimicked in the absence of bicarbonatewith phosphodiesterase inhibitors (inhibition ofcAMP breakdown, Fig. 5), PKA activators, proteinphosphatase 1 and 2a inhibitors and by addition ofcAMP analogs.

Merocyanine is believed to monitor disordered lip-id acyl chain packing of membranes [42]. The probeshows only faint £uorescence in non-capacitatedsperm cells where the lipid asymmetry is maximal,whereas after scrambling it becomes more interca-lated into the membrane which results in higher £uo-rescence. The use of the probe was ¢rst described forother cell types but con¢rmed for sperm cells byHarrison et al. [43]. These studies clearly showedthat only a discrete subpopulation of living spermcells actually picked up high merocyanine £uores-cence. The number of positively responding livingcells corresponded very well with the proportion ofsperm cells that showed aminophospholipid exposureat the apical sperm head region [26,41]. In fact, themerocyanine response mirrored the scrambling re-sponses, because it was dependent on the same bicar-bonate induced signalling cascade [44]. Moreover,the merocyanine response turned out to be independ-ent of activation of tyrosine kinases and protein ki-nase C (PKC), indicating that a signalling cross talkwith tyrosine kinase (normally occurring duringsperm capacitation) has no in£uence on the lipidasymmetry regulation.

2.2.3. Membrane £uidityAs mentioned above the plasma membrane of

mammalian sperm cells has a pronounced domainorganization (see Section 2.2.1 and Fig. 1A). It isnot clear whether or not this situation also appliesfor the lateral £uidity of lipids in the plasma mem-brane. Theoretically, the sperm plasma membraneshould be very £uid due to the unusually high pro-portion of long chain polyunsaturated fatty acids of



the main phospholipids. This prediction is supportedby Laurdan £uorescence spectroscopy revealing asingle liquid crystalline lipid phase and a lack ofthermotropic phase transitions in the plasma mem-brane of living human sperm cells [45]. Other bio-physical techniques, however, such as di¡erentialscanning calorimetry, electron spin resonance andFourier transform infrared spectroscopy, have de-tected thermotropic phase transition temperatures

in membrane vesicles and lipid extracts from variousmammalian sperm cells [11,30,46]. The multiple tran-sition temperatures detected are consistent with thesperm plasma membrane's specializations into lateralsubdomains. In fact, it has been suggested that thecoexisting gel and £uid phases of lipids are a majordriving force in the organization of the lateral mem-brane heterogeneity (Fig. 3A) [30]. However, itshould be kept in mind that these studies have

Fig. 5. Proposed sequences of mammalian sperm capacitation. (A) Bicarbonate may enter sperm cells via ion channels or via di¡usionas carbon dioxide. Intracellular bicarbonate switches on AC and concomitant production of cAMP activates PKA. The role of choles-terol e¥ux in the activation of PKA is unclear. Cholesterol e¥ux may induce increased bicarbonate entry or may a¡ect AC. (B) PKAinduces tyrosine (Y) phosphorylation of several substrates (S) most likely via activation of PTK or inhibition of protein tyrosine phos-phatases (PTP). (C) Sperm^ZP binding proteins and other plasma membrane proteins become tyrosine phosphorylated via the bicar-bonate induced activation of PKA. (D) Cytosolic PLC is tyrosine phosphorylated via the bicarbonate^PKA pathway. Tyrosine phos-phorylated PLC is subsequently translocated to the plasma membrane. (E) PKA activation induces plasma membrane changes likelateral redistribution of seminolipid and translocation of aminophospholipids. (F) Aminophospholipids are translocated by the PKAdependent activation of a postulated scramblase. Most likely the e¥ux of cholesterol is involved in these plasma membrane transi-tions. (G) The entry of small amounts of calcium into sperm cells plays possibly an important role in capacitation. (H) Decapacitationfactors (DF) are removed from the sperm cell surface, uncovering receptors like the postulated progesterone receptor (P4R).



been performed on isolated membranes and vesiclemembranes reconstituted from lipid extracts, whichis not necessarily indicative for the intact plasmamembrane in the living sperm cell [47].

One technique which can be employed to detectthe lateral £uidity organization in the plasma mem-brane of the living sperm cell is £uorescence recoveryafter photobleaching (FRAP), in which a £uorescentreporter probe is inserted into the bilayer. The probedi¡uses over the membrane surface but a well de¢nedspot on the surface is illuminated with a high pow-ered laser beam. Probe molecules that are in the laserspot are bleached (photo destructed) by the high en-ergy of the laser excitation light pulse. The still intactprobes from the unbleached environment of thebleached spot can now di¡use into the bleachedspot. The rate of £uorescence recovery is proportion-al to the lateral £uidity of the membrane, whereas,the proportion of mobile lipids in the bleached spotcan be calculated by the proportion of total £uores-cence recovery. The technique is highly dependenton the probe that is used. For instance, Wolf et al.used 1,1P-dihexadecyl-3,3,3P,3P-tetramethylindocarbo-cyanin (DiI16) stains to monitor lateral di¡usionproperties of ram and rodent sperm cells [48,49].With this probe, marked di¡erences were found inlateral di¡usion rates within the di¡erent regions ofthe sperm plasma membrane. Moreover, rather lowrecovery percentages of 40^50%, indicative for theexistence of gel phase domains, were found. Both£uorescence recovery rates as well as recovery per-centages within the sperm membrane specializationschanged upon sperm maturation as well as spermcapacitation. Interestingly, Jones et al. demonstratedusing sperm of bull and other species that DiI16probes only stained deteriorated cells, whereas theprobe did not stain living cells [47]. In those studiesa more successful reporter probe 5-(N-octadecanoyl)amino£uorescein (ODAF) for FRAP studies wasused. ODAF stains living cells as well as deterioratedcells [47]. It is unlikely that ODAF stained intracel-lular membranes of living sperm cells because theentire cell became homogeneously labelled (in thecase of intracellular labelling more pronounced £uo-rescence would be expected in the acrosomal region aswell as the mid-piece). However, in deteriorated cellssuch labelling patterns were found which was also thecase for the DiI16 probe [47,50]. With ODAF the

recovery percentages were approximately 90% in thehead plasma membrane of living sperm cells [47,50].The ODAF results are in agreement with the dataobtained with Laurdan £uorescence spectroscopy onliving sperm cells and on the lack of gel phase patchesdetectable in EM specimens of capacitating spermcells (see Section 2.2.1). In this respect it is interestingfor andrologists that cooling of a sperm specimen forcryopreservation may lead to phase transitions as wellas protein clustering [51]. This is believed to be one ofthe major causes of cryo-damage in sperm cells be-sides crystallization of water [52].

In our view the group of Jones has solved thecontradiction that existed in literature about thepresence and absence of a gel phase in sperm cells.In living sperm cells the gel phase does not exist,whereas, deterioration, cooling or ¢xation leads torigidi¢cation of the membrane and low percentagesof photobleaching recovery will be detected [47].Similar artifacts probably occurred in the biophysicalstudies on extraction and reconstitution of spermmembranes explaining the detection of lipid transi-tion temperatures [11,53]. It should be noted that theODAF studies clearly demonstrated major di¡eren-ces in lateral di¡usion rates within regions of thesperm head plasma membrane as well as in the tailand mid-piece. This observation is in line with thelateral polarity detected for anionic phospholipids,cholesterol and glycolipids in sperm cells. The lateraldi¡usion rates of lipids in the di¡erent subdomainschanges upon sperm maturation [54,55] as well ascapacitation [32,56], although the latter has to beestablished with FRAP studies using the ODAFprobe. Probably the alterations in lateral di¡usionrates are due to the migration of certain lipidsfrom one subdomain to another (Figs. 3 and 4) aswell as the capacitation dependent scrambling ofphospholipids in the apical plasma membrane ofthe sperm cell (Figs. 3 and 5).

2.3. Lipid^protein interactions

Lipid molecules can be extracted or exchangedfrom the sperm plasma membrane by speci¢c lipo-proteins and lipid transfer proteins (carrier facilitatedlipid transport). This process seems to play an im-portant role in the modi¢cation of the lipid compo-sition at the extracellular site of the sperm plasma



membrane during capacitation [57,58]. Another typeof protein^lipid interaction, relevant for the fertiliz-ing capacity of sperm cells, is the protein^lipid inter-action that results in linkage of the protein to amembrane. This can be in the form of covalent pro-tein linkages to fatty acid chains (e.g. myristoylatedproteins), glycosylphosphatidylinositol (GPI) an-chors or via electrostatic protein interactions withlipid head groups (via the phosphocholine headgroup of PC or the glycosidic moiety of glycolipids).

2.3.1. Lipoprotein mediated cholesterol transportThe observation that albumin should be included

in in vitro fertilization (IVF) media in order to gete¤cient embryo production led Langlais and Robertsto propose a capacitation model leading to the acro-some reaction based on the possibility that lipopro-teins extract sterols from the sperm plasma mem-brane [59]. More recently it has been establishedthat cholesterol is indeed an important molecule inthe sperm plasma membrane. The cholesterol contentof sperm cells capacitated in vitro in a medium con-taining albumin decreased markedly (up to 40%) invarious mammalian species [60] (Flesch et al., unpub-lished results). We recently discovered that this albu-min mediated decrease in cholesterol content onlyoccurred when sperm cells were treated in a bicar-bonate enriched capacitation medium, but not in theabsence of bicarbonate under otherwise similar con-ditions. This decrease in cholesterol content has alsoimplications for the cholesterol topology in thesperm plasma membrane [31], which could indicatethat albumin mediated cholesterol extraction onlyoccurs in restricted surface areas of the sperm cell.The extraction of cholesterol by albumin was ratherspeci¢c because the phospholipid content remainedidentical (Flesch et al., unpublished results). Onlysperm cells with a low concentration of cholesterolgave a positive merocyanine response and a partialscrambling of phospholipid asymmetry indicatingchanges in the plasma membrane architecture (Fleschet al., unpublished results), whereas immature orcholesterol-rich sperm cells present in the same sus-pension did not show these events. This may explainsome di¡erences between studies on cholesterol inboar and human sperm [61,62] and the sperm ofmice or other rodents [63,64]. The sperm of rodentscannot be collected in ejaculated form because it is

secreted together with spermicidal coagulation plugcomponents [65]. Therefore, rodent sperm specimenshave to be collected by aspirating the epididymis,and a relative large fraction of sperm cells fromthis site is still immature (i.e. with relatively highcholesterol levels). It has been shown that albuminhas an additive e¡ect on the bicarbonate mediatedchanges in membrane £uidity and the induction of acollapse in phospholipid asymmetry (see Section2.2.2). Therefore, we now believe that a subpopula-tion of well matured sperm cells in ejaculated spermsamples already contains low enough cholesterol forbicarbonate induced phospholipid scrambling. Onthe other hand albumin mediated cholesterol extrac-tion is required for the remaining non-respondingsperm subpopulation to become responsive for bicar-bonate mediated changes in the plasma membranearchitecture and signalling.

In this respect an interesting new approach of ex-tracting cholesterol has recently been tested by Crosset al. [66] and by Visconti et al. [63]. They have usedcyclodextrins to remove a large proportion of choles-terol from the sperm surface. Cyclodextrin has a veryhigh a¤nity for cholesterol and, in contrast to albu-min, it can extract cholesterol in the absence of bi-carbonate (i.e. in non-capacitating sperm cells).When cyclodextrin treated sperm cells are incubatedin a capacitation environment, a very rapid and pro-nounced signalling activation of PKA and proteintyrosine kinase (PTK) can be observed [63]. Takentogether this demonstrates that only at relatively lowcholesterol concentrations in sperm cell membranescan activation of certain signalling pathways in-volved in fertilization processes proceed. Scienti¢csupport for this can be found in a report where cho-lesterol has been described as a modulator of recep-tor function in another cell type [67].

It is very well possible that the time required foroptimal sperm capacitation relates to the slow e¥uxof cholesterol from the non-responding subpopula-tion of sperm cells. Further support for that idea isthe fact that species with high cholesterol content(human and bull) require rather extended periodsfor optimal capacitation (respectively, 8 and 6 h)whereas boar and ram sperm with lower cholesterolcontents only require 1 or 2 h of capacitation [3]. Theseminal plasma of human, stallion and perhaps othermammals contain prostasomes which are cholesterol-



rich vesicles secreted by the prostate [68,69]. Theseprostasomes block cholesterol e¥ux from sperm cellsand probably serve to delay capacitation [68].

Although albumin may be a more physiologicalcomponent than cyclodextrin to study cholesterol ef-£ux from capacitating sperm cells, only a few groupshave tried to determine cholesterol extracting lipo-proteins from the female genital tract. In this respect,Ravnik et al. [58] have reported a stimulating e¡ectof human sperm capacitation by lipid transfer pro-tein I, a key component of high density lipoproteins(HDLs). The protein stimulates the acrosome reac-tion, is present in the female genital tract and origi-nates from follicular £uid [70]. Therefore, the con-centration of cholesterol extractor is optimal in theenvironment of the oocyte just after its ovulation.This scenario was predicted some time ago by Goand Wolf [71,72], who besides cholesterol extractionalso claimed a function in free fatty acid extractionfor albumin. However, the importance of fatty acidextraction in sperm capacitation has never been in-vestigated in further detail. Remarkably, also no at-tention has been paid in this respect to intracellularlipid transport proteins, despite the fact that the plas-ma membrane is in very close contact with intracel-lular membranes. In sperm cells, protein facilitatedintracellular transport would be the way for trans-porting lipids and proteins from one membrane toanother since vesicle mediated membrane transport islacking in these cells.

2.3.2. Phosphatidylcholine binding proteins andcapacitation

Bovine seminal plasma contains a series of pro-teins collectively called bovine seminal plasma pro-teins (BSP, A1/A2, A3, 30 kDa), which are secretedby the seminal vesicles [73]. These BSPs appear tohave a¤nity for the sperm plasma membrane[74,75] and more speci¢cally bind to phosphocholinemoieties and PC liposomes [76,77]. In line with this,BSPs have been shown to induce PC e¥ux fromepididymal sperm cells [78]. Furthermore, BSP com-plexes have been shown to support in vitro HDLmediated cholesterol e¥ux from the sperm cell sur-face and capacitation of bull sperm [79]. The exactworking mechanism of BSPs in this process is notclear, although it has been shown that BSP canbind to Apo A1 [80]. It appears that BSP binding

to sperm cells is lost upon their interaction with ep-ithelial cells of the oviduct [81]. Released sperm cellsfrom this site are considered to be fully capacitated,competent to bind to the ZP and hypermotile [82^84].

2.3.3. GPI anchored membrane proteins andZP binding

It is well known that membrane proteins can becovalently linked to membrane lipids. The mostprominent way seems to be via the GPI anchor.For instance, the protein PH20, later identi¢ed as amembrane bound hyaluronidase, plays a role inbinding to and the digestion of ZP structures [85].Although three soluble isoforms of this protein havebeen localized in the acrosome, at least one 68 kDaisoform is GPI linked to the plasma membrane ofmouse sperm [86]. All forms may be important forsuccessful penetration of the extracellular vestmentsthat surround the egg prior to fertilization [85]. Withrespect to GPI anchored proteins, Rooney et al.made interesting observations in seminal plasma ofdi¡erent mammalian species [87]. They observed thatseminal plasma contained a stable population ofmembrane free proteins that can be GPI linked tothe sperm cell surface (i.e. CD59, CD55 and Cdw52). This type of sperm surface modi¢cation prob-ably serves to make sperm cells resistant to comple-ment attack in the female genital tract [87]. GPI pro-tein modi¢cations at the sperm surface may well beassociated to polarity dynamics of the sperm plasmamembrane.

2.3.4. Glycolipid binding proteins and ZP bindingMale germ cells of mammals contain a sperm spe-

ci¢c glycolipid called seminolipid (for review see [2]).In mature germ cells and sperm cells this glycolipid isexclusively present in the sperm plasma membrane[88] and only at the outer lipid lea£et of this mem-brane [16]. From the moment that seminolipid is sur-face oriented, SLIP-1 is also found on male germcells (Fig. 3B) [89,90]. The protein is 68 kDa largeand therefore cannot immobilize the entire semino-lipid fraction (about 20% of the molar lipid fractionin the outer lipid lea£et of the sperm plasma mem-brane). Recently, Tanphaichitr et al. have demon-strated that SLIP has a role in binding to the ZP[91]. Free seminolipid (i.e. the proportion of semi-



nolipid that is not immobilized by SLIP) may alsobind to the ZP by electrostatic interactions [92],which may serve to further stabilize the SLIP ZPbinding.

2.4. Lipid metabolism and signalling

Several reports have been published on the role oflipases in sperm capacitation and subsequently in-duced acrosome reaction (for review see [93]). Thephospholipases cleave intact phospholipids into cer-amide (sphingomyelinase), diacylglycerol (DAG) andalkylacylglycerol (phospholipase C, PLC), phospha-tidic acid (phospholipase D, PLD) and in lysophos-pholipids and free fatty acids (phospholipase A2,PLA2). These phospholipid catabolites serve as intra-cellular signalling molecules and activate proteinphosphorylation cascades that are triggered in spermcells during acrosomal exocytosis. Special attentionhas been paid to the activation of phospholipasesA2, C and D in sperm cells whereas far less is knownabout sphingomyelinase although its activity in-creases upon capacitation [26]. Each of the followingsubsections will deal with the regulation of the activ-ity of one of the sperm phospholipases and the sub-sequently induced lipid signalling. In these subsec-tions, which protein phosphorylation cascades areactivated by lipid signalling and whether they areinvolved in signalling cross-talks will also be dis-cussed.

2.4.1. PLA2

PLA2 is the enzyme which is responsible for cleav-ing intact phosphoglycerolipids (e.g. PC) into freefatty acids (liberated from the sn-2 position of theglycerol backbone) and lysophospholipids (e.g. lyso-phosphatidylcholine, LPC). Lysophospholipids andfatty acids seem to serve as co-activators of PKC,but their major roles appear to relate to perturbationof the membrane structure (see Section 2.5 and Fig.6). Lysophospholipids may also modulate channelfunction by altering the mechanical properties of lip-id bilayers [94,95], which might be required for in£uxof extracellular Ca2�. In addition to this, it should benoted that Flaherty and Swann reported that pro-teases are not involved in the LPC mediated acro-some reaction in guinea pig sperm cells [96]. Fattyacids like arachidonic acid and docosahexaenoic acid

can be converted into leukotrienes or prostaglandins(by cyclooxygenase or lipoxygenase [97]), whereaslysophosphatidalcholine can be acetylated into plate-let activating factor (PAF; 1-alkyl, 2-acetyl,glycerol-3 phoshocholine) which are bioactive molecules [98].The generation of secondary bioactive fatty acid me-tabolites (and the presence of responsible enzymes) incapacitating sperm cells has so far received little at-tention. Schaefer et al. reported that the acrosomereaction can be induced by prostaglandin E [99].The formation and capability of PAF to induce theacrosome reaction during sperm capacitation havebeen reported [100,101].

PLA2 is activated in human [102] and boar sper-matozoa [103] in response to progesterone. Enhancedactivities were also induced by 15 mM bicarbonate[26]. Interestingly, reagents known to activate gua-nine nucleotide binding proteins (G-proteins) (atransduction event involved in ZP triggered acro-some reaction, Fig. 6) also induce PLA2 [104].PLA2 is a large superfamily of enzymes (for reviewsee [105]) divided into groups I to IX on bases ofsequence information. In general the phospholipasesA2 can be divided into secretory low molecular massenzymes of 13^18 kDa, and cytosolic high molecularmass enzymes of 85 kDa forms and 29 kDa forms).All secretory enzymes require millimolar levels ofCa2� for activation, whereas, some cytosolic formsdo not require any Ca2� and some require micromo-lar levels of this divalent cation. Information onwhich isoenzymes are present in sperm cells is farfrom complete. A 14^16 kDa sperm PLA2 has beenpartially puri¢ed and characterized, and its N-termi-nal region revealed some similarities with membersof the secretory group I and II PLA2 (from snakevenom and porcine/human pancreas), although theentire protein appears to represent a novel sequence[106]. Antibodies raised against cobra (Naja naja)venom recognize a 16 kDa protein in sodium dodecylsulfate extracts from bull sperm cells [107], whereas,antibodies raised against porcine pancreas PLA2 rec-ognize a protein in hamster and human sperm cells[108]. Immunostaining with these antibodies revealedthat PLA2 was present in the acrosomal region aswell as in other sperm compartments. No data areavailable on the ultrastructural localization of PLA2

using immunoelectronmicroscopy on ultrathin sec-tions. However, the observation that incubation of



sperm cells with Fab fragments from the abovementioned antibodies blocked acrosomal exocytosisat least indicates that the PLA2 is localized at thesperm surface and that its activity is involved inthe acrosome reaction (Fig. 6) [109]. The larger cy-tosolic types of phospholipase have not yet been dis-covered in sperm cells. Homozygous male mice witha gene coding for the mutated (non-active) 85 kDatype of PLA2 showed normal fertility [110,111],which suggests that this cytosolic PLA2 is not essen-tial in sperm function including exocytosis. On theother hand bovine seminal plasma contains a 100kDa isotype of secretory PLA2 [112], although itsrelevance for sperm physiology has not been reportedyet.

2.4.2. PLDPLD cleaves the polar head group from phospho-

lipids leaving phosphatidic acid as a lipid catabolitein the membrane. The formation of phosphatidicacid is only a minor pathway upon stimulation ofsperm cells [113]. In fact the low amounts of phos-phatidic acid were not a result of the induced PLDactivity but rather a result of DAG kinase, an en-zyme that phosphorylated DAG that was formedunder capacitating conditions from phosphatidylino-sitol-4,5-bisphosphate (PIP2) by the activation ofphosphatidylinositol phosphate phospholipase (PI-PLC) [114,115]. The phosphorylation of DAG maybe relevant for down regulating the PKC activationalthough phosphatidic acid has no e¡ect on the time

Fig. 6. Proposed sequence of the ZP and progesterone induced acrosome reaction. (A) ZP proteins (most likely ZPC) bind to spermZP receptors, leading to aggregation and tyrosine (Y) phosphorylation. (B) The direct environment of the ZP contains high levels ofprogesterone that can bind to its non-genomic receptor (P4R) on the sperm surface. Both ZP and progesterone have a dual e¡ect onsperm cells. (C) The intracellular pH (pHi) is increased via G-proteins (Gi) and (D) the plasma membrane potential depolarizes. (E)Both the increased pHi and depolarization induce the entry of calcium via a T-type voltage dependent Ca2� channel. (F) The higherintracellular Ca2� levels activate PLC that has been translocated to the plasma membrane during capacitation. PLC converts PIP2 toDAG and IP3. (G) Increased Ca2� levels activate PLA2, which degrades PC to LPC and free fatty acids (FFA). (H) The role of IP3

is unclear, but DAG, FFA and LPC activate PKC. Both the increased intracellular Ca2� levels and PKC activation are necessary forthe fusion of the plasma membrane with the underlying acrosomal membrane, which leads to the subsequent secretion of acrosomalenzymes.



course of exocytosis [116]. In conclusion, it is notlikely that PLD is involved in lipid signalling insperm cells.

2.4.3. PLCPLC cleaves DAG from the phosphorylated head

group of phosphoglycerolipids. Both the role offormed DAG and phosphorylated head groupshave been described extensively in the literature toserve as second lipid messengers in cell signalling. Incontrast to PLA2 the PLC isoforms are speci¢c forthe head groups of the phospholipids. Hydrolysis ofPIP2 and phosphatidylinositol-4-phosphate (PIP) ismediated by a speci¢c PIPLC, whereas PC break-down is mediated by a PC speci¢c isoform of PLC(PC-PLC). Both PC-PLC and PI-PLC activities havebeen identi¢ed in membrane preparations from bullsperm [117] but, although these enzymes seem to belocalized in di¡erent membrane subfractions, no at-tempts have been made to localize these enzymesultrastructurally. PC-PLC is activated in capacitatingsperm cells [118] and is probably responsible for thebulk formation of DAG. PC-PLC appears to haveno e¡ect on PE and PI [119]. Another metabolicroute to produce DAG is the degradation of phos-pholipids by concerted action of PLD and phospha-tidic acid phosphohydrolase, which was assumed tobe active in sperm cells [120,121]. However, recentdata show that this alternative route is quite unlikelyin capacitating sperm cells (see Section 2.4.2) [93]. Onthe other hand, sperm cells of a variety of mamma-lian species showed increased PI-PLC activity uponincubations that induce the acrosome reaction, suchas calcium ionophore (A23187), progesterone or ZPproteins [113,122,123]. It is not well known whichtypes of PI-PLC are involved in this process (for re-view see [124]). There is some evidence that PI-PLC-Qactivation is modulated by tyrosine phosphorylation[125,126]. On the other hand there is no clear evi-dence for the involvement of a PI-PLC-L that is ac-tivated by the pertussis toxin insensitive G-protein[124]. The presence of other types of PI-PLCs hasbeen suggested: sperm cells may have a PI-PLC ac-tivated by a pertussis toxin sensitive G-protein (Go

or Gi type), or a Ca2� dependent isoform, PI-PLC-N[124].

The DAG generated by either PC-PLC or PI-PLCprobably has a central role in downstream lipid sig-

nalling of the acrosome reaction [124]. The PC-PLCis like PI-PLC stimulated by calcium ionophore(A23187), progesterone or ZP [118,123,127]. DAGis now known to activate sperm PKC [115] andPLA2 [128] and also to have a positive e¡ect onPC speci¢c PLC [118]. PKC is a serine/threonine ki-nase with various isoforms that have been classi¢edinto three groups: (i) the conventional type (cPKC:K, LI, LII and Q isotypes), which are activated byCa2�, DAG, PS, free fatty acids and LPC [93], (ii)the second group of novel PKCs (nPKC: N, O, R, Wand a isotypes), which are activated by DAG and PSbut not by PLA2 products, and are Ca2� independ-ent, and (iii) the group of atypical PKC (aPKC: j, Vand I isotypes), which are DAG and Ca2� independ-ent but require PS and other activators such as freefatty acids [120]. Therefore, it should be noted thatthe scrambling of phospholipids (e.g. reorganizationof PS [26]) also may a¡ect the activation of PKC.Originally, no PKC activity was identi¢ed on ramsperm stimulated with calcium ionophore A23187and phorbol esters (a classical way to induce PKCactivity) [129]. However, phorbol esters have nowbeen established to induce PKC activity in spermcells of di¡erent mammalian species [130]. In fact,phorbol esters induced the acrosome reaction andPKC inhibitors were potent inhibitors of the ZP in-duced acrosome reaction [131]. Recent work hasshown that progesterone induces the activation ofPKC and phosphorylation of various substrates,while both e¡ects could be blocked by PKC inhib-itors [115]. Several immunocytochemical studies havelocalized PKC in the sperm head and tail. Bull spermhave PKCLI throughout the acrosomal region [132],whereas human sperm cells have PKCLII in the equa-torial region of the sperm head [133]. With the use ofan antibody recognizing the PKC K, L and Q iso-types, PKC was localized in the equatorial regionof the human sperm head and in the post-equatorialregion of bull sperm [133,134]. In mice an unidenti-¢ed PKC has been localized in the acrosomal regionof the sperm head [126]. Treatment of bovine spermwith phorbol esters resulted in the Ca2� dependenttranslocation of PKCK and PKCLI [132]. It has beensuggested that PKC activation by phorbol esters andthe stimulation of the acrosome reaction are Ca2�

independent [133]. However, up until now it hasbeen widely accepted that increased intracellular



free Ca2� levels are crucial for the acrosome reaction.Furthermore, the observations of Rottem et al. [133]do not correspond with the results of O'Toole et al.[115,135] who demonstrated that PKC mediatedphosphorylation of protein substrates is inhibitedby blocking Ca2� entry with verapamil. Another ob-servation that PKC activation controls Ca2� £uxesover the plasma membrane [130] has been disputedby the ¢ndings of Bonaccorsi et al. [136] who dem-onstrated that the progesterone and the ZP inducedCa2� in£ux in the sperm cells are not a¡ected byPKC inhibitors or phorbol esters. Roldan and Fraser[137] have demonstrated conclusively that PKC acti-vation is downstream of Ca2� in£ux. None of thesubstrates of PKC have been identi¢ed so faralthough one putative substrate for PKC could bea 40 kDa mitogen activated protein (MAP) kinase[138].

Besides its reputation as a signalling lipid, DAGmay also perturb the plasma membrane structureand may facilitate membrane fusion directly (see Sec-tion 2.5). The PI-PLC mediated breakdown of PIP2

not only results in the bioactive component DAG,but also in inositol-1,4,5-triphosphate (IP3, Fig. 6).In somatic cells this phosphorylated head group canbind to speci¢c IP3 receptors on the ER and therebymobilizes the intracellular Ca2� pool (transport ofCa2� ions from the ER lumen towards the cytosol).However, sperm cells do not contain ER and mobi-lizable intracellular Ca2� pools have not yet beendemonstrated. In addition, IP3 has no e¡ect onsperm activation or the acrosome reaction [93]. How-ever, the generated IP3 may have a function in intra-cellular Ca2� elevation in oocytes [139]. Initially itwas believed that a cytosolic factor from hamstersperm cells called oscillin induced oscillations of thelevel of free cytosolic Ca2� in the fertilized oocyte[140]. However, although a soluble factor fromthe cytosol of sperm cells seems to be involved, theCa2� releasing component(s) of mammalian spermextracts remains to be identi¢ed [140,141]. Oscillinis later identi¢ed as the glucosamine-6-phosphateisomerase enzyme, and does not appear to be in-volved in Ca2� release in mammalian oocytes[141]. Watson et al. have localized Ca2� at the outeracrosomal region by electron microscopical tech-niques. It should be mentioned, however, that thisCa2� is probably immobilized to proteins or crystal-

lized as salt due to the acidic pH of this organelle[142].

2.4.4. SphingomyelinaseSphingomyelinase is involved in the generation of

ceramide and phosphocholine from SM. Its activityin sperm cells is only poorly characterized. Neverthe-less, it was recently demonstrated that sphingomye-linase activities were enhanced up to six times bybicarbonate, whereas PLA2 and PLC were only mod-erately activated [26]. It is of interest to note thatceramide formation has been implied in the induc-tion of apoptosis in several types of somatic cells[143]. In sperm cells, classic apoptosis is impossiblebecause gene expression and nuclear fragmentationare not possible on the completely compactedDNA in the sperm head. However, it is still possiblethat an apoptosis-like signalling cascade is involvedin the induction of the acrosome reaction. In linewith this is the discovery that the lipid asymmetryin the apical sperm head plasma membrane scram-bles upon in vitro capacitation (see Section 2.2.2).This process is induced by bicarbonate dependentactivation of PKA [26].

2.4.5. PI3 kinasesA recent report suggested the presence of phos-

phoinositide 3 kinase (PI3 kinase) in sperm cellswhich converts PIP2 into PIP3. In analogy with so-matic cell types [144,145] it was found that wortman-nin (a PI3 kinase inhibitor) blocked exocytosis insperm cells [126]. Although it is attractive to specu-late that PI3 kinase plays an important role in theregulation of the acrosome reaction, information onthe mechanism involved is still lacking.

2.5. Lipid metabolism and fusion

Besides their potential as second messenger mole-cules in cell signalling processes, phospholipid break-down products are requisites for the promotion ofnon-bilayer lipid membrane structures that occurduring membrane fusions [146,147]. In line withthis concept, ceramide, DAG and/or phosphatidicacid formation in the inner lea£et of the sperm plas-ma membrane may be required for the acrosomereaction, because these phospholipid cataboliteshave only a small hydrophilic group combined with



a wider hydrophobic group. As a consequence theymay promote non-bilayer membrane structures sur-rounding and including the hemifusion micelle (aninverted micelle structure) at the spots where the fu-sions between the plasma membrane and the outeracrosomal membrane occur. On the other hand theformation of lysophospholipids and fatty acids pro-mote the inward bending curvature of the outer leaf-let of the sperm plasma membrane. In line with thisis the observation made by Roldan et al. that LPCand LPI (outer lea£et lipids [27]) induce the acro-some reaction, whereas LPS (formed in the innerlipid lea£et of the sperm plasma membrane) preventsthe acrosome reaction [100,148]. Similarly some fattyacids can enhance exocytosis, whereas others cannot[100]. The non-bilayer promoting catabolites are re-quired to stabilize the hemifusion structure and tocomplete the fusions between the plasma membraneand the outer acrosomal membrane (for review see[149]). Veterinarian and human fertility clinics pres-ently use an LPC test to observe the inducibility ofthe acrosome reaction [150]. A similar scenario asdescribed above for the acrosome reaction could bevalid for the fusion event between the plasma mem-brane of the sperm head's equatorial region and theoolemma. However, the production of phospholipidcatabolites should then be in the opposite lipidmonolayer of the sperm plasma membrane for thisintercellular fusion event. Now the outer lea£et ofthe plasma membrane from the adhered gametewill form hemifusion structures, whereas the innerlea£et will stabilize this structure and is involved incompletion of this fusion. The sperm speci¢c glyco-lipid, seminolipid, localized exclusively in the outerlea£et of the sperm head plasma membrane [16] isalso believed to play a role in the acrosome reactionand the fertilization fusion. In freshly ejaculatedsperm cells the glycolipid is concentrated in sulfatedform in the apical region probably preventing theacrosome reaction by stabilizing lamellar lipid bi-layer of the plasma membrane [2] and by preventingCa2� in£ux over the plasma membrane (both requi-sites for the acrosome reaction [3,93,151]). However,after capacitation seminolipid migrates into the equa-torial region of the plasma membrane of the spermhead (Figs. 4 and 5) and becomes partly desulfatedby arylsulfatases [16,152]. The desulfoseminolipid hasthe shape of an inverted cone and, therefore, prob-

ably facilitates the formation of a hemifusion struc-ture at the outer lea£et of the equatorial plasmamembrane of the sperm head. It is well known thatthe fertilization fusion with the oolemma only occursat this speci¢c area of the site sperm surface. Thecleavage of the sulfate moiety from seminolipidmay also interfere with Ca2� £uxes over the spermplasma membrane [2]. Nevertheless, despite all theseconsiderations, direct evidence for the involvementof lipid catabolites as non-bilayer lipids in the pro-cess of membrane fusion events remains to be pre-sented.

2.6. Lipid peroxidation

Sperm cells are in contact with radicals on theirjourney to meet the oocyte. Mammalian sperm cellsare sensitive for oxidative stress because of their highpolyunsaturated fatty acid content (see Section 2.2.3)and relative poor antioxidant defence [153,154]. Inaddition, these cells are constantly subjected to oxi-dative attack from the exoplasm where leukocytessecrete reactive oxygen species (ROS, [155]) butmay also be capable of producing intracellularROS [154,156]. The mechanism by which sperm cellswould produce ROS is still unknown although twoROS generating systems have been proposed, spermdiaphorase [157] and NADPH oxidase [158]. Theselatter activities could well be attributed to contami-nating neutrophils in semen [159]. Peroxidation isbelieved to regulate sperm function in two ways: (i)mild peroxidation might bene¢t capacitation in vitroby redox dependent activating sperm speci¢c PKA[160,161] and thereby also switching on tyrosine ki-nases [162] while superoxide anion induces spermhypermotility and results in a¤nity for the ZP. (ii)Excessive peroxidation will result in sperm deteriora-tion [163]. Although the detrimental e¡ects of lipidperoxidation on sperm cells have been extensivelydescribed and reviewed [164], the bene¢cial e¡ectsof peroxidation of sperm capacitation are still poorlycharacterized and under present debate [164^167].Peroxidative attack of tyrosine residues in proteins(formation of 3-nitro-tyrosine, tyrosyl radicals andO,OP-dityrosine [168,169]) could have an e¡ect onsperm tyrosine kinase induced signalling. However,the importance of peroxidation for e¡ective fertiliza-tion remains to be investigated.



3. Proteins involved in sperm^oocyte interaction

Sperm cells are extremely specialized cells that arenot able to synthesize proteins after spermatogenesis(lack of ribosomes and ER). Nevertheless, majorpost-translational modi¢cations and relocations ofsperm plasma membrane proteins (including the ex-change of proteins from genital £uids and the spermsurface) take place prior to and during fertilization[3]. In this section the involvement of sperm plasmamembrane proteins in mammalian fertilization willbe reviewed.

3.1. Capacitation

Freshly ejaculated spermatozoa bind to the ZPonly after activation in the female genital tract[3,170]. The activation of sperm cells (capacitation)is a widely investigated but very complicated subject.Obviously capacitation induces changes in the spermplasma membrane resulting in its increased a¤nityfor the ZP [3,171]. One of the complications in ca-pacitation research is the di¤culty to discriminatebetween induced changes at the plasma membraneand those in the remaining part of the sperm cell[172]. Furthermore, most research has been doneon whole sperm cell populations although it hasnow been established that speci¢c subpopulationsof sperm cells react or do not react on capacitationstimuli [43,172,173] or do deteriorate under theseconditions [174,175]. Flow cytometry has proved tobe a valuable tool to discriminate between di¡erentresponses of sperm subpopulations to capacitationconditions [173]. Flow cytometric studies revealedthat capacitated sperm cells should be kept at 38³Cduring analysis, since cooling to only 30³C alreadyinduces membrane deterioration in primed cells [26].In this respect published data on the induction of theacrosome reaction under capacitating conditionsshould be interpreted with caution: they might be aresult of post-capacitation temperature artifacts (e.g.in a £ow cytometer which was not pre-equilibratedto physiological temperature in order to study spermcapacitation at 38.5³C [26]).

3.1.1. Regulation of tyrosine phosphorylationIt has been shown for a number of mammalian

species, including man, that a considerable number

of proteins become tyrosine phosphorylated duringin vitro capacitation [151]. It is not known how ty-rosine phosphorylation is induced during in vivo ca-pacitation (i.e. in the oviduct) because it is extremelydi¤cult to evaluate oviductal in£uences on spermcells in situ and at the proper period in the repro-ductive cycle [176]. However, in vitro capacitationprotocols allow careful examination of the potentialof various components in the capacitation media toinduce protein tyrosine phosphorylation of spermproteins. The main component is bicarbonate, andits omission not only inhibits tyrosine phosphoryla-tion [177,178], but also diminishes the ability ofsperm cells to bind to solubilized ZP proteins [171],and inhibits hypermotility induction of sperm cells[179]. A second component is albumin, and its roleon cholesterol extraction is detailed in Section 2.3.1.However, the role of albumin in bicarbonate inducedtyrosine phosphorylation is debated [172,177,180,181]. A third capacitation component is the ionCa2�, although its direct role on protein tyrosinephosphorylation is di¤cult to examine (various pro-cesses need Ca2�) and di¡ers between various mam-malian species [180,182]. In conclusion, bicarbonateseems to be the key player in triggering tyrosinephosphorylation of proteins in capacitating mamma-lian sperm.

The bicarbonate concentration is very low (6 1mM) at the site of sperm storage in the cauda epi-didymidis and sperm cells are confronted with muchhigher bicarbonate levels (s 15 mM) in the femalegenital tract indicating a possible in vivo role forbicarbonate [183]. Intracellular bicarbonate levelscan be raised by bicarbonate entering the sperm cellsvia ion channels in the plasma membrane (Cl3/HCO3

3 exchanger [23,184], Na�/HCO33 exchanger

[185]). An alternative explanation for the entranceof bicarbonate into the sperm cell is that CO2/HCO3

3 levels are in equilibrium in the intracellularand extracellular compartments by gas di¡usionthrough the sperm plasma membrane [183]. Carbonicanhydrase, which is present in the sperm head, couldbe involved in the maintenance of high intracellularbicarbonate concentration by conversion of di¡usedCO2 [186]. So how can the increased level of intra-cellular bicarbonate induce protein tyrosine phos-phorylation? Most likely, bicarbonate is able tobind directly to a sperm speci¢c AC and thereby



activates the enzyme to produce increased levels ofcAMP [187^189]. Increased cAMP levels activatecAMP dependent protein kinases (PKA), and theactivated PKA induces protein tyrosine phosphory-lation via a yet to be discovered sperm speci¢c cross-talk signalling pathway: (i) mouse sperm capacitatedwith PKA speci¢c inhibitors also reduced tyrosinephosphorylation whereas (ii) stimulation of PKAby active cAMP-analogues [190] or by phosphodies-terase inhibitors induced tyrosine phosphorylation[191]. This may explain the supportive e¡ect in theinduction of sperm capacitation by including caf-feine, a routine component in IVF media [3]. In vitrostudies have shown signalling cross-talk betweenPKA and tyrosine phosphatases in somatic cells[192,193]. Inhibition of protein phosphatases thatare speci¢c for phosphoserine and phosphothreonineresidues (PP1 and PP2A), resulted in increased num-bers of capacitated sperm cells [194]. However, inhib-itors for phosphotyrosine speci¢c protein phospha-tases have not been tested. Vanadate, known toinhibit tyrosine phosphatases as well as some otherATP dependent enzymes, has been shown to signi¢-cantly inhibit the plateau phase of progesterone in-duced Ca2� entry [195] (see also Section 3.1.2), whichmay indicate that tyrosine phosphatases are involvedin capacitation.

Tyrosine phosphorylation of sperm proteins islinked with increased ZP a¤nity [196], acrosome re-action [197] and hypermotility [198]. Only a few stud-ies have been performed to investigate the tyrosinephosphorylation status of sperm plasma membraneproteins under capacitation conditions. Capacitationof boar sperm cells induced, in a bicarbonate depen-dent fashion, tyrosine phosphorylation of a plasmamembrane speci¢c set of proteins [172]. Interestinglyour results indicate that two speci¢c proteins fromthe apical sperm plasma membrane that became ty-rosine phosphorylated upon capacitation in vitroshowed high a¤nity for native ZP material [199].

3.1.2. Ion channelsThe initiation of the acrosome reaction is, like

other membrane fusion events, dependent on a mas-sive increase in intracellular Ca2� levels of spermcells [200,201]. Extracellular Ca2� is required forsperm capacitation and the ability to undergo in-duced acrosome reaction [177,202]. Low levels of

extracellular Ca2� (90 WM) in combination with cal-cium ionophore induce capacitation but not the acro-some reaction in mouse sperm, whereas, higher levelsof extracellular Ca2� (1.8 mM) are appropriate toinduce the acrosome reaction [203]. Addition of acalcium chelating agent (required to study the Ca2�

independent sperm capacitation) in combination withCa2� imaging techniques [173,204,205], radioactiveCa2� [206] and ion-selective microelectrodes [207]have shown the increase in intracellular Ca2� concen-tration in sperm cells during in vitro capacitation.

It is not clear how extracellular Ca2� passes theplasma membrane during sperm capacitation. Thesperm plasma membrane contains a variety of Ca2�

channels: voltage dependent Ca2� channel [208] (seeSection 3.3.4), Ca2�-ATPase [209], Na�/Ca2� ex-changer [210] and probably others. Besides theseCa2� channels, the presence of IP3-gated Ca2� chan-nels on acrosomes have been reported [211] as well asthe presence of a Ca2�-ATPase in the acrosome[212], suggesting that the acrosome could also serveas an intracellular sink for Ca2�. However, acroso-mal Ca2� most likely is immobilized to proteins or assalt crystals due to the acidic pH of the acrosome[213]. It should be noted that sperm cells lack theendoplasmic reticulum as a reservoir of immobiliz-able Ca2�, which is supported by the observationthat IP3 did not a¡ect cytosolic Ca2� levels in spermcells (see Section 2.4.3). Furthermore, mitochondriaare not localized in the sperm head and most likelydo not in£uence the Ca2� levels at the acrosomalregion of the sperm head. It has been postulatedthat free Ca2� levels in the cytoplasm are kept lowin freshly ejaculated sperm cells by calmodulin sensi-tive Ca2�-ATPase that inhibits capacitation. Inhibi-tion of calmodulin or calmodulin sensitive Ca2�-ATPase leads to increased intracellular Ca2� levels,which was re£ected in a higher proportion of capaci-tated cells [206,214,215]. These ¢ndings suggest thatthe continuous removal of cytoplasmic Ca2� byCa2�-ATPase is involved in the prevention of prema-ture capacitation of sperm cells. Furthermore, inhi-bition of a Na�/Ca2� exchanger by a 10 kDa seminalplasma peptide (caltrin) prevents Ca2� uptake byfreshly ejaculated bovine sperm cells [210,216]. Prob-ably bicarbonate is involved in elevation of theintracellular Ca2� levels since anion channel blocker4-acetamido-4P isothiocyano-stilbene-2,2P-disulfonic



acid reduced Ca2� uptake [207]. PKC and voltagedependent Ca2� channels also seem to be involvedin increased Ca2� uptake [205]. In this respect, depo-larization of the plasma membrane potential indeedis a prerequisite for the ZP induced acrosome reac-tion [217,218]. Na�/K�-ATPase could play a role incapacitation induced depolarization, since the incu-bation of sperm cells in capacitation medium with

low levels of sodium (less than 25 mM) inhibits ca-pacitation [219]. This phenomenon could be ex-plained by a requirement of intracellular sodium toactivate Na�/Ca2� exchangers and thus to increaseintracellular Ca2� concentrations.

3.1.3. GlycocalyxThe glycocalyx could play a major role in intercel-

lular gamete communication because it forms theextracellular coating of the sperm's surface. Besidesthis, the lateral polarized nature of the glycocalyxcould be relevant for the lateral organization of plas-ma membrane molecules since the carbohydrate net-work is in direct contact with the sperm plasmamembrane via integral membrane proteins and gly-colipids (Fig. 3B).

The glycocalyx alters during capacitation as can beshown by lectin binding studies (for review see [3]). Itis not known whether the changes in lectin bindingre£ect chemical modi¢cation of carbohydrate struc-tures, (un-)covering of carbohydrate structures, orrepositioning of glycoproteins or glycolipids. The re-positioning of the glycolipids during capacitation hasbeen shown (see Section 2.2.1 and Fig. 3B) as well asthe release of `decapacitation factors' during capaci-tation [220,221] and other factors bound to thesperm plasma membrane [222]. It has been postu-lated that the removal of decapacitation factors in-duces tyrosine kinase activity in transmembraneproteins (Fig. 7) [3]. Interaction of these transmem-brane proteins with the ZP leads to aggregation ofthe transmembrane proteins and subsequently fur-ther increases the tyrosine activities. Related to theremoval of surface coating components is the pro-posed uncovering of progesterone receptors on thesperm plasma membrane (see also Section 3.3.5).For instance in dogs it has been demonstrated thats 90% of the cauda epididymal sperm cells have af-¢nity for progesterone, whereas, freshly ejaculatedsperm do not have such a¤nity due to a coatingfactor that is secreted from the prostate [223]. How-ever, subpopulations of sperm cells regained the af-¢nity for progesterone when this factor is releasedduring in vitro capacitation or by density gradientwashing. The sperm cells that expose a functionalprogesterone receptor are the speci¢c cells that ini-tiate the acrosome reaction after a progesterone chal-lenge [223^225] (see also Section 3.3.5).

Fig. 7. Model for regulation of tyrosine kinase activity by ca-pacitation and ZP binding. (A) Decapacitation factors are com-ponents of the ECM and interact with transmembrane proteins.The decapacitation^transmembrane protein interactions preventtyrosine kinase activity. Tyrosine kinase domains may be inand/or coupled to the transmembrane proteins. (B) Upon ca-pacitation, the decapacitation factor is removed which activatestyrosine kinases and may enable the sperm cell to bind to theZP. ZP binding to sperm ZP receptors (transmembrane tyrosinekinases) induces aggregation and further increases tyrosine ki-nase activity. (Adapted from [3]).



3.2. ZP binding

Sperm binding to the ZP is important in fertiliza-tion since it is considered to be a species speci¢crecognition of the two gametes (in contrast to sperminteractions with the oolemma, see Sections 4.1 and4.2). Sperm binding to the ZP also evokes the acro-some reaction, which is required for successful pen-etration of the ZP (and thus for fertilization). De-spite the importance of sperm binding to the ZP,the proteins from the sperm plasma membrane in-volved in ZP recognition and adhesion have notbeen completely elucidated yet.

3.2.1. ZPNomenclature for ZP proteins is inconsistent be-

tween the di¡erent mammalian species. Therefore,we have followed the protein nomenclature as pro-posed by Harris [226], who divided the proteins ofthe ZP of a number of mammals into ZPA, ZPB andZPC based on sequence variations and homologies.

Besides the ZP amino acid sequences of severalspecies, information about the ZP carbohydratestructure has also been revealed [227,228]. The im-portance of N-linked and/or O-linked glycosidic res-idues on the ZP proteins in the primary sperm bind-ing is still a subject of debate. Enzymatic removal orpartial digestion of both O- and N-linked carbohy-drates have been reported to abolish sperm^ZP bind-ing [229^231]. ZPC seems to be the most importantcandidate to function as primary sperm receptor butalso as inducer of the acrosome reaction [228,232].However, indirect ZP a¤nity experiments indicatethat anti-ZPA could inhibit sperm^ZP binding,whereas, anti-ZPC did not inhibit the sperm^ZPbinding in human sperm [233]. In porcine sperm,ZPB and ZPC seem to be involved in sperm bindingto the ZP [234^236]. Finally, ZPB has been shown tobe the primary sperm receptor in the rabbit system[237,238]. Most likely, the complete native texture ofthe ZP is required for optimal sperm binding [199].

3.2.2. Primary sperm^ZP bindingRecognition of the ZP by the sperm cell can be

subdivided in two phases: (i) the primary bindingin which acrosome intact sperm cells bind with pro-teins that are situated only at the apical plasmamembrane of the sperm head [3] (intra-acrosomal

proteins are not exposed in acrosome intact cells,and proteins localized at other sites of the spermsurface will block ZP penetration) and (ii) the sec-ondary binding in which the acrosome reacted spermcell exposes a set of intra-acrosomal proteins withhigh a¤nity for the ZP (see Section 3.2.3). Mostlikely, the putative primary ZP receptors are modi-¢ed upon sperm capacitation since the binding a¤n-ity of the sperm cell for the ZP increases dramaticallyupon capacitation [3,171]. The increased a¤nity ofthe sperm cell for the oocyte could also be a resultof capacitation induced conformational changes ofZP receptors (including dimerization of receptors)or by decoating the ZP receptors. Sperm^ZP bindingresults almost instantaneously in the acrosome reac-tion [239]. The cellular signalling cascades involvedin this process are not completely resolved. It seemsplausible that an integral plasma membrane proteinis involved with at its extracellular domain a ZPbinding site and at its cytosolic domain a functionalsite for activation of intracellular signal transduction[240]. More than one entity may be involved in pri-mary sperm^ZP binding as is based on results fromknock-out mice lacking L1,4-galactosyltransferase[241] (see below).

The research on primary sperm^ZP binding hasfollowed several strategies. One of them is couplingof solubilized sperm proteins or ZP proteins to asolid phase. The solid phase can be column materiallike Sepharose, polystyrene, Western blot paper or inthe case of ZP, intact ZP [242]. Proteins with a¤nityfor the solid phase proteins can easily be separatedfrom proteins with no a¤nity. A drawback from thismethod is that proteins have to be solubilized, whicha¡ects the quaternary structure of the ZP proteinsand may even lead to protein denaturation. Bothe¡ects can reduce the bioactivity (sperm binding)dramatically. Therefore, the method of choice wouldbe to investigate ZP binding with native ZP material[242]. Extending this, primary ZP binding should beinvestigated with isolated apical plasma membranematerial as has been done with control and capaci-tation induced boar sperm material [199].

A second strategy is the inhibition of sperm^ZPbinding by either solubilized sperm proteins or anti-bodies directed against sperm proteins [243^245]. Aclear drawback of this method is that it does notprovide direct evidence for the involvement of the



protein under study in ZP binding. Alternative phe-nomena such as sterical hindrance of the antibody orartifactual immuno-aggregation (antibody capping)may explain the decreased a¤nity for the ZP. Theinhibition of sperm^ZP binding by solubilized spermproteins is an elegant method to show the a¤nity ofthe sperm protein for the ZP [246]. Solubilized spermproteins are sometimes used to show the inhibitorye¡ect on IVF. In such studies it is important to re-member that inhibitory e¡ects may be at a di¡erentlevel of fertilization than primary ZP binding (thetested protein could play a role in secondary binding[247], acrosome reaction, oolemma binding or fu-sion). These arguments are also valid for in vivoimmunization experiments [243], since antigens caninterfere at di¡erent stages of fertilization.

Although several proteins have been postulated assperm ZP receptors (for review see [248]), only twoimportant primary ZP receptor candidates are dis-cussed here. Both proteins bind to ZP and are local-ized at the apical plasma membrane and were ¢rstdescribed in mice: (i) a 95 kDa protein reported inseveral mammalian species [249^255]. The protein isphosphorylated on a tyrosine residue [177,252] andthe degree of tyrosine phosphorylation probably in-creases during in vitro capacitation [250,255,256],although some contradictory ¢ndings have been pub-lished [177,190]. Binding of ZPC to sperm cells in-duces (additional) tyrosine phosphorylation [249,252,257] and the p95 protein is also assumedto be involved in acrosome reaction [258]. Spermspeci¢c hexokinase is a tyrosine phosphorylated in-tegral plasma membrane protein that migrates onreducing gels as a 116 kDa protein and on non-re-ducing gels as a 95 kDa protein [259]. The possibilitythat p95 is the sperm speci¢c hexokinase has beenpostulated [260], although this possibility has beendebated [261]. (ii) The enzyme L1,4-galactosyltrans-ferase has been proposed as (a part of) a ZP receptorin the sperm plasma membrane of a variety of mam-malian species [241]. At least in mouse sperm cellsL1,4-galactosyltransferase binds to ZPC [262] andsubsequently the acrosome reaction is evoked bythis binding [263]. Sperm cells from L1,4-galactosyl-transferase de¢cient mice are still able to fertilizeoocytes [264], although these sperm cells are lessable to bind to ZPC and only insu¤ciently undergothe ZPC primed acrosome reaction (see Section 3.3)

compared to wild type sperm cells [264]. The generalconcept that sperm L1,4-galactosyltransferase bindsto ZPC which provokes the acrosome reaction andis subsequently followed by sperm ZP penetration isprobably oversimpli¢ed. Most likely other molecules(sperm as well as ZP proteins) are also involved inthe primary sperm^ZP interaction [240].

Small sperm associated proteins like pig sperm-ad-hesins probably contribute to a ¢rmer binding ofsperm cells to the ZP [222], although the direct in-volvement in ZP adhesion of these proteins to thesperm cell at the site of fertilization has not yetbeen con¢rmed. In fact it has been shown that thesetightly bound components are released from thesperm surface during sperm interactions with oviduc-tal epithelia [265]. A more likely scenario is thatsperm-adhesins are involved in suppressing antigenicactivity against sperm cells [266] or are just requiredfor interacting with the epithelial cells of the oviduct[267]. From studies in oviductal and primary cellcultures it appeared that sperm cells bind to the ovi-duct for a period of time. The released cells werecapacitated and had high a¤nity for the ZP [82^84].

3.2.3. Secondary sperm^ZP bindingIntra-acrosomal proteins become exposed after the

acrosome reaction and are involved in a more ¢rmattachment of the sperm cell to the ZP (secondaryZP binding) [268]. ZPA seems to be the secondarybinding partner in the ZP [269]. Several proteinshave been identi¢ed as secondary ZP binding pro-teins: PH-20 [270,271], sp38 [272^274], acrosin [275]and P-selectin [276,277]. Acrosin and PH-20 haveboth, besides the mentioned ZP a¤nity, also protein-ase [278] and hyaluronidase activities [86], respec-tively. Binding of these secondary ZP binding pro-teins may prevent the release of hyperactivated spermcells from the ZP. Meanwhile, their enzymatic activ-ities are used to digest the ZP matrix followed bybinding to a new substrate, ultimately enabling thesperm cell to pass the ZP [279].

3.3. Acrosome reaction

The acrosome reaction is initiated immediatelyafter primary binding of a sperm cell to the ZP ofthe oocyte [280]. The apical plasma membrane of thesperm head starts to fuse with the underlying outer



acrosomal membrane at multiple sites resulting in thedispersal of the acrosomal content (Figs. 1 and 2)[281]. During the acrosome reaction hydrolytic andproteolytic enzymes are secreted in order to release,hydrolyze and dissolve the ZP matrix locally in theimmediate direction of the penetrating sperm cellwhich ultimately ensures the entrance of that spermcell into the perivitelline space [282]. The acrosomereaction should not proceed prior to ZP binding be-cause the part of the enzymatic machinery of thisorganelle required for successful ZP penetrationwill be lost. Preliminary acrosome reacted sperm cellsare considered to be incompetent to fertilize the oo-cyte. Sperm cells that reach the perivitelline space arealways acrosome reacted and able to fuse with theoocyte [283,284]. Importantly, the equatorial plasmamembrane area of the sperm head has not partici-pated in the fusions with the acrosomal outer mem-brane and seems to be the speci¢c site involved inoolemma interaction (see Section 4).

The acrosome reaction has been widely studiedand a list of inducers is presented in Table 1. In

some reports the acrosome reaction is considered asan indicator of sperm capacitation. Indeed capaci-tated sperm cells have a destabilized plasma mem-brane (see Section 2.2) and therefore are sensitiveto even small environmental stress when comparedto non-capacitated sperm plasma membranes [26].Therefore, addition of Ca2� in combination with cal-cium ionophore e¡ectively induces the fusion of plas-ma membrane with the underlying acrosome mem-brane, whereas, non-capacitated sperm cells withmuch more rigid plasma membranes do not inducethese membrane fusions under the same conditions[285]. The destabilized plasma membranes of capaci-tated sperm cells also make them more vulnerable toeven slight temperature changes (slow cooling from38.5 to 30³C induces spontaneous acrosome reac-tions in capacitated but not in control sperm cells)[26]. However, when capacitated sperm cells are ex-amined under physiological conditions they fail toinduce the acrosome reaction [26]. It has been shownbefore that dramatic di¡erences exist between spon-taneous acrosome reaction (detrimental), calcium

Table 1The acrosome reaction can be induced in vitro by several agents

Inducer Possible mechanism Reference

Protein :Solubilized ZP receptor activation (physiological) [289]Progesterone receptor activation? (physiological?) [327]Glycoconjugate mimic ZP activation? [384]Glycosamineglycan sulfate mimic ZP activation? [385]Angiotensin II receptor mediated? [386]Atrial natriuretic peptide receptor mediated? [387]Trypsin inhibitor ? [388]Sialic acid binding protein ? [389]Lipid :Arachidonic acid membrane perturbation? receptor mediated? [390]Platelet activating factor membrane perturbation? receptor mediated? [391]Lysophospholipids membrane perturbation? receptor mediated? [150]Fluid :Follicular £uid multiple? activators including progesterone [392]Chemical agents :Calcium ionophore Introduction of Ca2� into the cell [393]cAMP analogue capacitation e¡ect? [394]cGMP analogue capacitation e¡ect? [318]Other :Ethanol membrane perturbation [393]Low temperature `cold shock' membrane perturbation [394]Electropermeabilization introduction of Ca2� into the cell [395]

The mechanism of action of these agents is very diverse, ranging from the physiological relevant activation of sperm ZP receptors bysolubilized ZP proteins, to perturbation of membranes by ethanol.



ionophore induced (by-passing physiological inducedCa2� in£ux required for membrane fusion) and thephysiologically induced acrosome reaction (by theZP) [196,286,287]. Nevertheless, useful clinical assaysto predict the fertilizing potential of semen have beendeveloped in which the calcium ionophore challengeis used to detect the relative amount of induced acro-some reaction indirectly indicating to the relativeamount of capacitated sperm cells [288].

3.3.1. Receptor aggregationSoluble ZPC induces the acrosome reaction in

sperm cells of several mammalian species [289]. Theacrosome reaction is probably induced by ZP recep-tor aggregation which is mediated by primary ZPbinding (Figs. 6 and 7). In vitro experiments revealedthat p95 and L1,4-galactosyltransferase may aggre-gate after ZP binding: ZP peptides bind to p95and, subsequently, addition of anti-ZP antibodies re-sults in aggregation [290]. Anti-L1,4-galactosyltrans-ferase Fab fragments and, subsequently, additionof antibodies against these Fab fragments resultedin aggregation of L1,4-galactosyltransferase [291].Although these experiments were performed in vitroin both cases the ZP binding event did not induce theacrosome reaction, whereas the antibody induced ag-gregation coincided with the induction of the acro-some reaction. Aggregation of proteinase inhibitorbound to the mouse sperm surface in the absenceof ZP glycoproteins [292], as well as immuno-aggre-gation of seminolipid [29], also coincided with theacrosome reaction. Therefore, clustering of proteinsor lipids by immuno-aggregation in vitro already co-incides with the acrosome reaction. Most likely, theZP induces the acrosome reaction in a comparablemanner.

The aggregation of receptors is a common themein signal transduction. Growth hormone for instancebinds to two growth hormone receptors, inducingdimerization of these receptors. Dimerization resultsin the activation of an associated tyrosine kinase,resulting in tyrosine phosphorylation of the receptoras well as the tyrosine kinase. Subsequently, a varietyof signalling molecules are recruited and/or activated[293]. G-proteins have also been suggested to be im-plicated in receptor dimerization induced signaltransduction [294]. Several models exist for signallingpathways including the involvement of G-protein re-

ceptors which could play an important role in ZPinduced acrosome reaction [295,296]. However,more research is needed to unravel the ZP inducedaggregation of sperm ZP receptors and the subse-quent signal transduction.

3.3.2. Protein phosphorylationProtein tyrosine phosphorylation is not only in-

volved in capacitation (see Section 3.1.2) but alsoin the acrosome reaction: mouse sperm p95 tyrosinephosphorylation is enhanced after ZPC binding (Fig.6) [249], which is due to autophosphorylation in-duced by ZPC mediated p95 aggregation. Tyrosinekinase inhibitors blocked the ZP induced tyrosinephosphorylation and the ZP induced acrosome reac-tion [258]. Coupling of ZPC to p95 also stimulatestyrosine phosphorylation of PLCQ in capacitatedmouse sperm cells (for PLC signalling see Section2.4.3) and this e¡ect is abolished in the presence oftyrosine kinase inhibitors [125]. Sperm capacitationresulted in the translocation of PLCQ from the cyto-sol to the membrane which is believed to be a resultof tyrosine phosphorylation [125]. Therefore, a bi-phasic activation of tyrosine kinase has been sug-gested (see Fig. 7). The ZP induced acrosome reac-tion can be blocked by tyrosine kinase inhibitors butthese inhibitors did not a¡ect motility nor the iono-phore induced acrosome reaction, indicating that ca-pacitation was not a¡ected [196]. The induction ofCa2� entry into sperm cells challenged with solubi-lized ZP proteins is blocked by tyrosine kinase inhib-itors [200]. Furthermore, Brewis et al. [252] showedthat recombinant human ZPC induced acrosome re-action in capacitated human sperm cells that coin-cided with an increased tyrosine phosphorylationstate of a 95 kDa protein. Progesterone, which isalso able to induce the acrosome reaction (see Sec-tion 3.3.5), induces a Ca2� in£ux in human spermcells. Tyrosine kinase inhibition reduces the plateauphase of the progesterone induced Ca2� increase[195].

Other protein kinases may also be involved in theinduction of the acrosome reaction. Staurosporine,an inhibitor of PKC, blocked the ZP induced acro-some reaction in sperm cells of human and variousother mammalian species but did not a¡ect motility[131,134,297^300] (Fig. 6). The activation of varioustypes of PKC is detailed in Section 2.4.3. The PAF



induced acrosome reaction is a¡ected by inhibitorsfor PKC, PTK and PKA in human sperm cells [299],possibly because signalling cross-talk is involved inthe activation of the three protein kinases. In factcross-talk of PKA and PKC has been proposed toinduce the human acrosome reaction [298].

3.3.3. G-proteinsG-proteins are involved in signal transduction in

virtually all mammalian cells [301] and most likelyare involved in the induction of the acrosome reac-tion. Mammalian sperm cells possess a subset of G-proteins distributed over di¡erent regions includingthe acrosome and the equatorial segment [302^304].The ZP induced acrosome reaction can be blockedby pertussis toxin (a Gi-proteins inhibitor) withouta¡ecting ZP binding itself [295,296,305^307]. Gi-pro-teins are directly activated by ZPC [295,308^310]. Infact, the non-physiological stimulation of G-proteinswith GTPQS, GppNHp, mastoparan and AlF3

4 alsoinduced the acrosome reaction [311]. The ZP inducedaggregation of L1,4-galactosyltransferase (see Section3.2.3) has been suggested to activate G-proteins inmouse sperm cells [312]. The activation of G-proteinsby ZP binding seems to increase AC activity [313].Furthermore, ZPC binding induces alkalinization viaa Gi-protein and activates a poorly selective cationchannel (pertussis toxin insensitive) [314]. The con-certed e¡ects of depolarization and alkalinization ap-pear to open T-type voltage sensitive Ca2� channels(Fig. 6) [314].

3.3.4. Ion channelsThe acrosome reaction is a membrane fusion event

requiring rather high (mM range) cytosolic Ca2� lev-els. In capacitated sperm cells the intracellular Ca2�

concentration is considerably lower (WM range) and,as is mentioned before (Section 3.1.2), sperm cells donot possess an intracellular pool of mobilizable Ca2�.Therefore, extracellular Ca2� has to pass the plasmamembrane prior to the initiation of the acrosomereaction. A signi¢cant in£ux of Ca2� into sperm cellsis accomplished by binding to the ZP [200,201]. Lowvoltage activated Ca2� channels are fully active afterZP interaction leading to Ca2� in£ux necessary forthe acrosome reaction [218]. ZPC binding to spermcells induces a membrane depolarization, from ap-proximately 360 mV to between 325 and 320

mV, via a poorly selective cation channel, and aGi-protein dependent alkalinization which synergisti-cally results in the opening of voltage dependentCa2� channels (Fig. 6) [314]. However, Gi dependentopening of voltage dependent Ca2� channels in hu-man sperm cells seems to be independent of alkalin-ization [315]. The opening of T-type voltage depen-dent Ca2� channels is believed to be responsible forthe Ca2� in£ux required for the acrosome reaction.Other types of voltage dependent Ca2� channels(subunits) are present in the sperm cell at speci¢clocations [316], however, a function for non-T-typevoltage dependent Ca2� channels in the induction ofthe acrosome reaction is not evidenced (for reviewsee [317]). Progesterone has been shown to inducethe acrosome reaction in several mammalian species,via an increase in intracellular Ca2�. Possibly proges-terone directly activates Ca2� channels [318] or indi-rectly via voltage dependent Ca2� channels (see Sec-tion 3.3.5).

Sodium channels may also be involved in the in-duction of the acrosome reaction. The sodium chan-nel inhibitor, amiloride, inhibited the acrosome reac-tion, whereas ionophore monensin enhanced theacrosome reaction [319]. A role for Na�/Ca2� ex-change was postulated, although others claim a rolefor Ca2� dependent potassium channels in the ham-ster acrosome reaction [320].

3.3.5. ProgesteroneSperm cells like other mammalian cells [321,322]

possess progesterone receptors at the plasma mem-brane (non-genomic progesterone receptor) [223,225,323]. These receptors di¡er considerably from thegenomic progesterone receptor located in the cytosolof somatic cells [324]. The sperm receptor has arather low a¤nity for progesterone when comparedto the genomic progesterone receptor in the cytosolof somatic cells (WM and pM, respectively [225,325]).However, follicular £uid contains an enormousamount of progesterone (V6 Wg/ml in follicular £uidversus V10 ng/ml in serum [326]) and this £uid isreleased at ovulation (the ZP is impregnated withhigh levels of progesterone as well).

The calculated physiological doses of 0.3 Wg/mlprogesterone will surround the sperm cell in the ovi-duct just after ovulation (i.e. the fertilization period).These doses of progesterone induce the acrosome



reaction in several mammalian species: [327,328] byrising the intracellular Ca2� levels [329]. The signaltransduction involved in the progesterone inducedCa2� in£ux is uncertain (Fig. 6). The binding of pro-gesterone to and stimulation of a Q-aminobutyricacid (GABA) receptor/Cl3 channel has been re-ported in the brain [330]. The idea that progesteroneacts via a GABAA receptor/Cl3 channel in spermcells was strengthened by the ¢nding that GABAbinds to human sperm cells and that progesteronestimulation induced a chloride e¥ux [224], whereasthe e¡ect was blocked by GABAA receptor/Cl3

channel antagonists [331]. Antagonists also inhibitedthe progesterone induced acrosome reaction [123].Progesterone and ZP seem to induce the acrosomereaction in a synergistic and comparable way [123].Progesterone primed calcium in£ux which was notenough to induce the human acrosome reaction,the addition of ZP was required [332]. Activationof the sperm GABAA receptor by progesterone orGABA resulted in elevated activity of PLC resultingin increased sperm DAG production [333] and induc-tion of chloride e¥ux from sperm cells [334,335].DAG probably induces Ca2� release in sperm cells[336] and the chloride e¥ux is likely involved inmembrane depolarization and subsequent Ca2� in-£ux through voltage dependent Ca2� channels[315,337]. For more details on the regulations andsignalling of PLC and DAG see Section 2.4.3.

Progesterone mediated induction of the acrosomereaction can be blocked by tyrosine kinase inhibitors,whereas progesterone itself stimulates protein tyro-sine phosphorylation [195,224,338]. This suggeststhat progesterone mediated signalling is transducedvia protein tyrosine phosphorylation. The PTK sub-strates that become phosphorylated after progester-one remain to be investigated in order to get a betterlink between progesterone binding and the subse-quent acrosome reaction. It is important to remem-ber here, that tyrosine kinase inhibitors not onlyblock progesterone e¡ects, but also sperm capacita-tion, which physiologically has been completed be-fore sperm cells are sensitized for progesterone [338](see Section 3.1.1). This is also demonstrated by the¢nding that progesterone induced acrosome reactionis inhibited by speci¢c blocking of capacitation in-duced tyrosine phosphorylation of two kinases(ERK-1 and ERK-2) [339]. Similar arguments can

be raised against the postulated role for A-kinaseanchoring protein and PKA in the progesterone in-duced signal transduction pathway (inhibitors for an-choring and direct PKA inhibitors block capacitationas well as progesterone induced acrosome reaction[340]).

Progesterone exhibits two distinct binding sites onthe human sperm membrane [341]. Isolation andidenti¢cation of the plasma membrane receptor(s)for progesterone remain to be performed and prob-ably is one of the major goals in further detailingsignalling events leading to the acrosome reaction.It may prelude the discovery of a new family ofhormone receptors oriented at the plasma mem-brane, with low a¤nity for steroids eliciting rapidnon-genomic physiological changes.

4. The ¢nal goal: binding and fusion with theoolemma

By the time the sperm reaches the egg plasmamembrane (oolemma) it has passed the processes de-scribed in Sections 2 and 3. Brie£y, the sperm cellwas capacitated in the female genital tract, it boundto the ZP and after the acrosome reaction it hassuccessfully penetrated the ZP and reached the peri-vitelline space, where it now will meet the oolemma(Fig. 2). Therefore, the sperm cell is dramaticallyaltered from its original ejaculated physiological stateupon oolemma binding. It is not clear whether theZP induced alterations are required to achieve fertil-ization, since ZP denuded oocytes can be fertilized bycapacitated acrosome-intact sperm cells [342]. How-ever, it should be noted that such an approach easilyleads to polyspermy and that such sperm^oolemmafusions can be achieved with sperm from di¡erentmammalian species with oocytes from heterologousmammals [342]. This is illustrated by the fact thatnude hamster oocytes are used to test the fusioncompetence of human sperm cells in the IVF clinic[343]. Sperm binding to the oolemma is a multistepprocess which has been most extensively studied inrodents [344,345].

The sperm ¢rst seems to approach the oolemma bybinding with its apical tip of the sperm head to theoolemma (Fig. 8) [346,347]. In golden hamsters, itwas shown that this preliminary binding of sperm



Fig. 8. Sperm egg binding and subsequent fusion is a three-step event. (A) Sperm initially binds with the apical tip of the inner acro-somal membrane to the oolemma. (B) After proper positioning, the apical binding is diminished and has facilitated a lateral bindingof the sperm cell to the oolemma. The equatorial region of the sperm head plasma membrane is involved in this process. (C) Thesperm cell is now capable of fusing its equatorial plasma membrane with the oolemma. The tail stops beating and sti¡ens after equa-torial sperm binding. (D) The multidomain organization of fertilin (heterodimer protein from the ADAM family) has been proposedto be directly involved in processes (B) and (C). (A^C adapted from [3] and D from [383]).



to the oolemma was maintained for an extended pe-riod of time in the presence of protease inhibitors[348]. In this particular experiment, sperm cells re-mained attached with the apical tip to the oolemma.Neither binding of the lateral side of the sperm headto nor fusion with the oolemma could be observed[348]. Therefore, this preliminary binding to the oo-lemma seems to be important for positioning thesperm head to the oolemma. Most likely this prelimi-nary binding involves species speci¢c sperm oolemmabinding components. Subsequently, proteases enablethe sperm cell to go to the second stage of spermbinding. The preliminary binding sites are degradedand the sperm cell is now competent to turn parallelwith the oolemma [348]. During this stage, spermcells lie £at on the surface of the oocyte; the equa-torial region of the sperm head plasma membrane isattached to the oolemma while the sperm tail contin-ues to beat vigorously. After a period of time, thetwo adhered membranes fuse, a process that coin-cides with a sti¡ening of the sperm tail [349]. Onlythe equatorial sperm head plasma membrane is com-petent to fuse with the oolemma [3,350].

In all three steps proteins may play a role thatbelong to the ADAM family of membrane proteinsthat contain a disintegrin and metalloprotease do-main [351,352]. These are multidomain proteins(Fig. 8) of which the protease domain and the dis-integrin domains are probably functional in sperm^oolemma interactions as will be detailed below.

4.1. Sperm tip binding to the oolemma

As mentioned above the ZP penetrated sperm cellinitially binds with the tip of its head to the oolem-ma. This attachment is an association between theinner acrosomal membrane or with an acrosomalmatrix protein and the egg surface [353,354]. A can-didate adhesion molecule for this binding step is thesperm protein cyritestin. Cyritestin is a membraneprotein which was ¢rst identi¢ed as a testis speci¢cmouse gene belonging to the ADAM family [355].Incubating oocytes with peptides from the bindingsite of cyritestin blocked the tip binding as well asfusion of mouse sperm to the oolemma [356,357].The protein has been reported to be associatedwith the inner acrosomal membrane of mouse sperm[353,354]. The tip location of cyritestin would indi-

cate that it is in the correct position for the initialsperm binding to the oolemma. The apical tip bind-ing has to be diminished in order to facilitate theequatorial binding (see Section 4.2). Most likely theprotease domain of cyritestin is involved in diminish-ing this binding. After £attening to the oolemma theinner acrosomal membrane is excluded from mem-brane fusion and is not incorporated into the mem-brane that surrounds the developing zygote. Insteadthe inner acrosomal membrane is incorporated intothe egg cytoplasm by a process that resembles phago-cytosis [358]. The cyritestin binding component onthe oolemma has not been determined yet, but onelikely candidate is one or more of the oolemma'sintegrins (see Section 4.2).

4.2. Equatorial sperm binding to the oolemma

After the relatively short time that sperm cells areattached to the oolemma with the tip, the spermhead binds laterally with its equatorial region (Fig.8). However, this lateral binding is soon followed bythe fertilization fusion which complicates the molec-ular dissectioning of these two processes. The mostextensively studied protein from the sperm plasmamembrane, with an apparent role in this bindingand fusion, is fertilin. Fertilin, also indicated asPH-30, is a heterodimer [359]. Both the K and L sub-units are members of the ADAM family [360] andare non-covalently associated in guinea-pig sperm[359] and are also found in a variety of other speciesincluding the mouse [361]. The expression and pro-cessing of fertilin have been studied extensively (forreview see [345]). Fertilin is localized at the apicalhead region of the guinea-pig [359] and on the equa-torial region in mouse sperm [357]. In mouse, spermfertilin L is additionally observed on the inner acro-somal membrane [357], which may indicate that thisfraction is involved in the initial sperm tip binding tothe oolemma (see Section 4.1). By sequence align-ment of fertilin L with the related snake disintegrin,a binding site had been predicted for integrins [362].Homologous recombination experiments were de-signed to generate mice lacking fertilin L [363]. In-deed sperm produced by mice lacking the fertilin Lgene had severely reduced (eight-fold) ability to bindto the oolemma in vitro. Like cyritestin, fertilin L hasprobably binding a¤nity for the oolemma's integrins



[362]. It is not clear whether fertilin L and cyritestinbind to the same integrins or if they di¡er in integrinspeci¢city. Several di¡erent integrins have beenfound on the oolemma of various mammalian species(for review see [364] and references therein). Consis-tent data are available for the presence of K5L1,K6L1 and KvL3 integrins at the surface of humanand hamster oocytes [365]. In in vitro assays onmouse oocytes it has been demonstrated that fertilinL has a¤nity for K6L1 [365]. However, it is likelythat the other integrins are also involved in fertilinL binding (for review see [345]). A 94 kDa protein onthe mouse oolemma protein has been shown to beinvolved in spermôocyte interaction [366], but it isnot clear whether this protein has a¤nity for fertilin.

Recently, it has been demonstrated that SLIP (seeSection 2.3.4) may be involved in sperm binding tothe oolemma [367,368]. SLIP is probably a mem-brane bound isoform of arylsulfatase A, which nor-mally is a lysosomal enzyme involved in the desulfa-tion of seminolipid [16,91]. SLIP has been detectedon the oolemma and therefore may bind sperm cellsby immobilizing seminolipid. In capacitated, but alsoin acrosome reacted viable sperm cells, seminolipid ispredominantly localized at the equatorial region inthe outer lipid lea£et of the sperm plasma membrane,which is the right position for lateral binding to theoolemma [28,29]. A particular molecule of interest isFertPlus, which is a synthetic peptide derived fromprosaposin (also called sulfated glycoprotein 1, SGP-1) [369]. Because the peptide includes a region ofsaposin B (an activator protein for arylsulfatase), itmay activate SLIP to desulfate seminolipid. If so, theinterplay between SLIP and FertPlus induces the for-mation of desulfoseminolipid, which due to its rela-tively small head group can induce inverted micellesthat facilitate fusion [2].

4.3. Spermôolemma fusion

Currently, there are two basic models that explainhow the fusion between the two gametes may occur.Basically, the ¢rst model describes this event analo-gous to membrane fusion between membrane envel-oped viruses and host cell membranes [370,371],whereas the other is analogous to the fusion thatoccurs between transport vesicles in the cytoplasmand target membranes [372]. The virus model pre-

dicts that a fusion peptide stretches from the virionto the host cell membrane, where it exposes a hydro-phobic domain. This insertion enables conformation-al changes of membrane proteins in the host cell andthe virion, destabilizes the membrane bilayer struc-ture and generates the fusion pore. The fusion poreexpands so that the viral and the host membranesbecome continuous. The transport vesicle model pre-dicts that the two membranes contain two interactingproteins: for the transport vesicle v-snare and for thetarget membrane t-snare. After binding, these twoproteins assemble more complex linkages betweenthe two membranes in such a way that fusion hasbecome energetically favorable. A clear understand-ing of the molecular nature of membrane fusion be-tween sperm and egg plasma membranes is still lack-ing. Fertilin K has been proposed to play a role inthis fusion event, since it contains a 22 amino acidsequence in the cysteine-rich domain with an K helixstructure exposing its bulky hydrophobic residues atone face of the protein (Fig. 8) [360]. This may implythat fertilin is required for spermôolemma binding(fertilin L) as well as fusion (fertilin K), which wouldmake sense because after binding the hydrophobicdomain of fertilin K is well oriented for inserting itsputative hydrophobic region into the lipid moiety ofthe oolemma. Some support for this idea can befound in literature [345,360]. A synthetic peptide rep-resenting the hydrophobic region of fertilin K fromguinea-pig can bind to vesicles and can induce fusionbetween large unilamellar vesicles [373], althoughsuch properties have not yet been described for theentire protein. However, bovine fertilin K contains ahydrophobic region at a di¡erent region [374] andrabbit fertilin contains K helix breaking amino acids[375,376]. Furthermore, mice lacking the fertilin Lgene (see Section 4.2) have a marked reducedamount of fertilin K on the sperm surface, but still(despite the absence of fertilin L) have a limited po-tential to fuse with the oolemma [363]. Both ¢ndingsindicate that fertilin is not absolutely required for thefertilization fusion.

Only a limited number of studies have been dedi-cated to sperm fusion factors besides fertilinalthough unidenti¢ed proteins that facilitate porcine[377], hamster [378] and bovine spermôolemma fu-sions [379] have been reported.

Interestingly, capacitated human, bovine [380,381]



and boar sperm cells [27] spontaneously fuse in aCa2� dependent manner with £uorescently labelledliposomes. The fusion occurred in living capacitatedsperm cells at the equatorial region of the spermhead at 38³C. This is exactly the site of the spermcell that is speci¢ed in the fusion with the oolemmaand therefore we believe that with the £uorescentliposomes, a new assay has been developed to mon-itor the fusability of the sperm cell with the oolem-ma. Since the liposomes did not contain any mem-brane proteins, these observations would be in favorof the idea that sperm contain a virus-like fusionprotein. Indeed a sperm protein seems to be in-volved, since the liposome fusions were protease sen-sitive [382].

5. Conclusions

An overview has been given on the dynamical as-pects of the mammalian sperm plasma membrane inthe process of fertilization. Special attention to theparticular features of the sperm plasma membrane isof interest for reproductive biologists, but also forresearchers that are more generally interested in bio-membranes. The plasma membrane is highly polar-ized and dynamically reorganized during capacita-tion and other fertilization processes. Furthermore,it is involved in various membrane adhesion and fu-sion processes.

Many con£icting studies have emerged on thepresence and the involvement of surface componentsin sperm cells. Probably this is related to the fragilityof the sperm plasma membrane after capacitation.Artifacts are easily introduced when primed cellsare labelled or monitored under detrimental condi-tions (e.g. at temperatures lower then 30³C), orunder conditions where the incubation medium isnot equilibrated with 5% CO2 to maintain the appro-priate bicarbonate concentrations for capacitation invitro. Capacitation and the acrosome reaction aretwo speci¢c events that are required for fertilization.However, due to improper experimentation, in vitrocapacitation itself has been reported to induce theacrosome reaction. Under proper conditions the in-duction of the acrosome reaction only proceeds afterZP or progesterone mediated stimulation of capaci-tated sperm cells.

At least three intracellular signalling routes thathave been described for sperm cells are relevant forfurther study and in other cell systems (Fig. 5): (i)bicarbonate activates AC directly by binding 1:1 tothis protein. The subsequent induction of PKA seemsto cross-talk with tyrosine kinase and also is involvedin the collapse of phospholipid asymmetry in theapical plasma membrane. The e¡ects seem to be im-portant for sperm binding to the ZP, but also for theacrosome reaction. (ii) Cholesterol e¥ux further ac-tivates membrane mediated signalling upon capacita-tion. The picture of the regulation of cholesterol ef-£ux as well as the identi¢cation of proteins involvedin this process is far from complete. (iii) Sperm cellscontain a speci¢c non-genomic progesterone receptorthat enables extracellular Ca2� to pass the spermplasma membrane by opening voltage dependentCa2� channels (Fig. 6). The identi¢cation of this re-ceptor may open a new research ¢eld of an entirelynew family of steroid receptors.

Sperm binding to the ZP as well as the oolemma isdivided into more than one phase and in each phasedi¡erent proteins seem to be involved at di¡erentlocations of the sperm plasma membrane (Fig. 8).The various lateral plasma membrane specializationsand reorganizations thereof may not only be impor-tant for the regionalization of gamete adhesion andfusion processes, but may also be functional as sig-nalling compartments. ZP binding is initiated by thesperm plasma membrane proteins, whereas, second-ary binding is mediated by acrosomal matrix pro-teins. Therefore, it is important to isolate the spermplasma membrane from acrosomal components inorder to study primary ZP binding. Plasma mem-brane isolation might also be important in order todissect the primary sperm tip binding to the oolem-ma, which is probably initiated by an inner acroso-mal membrane protein, whereas the secondary oo-lemma binding is mediated by a plasma membraneprotein located in the equatorial sperm head. Fur-thermore, it seems to be important to study primaryZP binding proteins from the sperm plasma mem-brane by using native ZP material rather then solu-bilized ZP proteins.

The regulation of the sperm plasma membranefusions with either the outer acrosomal membraneor the oolemma is far from understood. In fact nocandidate fusion protein for the acrosome fusions



has been described yet, whereas, the involvement offertilin in oolemma fusion has only been evidencedby circumstantial data. Therefore, future researchshould be focussed on the components that inducefusions of the sperm plasma membrane with one ofthese two membranes.

Taking all these considerations into account, webelieve that this review may help to introduce futuremembrane researchers as well as reproductive biolo-gists into the complex processes that prelude the fer-tilization of the oocyte. Basic understanding of thesperm plasma membrane organization and the rele-vance of its structural rearrangements in signallingevents that leads to fertilization of the oocyte areof crucial importance. After passing all challengesdescribed in this review the sperm cell may success-fully activate the fertilized oocyte. New challenges inearly life are now switched on and the processes thatoccur in the developing zygote are several orders ofmagnitudes more complex than the processes re-viewed here.

Acknowledgements

The authors would like to acknowledge Dr. T.W.J.Gadella, Jr., for his help with Fig. 4; Professor Dr.B. Colenbrander and Professor Dr. L.M.G. vanGolde for their expert comments and critical readingof the manuscript. F.M.F. is a recipient of a Ph.D.fellowship from the Graduate School of AnimalHealth, B.M.G. is a senior fellow of the Dutch RoyalAcademy of Sciences and Arts (KNAW).

References

[1] E.M. Eddy, D.A. O'Brien, in: E. Knobil, J.D. Neill (Eds.),The Physiology of Reproduction, Raven Press, New York,1994, pp. 29^77.

[2] J.P. Vos, M. Lopez-Cardozo, B.M. Gadella, Biochim. Bio-phys. Acta 1211 (1994) 125^149.

[3] R. Yanagimachi, in: E. Knobil, J.D. Neill (Eds.), The Phys-iology of Reproduction, Raven Press, New York, 1994, pp.189^317.

[4] J.E. Flechon, D.C. Kraemer, E.S. Hafez, Folia Primatol.Basel 26 (1976) 24^35.

[5] J.K. Koehler, Arch. Androl. 6 (1981) 197^217.[6] M. Mattioli, B. Barboni, P. Lucidi, E. Seren, Theriogenology

45 (1996) 373^381.

[7] F. Szasz, S. Sirivaidyapong, F.P. Cheng, W.F. Voorhout, A.Marks, B. Colenbrander, L. Solti, B.M. Gadella, Mol. Re-prod. Dev. 55 (2000) 289^298.

[8] C. Yeaman, K.K. Grindsta¡, W.J. Nelson, Physiol. Rev. 79(1999) 73^98.

[9] A. Dunina-Barkovskaya, Membr. Cell Biol. 11 (1998) 555^589.

[10] T. Mann. C. Lutwak-Mann, Male Reproductive Functionand Semen, Springer, Berlin, 1981.

[11] J.E. Parks, D.V. Lynch, Cryobiology 29 (1992) 255^266.[12] R.W. Evans, D.E. Weaver, E.D. Clegg, J. Lipid Res. 21

(1980) 223^228.[13] J.F.H.M. Brouwers, B.M. Gadella, L.M.G. Van Golde,

A.G.M. Tielens, J. Lipid Res. 39 (1998) 344^353.[14] D.P. Selivonchick, P.C. Schmid, V. Natarajan, H.H. Schmid,

Biochim. Biophys. Acta 618 (1980) 242^254.[15] B.M. Gadella, B. Colenbrander, L.M.G. Van Golde, M.

Lopez-Cardozo, Biochim. Biophys. Acta 1128 (1992) 155^162.

[16] B.M. Gadella, B. Colenbrander, L.M.G. Van Golde, M.Lopez-Cardozo, Biol. Reprod. 48 (1993) 483^489.

[17] N. Saxena, R.N. Peterson, S. Sharif, N.K. Saxena, L.D.Russell, J. Reprod. Fertil. 78 (1986) 601^614.

[18] A.I. Mahmoud, J.J. Parrish, Mol. Reprod. Dev. 43 (1996)554^560.

[19] I. Revah, B.M. Gadella, F.M. Flesch, B. Colenbrander, S.S.Suarez, Biol. Reprod. 62 (2000) 1010^1015.

[20] A.P. Aguas, P. Pinto-Da-Silva, J. Cell Sci. 93 (1989) 467^479.

[21] E.L. Bearer, D.S. Friend, J. Electron Microsc. Tech. 16(1990) 281^297.

[22] A.I. Yudin, G.N. Cherr, C.A. Vandevoort, J.W. Overstreet,Mol. Reprod. Dev. 50 (1998) 207^220.

[23] S. Parkkila, H. Rajaniemi, S. Kellokumpu, Biol. Reprod. 49(1993) 326^331.

[24] F. Suzuki, R. Yanagimachi, Gamete Res. 23 (1989) 335^347.[25] E.L. Bearer, D.S. Friend, J. Cell Biol. 92 (1982) 604^615.[26] B.M. Gadella, R.A.P. Harrison, Development 127 (2000)

2407^2420.[27] B.M. Gadella, N.G.A. Miller, B. Colenbrander, L.M.G. Van

Golde, R.A.P. Harrison, Mol. Reprod. Dev. 53 (1999) 108^125.

[28] B.M. Gadella, T.W.J. Gadella, B. Colenbrander, L.M.G.Van Golde, M. Lopez-Cardozo, J. Cell Sci. 107 (1994)2151^2163.

[29] B.M. Gadella, M. Lopez-Cardozo, L.M.G. Van Golde, B.Colenbrander, T.W.J. Gadella, J. Cell Sci. 108 (1995) 935^945.

[30] E.Z. Drobnis, L.M. Crowe, T. Berger, T.J. Anchordoguy,J.W. Overstreet, J.H. Crowe, J. Exp. Zool. 265 (1993) 432^437.

[31] B.M. Gadella, P.F.E.M. Nievelstein, B. Colenbrander, R.Van Kooij, Hum. Reprod. 11 (1996) 43. Abstract book 1

[32] S. Ladha, J. Membr. Biol. 165 (1998) 1^10.[33] T. Harder, K. Simons, Curr. Opin. Cell Biol. 9 (1997) 534^

542.



[34] K. Muller, T. Pomorski, P. Muller, A. Zachowski, A. Herr-mann, Biochem. 33 (1994) 9968^9974.

[35] A.P. Rana, S. Misra, G.C. Majumder, A. Ghosh, Biochim.Biophys. Acta 1210 (1993) 1^7.

[36] V.T. Hinkovska, G.P. Dimitrov, K.S. Koumanov, Int.J. Biochem. 18 (1986) 1115^1121.

[37] A.J. Verkleij, P.H. Ververgaert, B. De Kruy¡, L.M. VanDeenen, Biochim. Biophys. Acta 373 (1974) 495^501.

[38] J.P. Nolan, S.F. Magargee, R.G. Posner, R.H. Hammer-stedt, Biochem. 34 (1995) 3907^3915.

[39] S.J. Martin, C.P. Reutelingsperger, A.J. McGahon, J.A.Rader, R.C. Van Schie, D.M. La Face, D.R. Green,J. Exp. Med. 182 (1995) 1545^1556.

[40] K. Emoto, N. Toyama-Sorimachi, H. Karasuyama, K. In-oue, M. Umeda, Exp. Cell Res. 232 (1997) 430^434.

[41] B.M. Gadella, R.A.P. Harrison, Biol. Reprod. 60 ((Suppl.1)) (1999) 207.

[42] L. McEvoy, R.A. Schlegel, P. Williamson, B.J. Del Buono,J. Leukoc. Biol. 44 (1988) 337^344.

[43] R.A.P. Harrison, P.J.C. Ashworth, N.G.A. Miller, Mol. Re-prod. Dev. 45 (1996) 378^391.

[44] R.A.P. Harrison, N.G.A. Miller, Mol. Reprod. Dev. 55(2000) 220^228.

[45] S. Palleschi, L. Silvestroni, Biochim. Biophys. Acta 1279(1996) 197^202.

[46] W.V. Holt, R.D. North, J. Reprod. Fertil. 78 (1986) 447^457.

[47] S. Ladha, P.S. James, D.C. Clark, E.A. Howes, R. Jones,J. Cell Sci. 110 (1997) 1041^1050.

[48] D.E. Wolf, J.K. Voglmayr, J. Cell Biol. 98 (1984) 1678^1684.[49] D.E. Wolf, S.S. Hagopian, S. Ishijima, J. Cell Biol. 102

(1986) 1372^1377.[50] P.S. James, C.A. Wolfe, A.R. Mackie, S. Ladha, A. Prentice,

R. Jones, Hum. Reprod. 14 (1999) 1827^1832.[51] F.E. De Leeuw, H.C. Chen, B. Colenbrander, A.J. Verkleij,

Cryobiology 27 (1990) 171^183.[52] W.V. Holt, R.D. North, J. Exp. Zool. 230 (1984) 473^483.[53] W.V. Holt, R.D. North, J. Reprod. Fertil. 73 (1985) 285^

294.[54] C.A. Wolfe, P.S. James, A.R. Mackie, S. Ladha, R. Jones,

Biol. Reprod. 59 (1998) 1506^1514.[55] P.S. James, C.A. Wolfe, S. Ladha, R. Jones, Mol. Reprod.

Dev. 52 (1999) 207^215.[56] D.E. Wolf, Mol. Membr. Biol. 12 (1995) 101^104.[57] E. Leblond, L. Desnoyers, P. Manjunath, Mol. Reprod.

Dev. 34 (1993) 443^449.[58] S.E. Ravnik, J.J. Albers, C.H. Muller, J. Exp. Zool. 272

(1995) 78^83.[59] J. Langlais, K.D. Roberts, Gamete Res. 12 (1985) 183^224.[60] A. Iborra, M. Companyo, P. Mart|nez, A. Morros, Biol.

Reprod. 62 (2000) 378^383.[61] N.L. Cross, Mol. Reprod. Dev. 45 (1996) 212^217.[62] N.L. Cross, Biol. Reprod. 59 (1998) 7^11.[63] P.E. Visconti, H. Galantino-Homer, X.P. Ning, G.D.

Moore, J.P. Valenzuela, C.J. Jorgez, J.G. Alvarez, G.S.Kopf, J. Biol. Chem. 274 (1999) 3235^3242.

[64] P.E. Visconti, X.P. Ning, M.W. Fornes, J.G. Alvarez, P.Stein, S.A. Connors, G.S. Kopf, Dev. Biol. 214 (1999)429^443.

[65] N. Tanphaichitr, Y.S. Zheng, M. Kates, N. Abdullah, A.Chan, Mol. Reprod. Dev. 43 (1996) 187^195.

[66] N.L. Cross, Mol. Reprod. Dev. 53 (1999) 92^98.[67] G. Gimpl, K. Burger, F. Fahrenholz, Biochem. 36 (1997)

10959^10974.[68] N.L. Cross, P. Mahasreshti, Arch. Androl. 39 (1997) 39^44.[69] A. Minelli, M. Moroni, E. Matinez, I. Mezzasoma, G. Ron-

quist, J. Reprod. Fertil. 114 (1999) 237^243.[70] S.E. Ravnik, P.W. Zarutskie, C.H. Muller, J. Androl. 11

(1990) 216^226.[71] K.J. Go, D.P. Wolf, Adv. Lipid Res. 20 (1983) 317^330.[72] K.J. Go, D.P. Wolf, Biol. Reprod. 32 (1985) 145^153.[73] E. To«pfer-Petersen, J.J. Calvete, L. Sanz, F. Sinowatz, An-

drologia 27 (1995) 303^324.[74] L. Desnoyers, P. Manjunath, J. Biol. Chem. 267 (1992)

10149^10155.[75] J.J. Calvete, K. Mann, L. Sanz, M. Raida, E. To«pfer-Peter-

sen, FEBS Lett. 399 (1996) 147^152.[76] I. Therien, G. Bleau, P. Manjunath, Biol. Reprod. 52 (1995)

1372^1379.[77] P. Muller, K.R. Erlemann, K. Muller, J.J. Calvete, E. To«p-

fer-Petersen, K. Marienfeld, A. Herrmann, Eur. Biophys.J. Biophys. 27 (1998) 33^41.

[78] I. Therien, R. Moreau, P. Manjunath, Biol. Reprod. 61(1999) 590^598.

[79] I. Therien, R. Moreau, P. Manjunath, Biol. Reprod. 59(1998) 768^776.

[80] P. Manjunath, Y.L. Marcel, J. Uma, N.G. Seidah, M. Chre-tien, A. Chapdelaine, J. Biol. Chem. 264 (1989) 16853^16857.

[81] E. To«pfer-Petersen, F. Sinowatz, S. Hein, P. Schwartz, L.A.Johnson, H.D. Guthrie (Eds.), Boar Semen Preservation III,Allen Press Inc., Lawrence, KS, 2000, pp. 3^11.

[82] R. Lefebvre, S.S. Suarez, Biol. Reprod. 54 (1996) 575^582.[83] A.R. Fazeli, A.E. Duncan, P.F. Watson, W.V. Holt, Biol.

Reprod. 60 (1999) 879^886.[84] S.S. Suarez, in: C. Gagnon (Ed.), The Male Gamete, Cache

River Press, Vienna, IL, 1999, pp. 71^80.[85] C.D. Thaler, R.A. Cardullo, Biochem. 34 (1995) 7788^7795.[86] M. Gmachl, S. Sagan, S. Ketter, G. Kreil, FEBS Lett. 336

(1993) 545^548.[87] I.A. Rooney, J.E. Heuser, J.P. Atkinson, J. Clin. Invest. 97

(1996) 1675^1686.[88] M.A. Shirley, H. Schachter, Can. J. Biochem. 58 (1980)

1230^1239.[89] H. Law, O. Itkonnen, C.A. Lingwood, J. Cell Physiol. 137

(1988) 462^468.[90] C. Lingwood, A. Nutikka, J. Cell Physiol. 146 (1991) 258^

263.[91] N. Tanphaichitr, C. Moase, T. Taylor, K. Surewicz, C. Han-

sen, M. Namking, B. Berube, N. Kamolvarin, C.A. Ling-wood, R. Sullivan, M. Rattanachaiy-Anont, D. White,Mol. Reprod. Dev. 49 (1998) 203^216.



[92] D. White, W. Weeachatyanukul, B.M. Gadella, N. Kamol-varin, M. Attar, N. Tanphaichitr, Biol. Reprod. 63 (2000)147^155.

[93] E.R.S. Roldan, in: C. Gagnon (Ed.), The Male Gamete,Cache River Press, Vienna, IL, 1999, pp. 127^138.

[94] J.A. Lundbaek, O.S. Andersen, J. Gen. Physiol. 104 (1994)645^673.

[95] N.V. Prokazova, N.D. Zvezdina, A.A. Korotaeva, Bio-chem. Mosc. 63 (1998) 31^37.

[96] S.P. Flaherty, N.J. Swann, J. Cell Sci. 104 (1993) 163^172.[97] W.L. Smith, F.A. Fitzpatrick, in: D.E. Vance, J. Vance

(Eds.), Biochemistry of Lipids, Lipoproteins and Mem-branes, Elsevier, Amsterdam, 1996, pp. 283^308.

[98] F. Snyder, Biochem. J. 305 (1995) 689^705.[99] M. Schaefer, T. Hofmann, G. Schultz, T. Gudermann,

Proc. Natl. Acad. Sci. USA 95 (1998) 3008^3013.[100] E.R.S. Roldan, C. Fragio, J. Biol. Chem. 268 (1993) 13962^

13970.[101] M. Luconi, L. Bonaccorsi, C. Krausz, G. Gervasi, G. Forti,

E. Baldi, Mol. Cell. Endocrinol. 108 (1995) 35^42.[102] E. Baldi, C. Falsetti, C. Krausz, G. Gervasi, V. Carloni, R.

Casano, G. Forti, Biochem. J. 292 (1993) 209^216.[103] E.R.S. Roldan, J.M. Vazquez, FEBS Lett. 396 (1996) 227^

232.[104] L. Dominguez, R.M.F. Yunes, M.W. Fornes, L.S. Mayor-

ga, Int. J. Androl. 19 (1996) 248^252.[105] E.A. Dennis, Trends. Biochem. Sci. 22 (1997) 1^2.[106] J. Langlais, J.G. Chafouleas, R. Ingraham, N. Vigneault,

K.D. Roberts, Biochem. Biophys. Res. Commun. 182(1992) 208^214.

[107] S. Weinman, C. Ores-Carton, D.P. Rainteau, S. Puszkin,J. Histochem. Cytochem. 34 (1986) 1171^1179.

[108] M.S. Ri¡o, M. Parraga, J. Exp. Zool. 279 (1997) 81^88.[109] M.S. Ri¡o, M. Parraga, J. Exp. Zool. 275 (1996) 459^468.[110] N. Uozumi, K. Kume, T. Nagase, N. Nakatani, S. Ishii, F.

Tashiro, Y. Komagata, K. Maki, K. Ikuta, Y. Ouchi, J.Miyazaki, T. Shimizu, Nature 390 (1997) 618^622.

[111] J.V. Bonventre, Z. Huang, M.R. Taheri, E. O'Leary, E. Li,M.A. Moskowitz, A. Sapirstein, Nature 390 (1997) 622^625.

[112] S. Soubeyrand, A. Khadir, Y. Brindle, P. Manjunath,J. Biol. Chem. 272 (1997) 222^227.

[113] E.R.S. Roldan, R.A.P. Harrison, Biochem. J. 259 (1989)397^406.

[114] E.R.S. Roldan, R.A.P. Harrison, Biochem. Biophys. Res.Commun. 172 (1990) 8^15.

[115] C.M.B. O'Toole, E.R.S. Roldan, L.R. Fraser, Mol. Hum.Reprod. 2 (1996) 921^927.

[116] E.R.S. Roldan, R.A.P. Harrison, Biochem. J. 281 (1992)767^773.

[117] V. Hinkovska-Galchev, P.N. Srivastava, Mol. Reprod.Dev. 33 (1992) 281^286.

[118] E.R.S. Roldan, T. Murase, J. Biol. Chem. 269 (1994)23583^23589.

[119] R.G. Sheikhnejad, P.N. Srivastava, J. Biol. Chem. 261(1986) 7544^7549.

[120] Y. Asaoka, Y. Tsujishita, Y. Nishizuka, in: R.M. Bell, J.H.Exton, S.M. Prescott (Eds.), Lipid Second Messengers, Ple-num Press, New York, 1996, pp. 59^74.

[121] M.N. Hodgkin, T.R. Pettitt, A. Martin, R.H. Michell, A.J.Pemberton, M.J. Wakelam, Trends Biochem. Sci. 23 (1998)200^204.

[122] P. Thomas, S. Meizel, Biochem. J. 264 (1989) 539^546.[123] E.R.S. Roldan, T. Murase, Q.X. Shi, Science 266 (1994)

1578^1581.[124] E.R.S. Roldan, in: P. Fenichel, J. Parinaud (Eds.), Human

Sperm Acrosome Reaction, John Libbey Eurotext, London,1995, pp. 225^243.

[125] C.N. Tomes, C.R. McMaster, P.M. Saling, Mol. Reprod.Dev. 43 (1996) 196^204.

[126] H.L. Feng, J.I. Sandlow, A. Sandra, Mol. Reprod. Dev. 49(1998) 317^326.

[127] C.M.B. O'Toole, E.R.S. Roldan, P. Hampton, L.R. Fraser,Mol. Hum. Reprod. 2 (1996) 317^326.

[128] E.R.S. Roldan, C. Fragio, Biochem. J. 297 (1994) 225^232.[129] E.R.S. Roldan, R.A.P. Harrison, Biochem. Biophys. Res.

Commun. 155 (1988) 901^906.[130] Z. Naor, H. Breitbart, Trends Endocrinol. Metabol. 8

(1997) 337^342.[131] D.Y. Liu, H.G. Baker, Mol. Hum. Reprod. 3 (1997) 1037^

1043.[132] Y. Lax, S. Rubinstein, H. Breitbart, Biol. Reprod. 56

(1997) 454^459.[133] R. Rotem, G.F. Paz, Z.T. Homonnai, M. Kalina, J. Lax,

H. Breitbart, Z. Naor, Endocrinology 131 (1992) 2235^2243.

[134] H. Breitbart, J. Lax, R. Rotem, Z. Naor, Biochem. J. 281(1992) 473^476.

[135] C.M.B. O'Toole, E.R.S. Roldan, L.R. Fraser, Mol. Re-prod. Dev. 45 (1996) 204^211.

[136] L. Bonaccorsi, C. Krausz, P. Pecchioli, G. Forti, E. Baldi,Mol. Hum. Reprod. 4 (1998) 259^268.

[137] E.R.S. Roldan, L.R. Fraser, Trends Endocrinol. Metabol. 9(1998) 296^297.

[138] M. Luconi, C. Krausz, T. Barni, G.B. Vannelli, G. Forti, E.Baldi, Mol. Hum. Reprod. 4 (1998) 251^258.

[139] R.A. Fissore, J.M. Robl, Dev. Biol. 159 (1993) 122^130.[140] R.A. Fissore, M.M. Reis, G.D. Palermo, Mol. Hum. Re-

prod. 5 (1999) 189^192.[141] Y.M. Wolny, R.A. Fissore, H. Wu, M.M. Reis, L.T. Co-

lombero, B. Ergun, Z. Rosenwaks, G.D. Palermo, Mol.Reprod. Dev. 52 (1999) 277^287.

[142] P.F. Watson, J.M. Plummer, P.S. Jones, J.C. Bredl, Mol.Reprod. Dev. 41 (1995) 513^520.

[143] A.D. Tepper, E. De Vries, W.J. Van Blitterswijk, J. Borst,J. Clin. Invest. 103 (1999) 971^978.

[144] H. Umehara, J.Y. Huang, T. Kono, F.H. Tabassam, T.Okazaki, E.T. Bloom, N. Domae, J. Immunol. 159 (1997)1200^1207.

[145] S. Chasserot-Golaz, P. Hubert, D. Thierse, S. Dirrig, C.J.Vlahos, D. Aunis, M.F. Bader, J. Neurochem. 70 (1998)2347^2356.



[146] P.L. Yeagle, F.T. Smith, J.E. Young, T.D. Flanagan, Bio-chem. 33 (1994) 1820^1827.

[147] A.J. Verkleij, Biochim. Biophys. Acta 779 (1984) 43^63.[148] E.R.S. Roldan, A.D. Fleming, J. Reprod. Fertil. 85 (1989)

297^308.[149] P. Mart|nez, A. Morros, Front. Biosci. 1 (1996) 103^117.[150] J.E. Dravland, M.N. Llanos, R.J. Munn, S. Meizel, J. Exp.

Zool. 232 (1984) 117^128.[151] P.E. Visconti, G.S. Kopf, Biol. Reprod. 59 (1998) 1^6.[152] N. Tanphaichitr, D. White, T. Taylor, M. Attar, M. Rat-

tanachaiy-Anont, D. D'Amours, M. Kates, in: C. Gagnon(Ed.), The Male Gamete, Cache River Press, Vienna, IL,1999, pp. 227^236.

[153] R. Jones, T. Mann, R. Sherins, Fertil. Steril. 31 (1979) 531^537.

[154] J.F. Griveau, D. Le Lannou, Int. J. Androl. 20 (1997) 61^69.

[155] H. Wol¡, J.A. Politch, A. Martinez, F. Haimovici, J.A.Hill, D.J. Anderson, Fertil. Steril. 53 (1990) 528^536.

[156] E. De Lamirande, C. Gagnon, Hum. Reprod. 10 ((Suppl.1)) (1995) 15^21.

[157] M. Gavella, V. Lipovac, Arch. Androl. 28 (1992) 135^141.[158] R.J. Aitken, H.M. Fisher, N. Fulton, E. Gomez, W. Knox,

B. Lewis, S. Irvine, Mol. Reprod. Dev. 47 (1997) 468^482.[159] M. Plante, E. De Lamirande, C. Gagnon, Fertil. Steril. 62

(1994) 387^393.[160] P. Leclerc, E. De Lamirande, C. Gagnon, J. Androl. 19

(1998) 434^443.[161] R.J. Aitken, D. Harkiss, W. Knox, M. Paterson, D.S. Ir-

vine, J. Cell Sci. 111 (1998) 645^656.[162] P. Leclerc, E. De Lamirande, C. Gagnon, Free Radic. Biol.

Med. 22 (1997) 643^656.[163] J.G. Alvarez, B.T. Storey, J. Androl. 14 (1993) 199^209.[164] B.T. Storey, Mol. Hum. Reprod. 3 (1997) 203^213.[165] E. De Lamirande, C. Gagnon, Int. J. Androl. 16 (1993) 21^

25.[166] E. De Lamirande, C. Gagnon, Fertil. Steril. 59 (1993)

1291^1295.[167] R.J. Aitken, E. Gordon, D. Harkiss, J.P. Twigg, P. Milne,

Z. Jennings, D.S. Irvine, Biol. Reprod. 59 (1998) 1037^1046.

[168] E. De Lamirande, H. Jiang, A. Zini, H. Kodama, C. Gag-non, Rev. Reprod. 2 (1997) 48^54.

[169] Y.J. Suzuki, H.J. Forman, A. Sevanian, Free Radic. Biol.Med. 22 (1997) 269^285.

[170] L.R. Fraser, Hum. Reprod. 13 ((Suppl. 1)) (1998) 9^19.[171] W. Harkema, R.A.P. Harrison, N.G.A. Miller, E.K. Top-

per, H. Woelders, Biol. Reprod. 58 (1998) 421^430.[172] F.M. Flesch, B. Colenbrander, L.M.G. Van Golde, B.M.

Gadella, Biochem. Biophys. Res. Commun. 262 (1999) 787^792.

[173] R.A.P. Harrison, B. Mairet, N.G.A. Miller, Mol. Reprod.Dev. 35 (1993) 197^208.

[174] M.A. Lee, G.S. Trucco, K.B. Bechtol, N. Wummer, G.S.Kopf, L. Blasco, B.T. Storey, Fertil. Steril. 48 (1987) 649^658.

[175] L.R. Fraser, J.E. Herod, J. Reprod. Fertil. 88 (1990) 611^621.

[176] T.T. Smith, Biol. Reprod. 58 (1998) 1102^1104.[177] P.E. Visconti, J.L. Bailey, G.D. Moore, D. Pan, P. Olds-

Clarke, Development 121 (1995) 1129^1137.[178] P. Leclerc, E. De Lamirande, C. Gagnon, Biol. Reprod. 55

(1996) 684^692.[179] D.E. Boatman, R.S. Robbins, Biol. Reprod. 44 (1991) 806^

813.[180] C. Emiliozzi, P. Fenichel, Biol. Reprod. 56 (1997) 674^679.[181] J.E. Oshero¡, P.E. Visconti, J.P. Valenzuela, A.J. Travis, J.

Alvarez, G.S. Kopf, Mol. Hum. Reprod. 5 (1999) 1017^1026.

[182] H. Zayas, E. Bonilla, Y. Ducolomb, E. Casas, A. Cordova,M. Betancourt, Med. Sci. Res. 23 (1995) 831^832.

[183] R.A.P. Harrison, Reprod. Fertil. Dev. 8 (1996) 581^594.[184] R. Ain, K. Uma-Devi, S. Shivaji, P.B. Seshagiri, Mol.

Hum. Reprod. 5 (1999) 618^626.[185] L.J. Jensen, B.M. Schmitt, U.V. Berger, N.N. Nsumu, W.F.

Boron, M.A. Hediger, D. Brown, S. Breton, Biol. Reprod.60 (1999) 573^579.

[186] S. Parkkila, K. Kaunisto, S. Kellokumpu, H. Rajaniemi,Histochemistry 95 (1991) 477^482.

[187] N. Okamura, Y. Sugita, J. Biol. Chem. 258 (1983) 13056^13062.

[188] N. Okamura, Y. Tajima, A. Soejima, H. Masuda, Y. Sugi-ta, J. Biol. Chem. 260 (1985) 9699^9705.

[189] N.B. Garty, Y. Salomon, FEBS Lett. 218 (1987) 148^152.[190] P.E. Visconti, G.D. Moore, J.L. Bailey, P. Leclerc, S.A.

Connors, D. Pan, P. Olds-Clarke, G.S. Kopf, Development121 (1995) 1139^1150.

[191] P. Kalab, J. Peknicova, G. Geussova, J. Moos, Mol. Re-prod. Dev. 51 (1998) 304^314.

[192] D.L. Brautigan, F.M. Pinault, Proc. Natl. Acad. Sci. USA88 (1991) 6696^6700.

[193] C. Blanco-Aparicio, J. Torres, R. Pulido, J. Cell Biol. 147(1999) 1129^1136.

[194] S. Furuya, Y. Endo, K. Osumi, M. Oba, S. Nozawa, S.Suzuki, Fertil. Steril. 59 (1993) 216^222.

[195] L. Bonaccorsi, M. Luconi, G. Forti, E. Baldi, FEBS Lett.364 (1995) 83^86.

[196] B.S. Pukazhenthi, D.E. Wildt, M.A. Ottinger, J. Howard,Mol. Reprod. Dev. 49 (1998) 48^57.

[197] S. Beno¡, Mol. Hum. Reprod. 4 (1998) 453^471.[198] A. Nassar, M. Mahony, M. Morshedi, M.H. Lin, C. Sri-

sombut, S. Oehninger, Fertil. Steril. 71 (1999) 919^923.[199] F.M. Flesch, B. Colenbrander, L.M.G. Van Golde, B.M.

Gadella, Biol. Reprod. 60 ((Suppl. 1)) (1999) 207.[200] J.L. Bailey, B.T. Storey, Mol. Reprod. Dev. 39 (1994) 297^

308.[201] I.A. Brewis, R. Clayton, C.L. Barratt, D.P. Hornby,

H.D.M. Moore, Mol. Hum. Reprod. 2 (1996) 583^589.[202] G. Kaul, S. Singh, K.K. Gandhi, S.R. Anand, Andrologia

29 (1997) 243^251.[203] L.R. Fraser, C.A. McDermott, J. Reprod. Fertil. 96 (1992)

363^377.



[204] S.S. Suarez, X.B. Dai, Mol. Reprod. Dev. 42 (1995) 325^333.

[205] M. Cordoba, T.A. Santa-Coloma, N.B. Beorlegui, M.T.Beconi, Biochem. Mol. Biol. Int. 41 (1997) 725^733.

[206] S.A. Adeoya-Osiguwa, L.R. Fraser, J. Reprod. Fertil. 99(1993) 187^194.

[207] A. Ruknudin, I.A. Silver, Mol. Reprod. Dev. 26 (1990) 63^68.

[208] S. Beno¡, Front. Biosci. 3 (1998) D1220^D1240.[209] M. Ashraf, R.N. Peterson, L.D. Russell, Biol. Reprod. 31

(1984) 1061^1071.[210] G.A. Rufo, P.K. Scho¡, H.A. Lardy, J. Biol. Chem. 259

(1984) 2547^2552.[211] L.D. Walensky, S.H. Snyder, J. Cell Biol. 130 (1995) 857^

869.[212] J.J. Parrish, J.L. Susko-Parrish, J.K. Graham, Theriogenol-

ogy 51 (1999) 461^472.[213] J.A. Martinez-Menarguez, H.J. Geuze, J. Ballesta, J. His-

tochem. Cytochem. 44 (1996) 313^320.[214] L.R. Fraser, L.R. Abeydeera, K. Niwa, Mol. Reprod. Dev.

40 (1995) 233^241.[215] R.L. Perry, C.L.R. Barratt, M.A. Warren, I.D. Cooke,

J. Exp. Zool. 279 (1997) 284^290.[216] G.A. Rufo, J.P. Singh, D.F. Babcock, H.A. Lardy, J. Biol.

Chem. 257 (1982) 4627^4632.[217] Y. Zeng, E.N. Clark, H.M. Florman, Dev. Biol. 171 (1995)

554^563.[218] C. Arnoult, I.G. Kazam, P.E. Visconti, G.S. Kopf, M.

Villaz, H.M. Florman, Proc. Natl. Acad. Sci. USA 96(1999) 6757^6762.

[219] L.R. Fraser, G. Umar, S. Sayed, J. Reprod. Fertil. 97(1993) 539^549.

[220] S.A. Adeoya-Osiguwa, L.R. Fraser, Mol. Reprod. Dev. 44(1996) 111^120.

[221] L.R. Fraser, Mol. Reprod. Dev. 51 (1998) 193^202.[222] E. To«pfer-Petersen, A. Romero, P.F. Varela, M. Ekhlasi-

Hundrieser, Z. Dostalova, L. Sanz, J.J. Calvete, Andrologia30 (1998) 217^224.

[223] S. Sirivaidyapong, M.M. Bevers, B. Colenbrander, J. An-drol. 20 (1999) 537^544.

[224] S. Meizel, K.O. Turner, J. Androl. 17 (1996) 327^330.[225] F.P. Cheng, B.M. Gadella, W.F. Voorhout, A.R. Fazeli,

M.M. Bevers, B. Colenbrander, Biol. Reprod. 59 (1998)733^742.

[226] J.D. Harris, D.W. Hibler, G.K. Fontenot, K.T. Hsu, E.C.Yurewicz, A.G. Sacco, DNA Seq. 4 (1994) 361^393.

[227] J.D. Bleil, P.M. Wassarman, Dev. Biol. 76 (1980) 185^202.

[228] M. Nakano, N. Yonezawa, Y. Hatanaka, S. Noguchi,J. Reprod. Fertil. 50 ((Suppl.)) (1996) 25^34.

[229] E. Skutelsky, E. Ranen, R. Shalgi, J. Reprod. Fertil. 100(1994) 35^41.

[230] E.S. Litscher, P.M. Wassarman, Biochem. 35 (1996) 3980^3985.

[231] K. Ozgur, M.S. Patankar, S. Oehninger, G.F. Clark, Mol.Hum. Reprod. 4 (1998) 318^324.

[232] H. Tsubamoto, A. Hasegawa, Y. Nakata, S. Naito, N.Yamasaki, K. Koyama, Biol. Reprod. 61 (1999) 1649^1654.

[233] K.D. Hinsch, E. Hinsch, Andrologia 31 (1999) 320^322.[234] E.C. Yurewicz, A.G. Sacco, S.K. Gupta, N.X. Xu, D.A.

Gage, J. Biol. Chem. 273 (1998) 7488^7494.[235] A.G. Sacco, E.C. Yurewicz, M.G. Subramanian, P.D. Mat-

zat, Biol. Reprod. 41 (1989) 523^532.[236] N. Blase, A.R. Fazeli, E. Topper, M.M. Bevers, H. Woel-

ders, E. To«pfer-Petersen, B. Colenbrander, Reprod. Dom.Anim. 33 (1998) 21^25.

[237] S.V. Prasad, B. Wilkins, S.M. Skinner, B.S. Dunbar, Mol.Reprod. Dev. 43 (1996) 519^529.

[238] S.V. Prasad, B. Wilkins, B.S. Dunbar, J. Reprod. Fertil. 50((Suppl.)) (1996) 143^149.

[239] A.R. Fazeli, W.J. Hage, F.P. Cheng, W.F. Voorhout, A.Marks, M.M. Bevers, B. Colenbrander, Biol. Reprod. 56(1997) 430^438.

[240] I.A. Brewis, C.H. Wong, Rev. Reprod. 4 (1999) 135^142.[241] B.D. Shur, Biochem. Biophys. Res. Commun. 250 (1998)

537^543.[242] D.M. Hardy, D.L. Garbers, J. Biol. Chem. 269 (1994)

19000^19004.[243] B. Berube, R. Sullivan, Biol. Reprod. 51 (1994) 1255^1263.[244] A.L. Kadam, M. Fateh, R.K. Naz, J. Reprod. Immunol. 29

(1995) 19^30.[245] R.N. Peterson, W.P. Hunt, Gamete Res. 23 (1989) 103^118.[246] L.C. Lopez, E.M. Bayna, D. Lito¡, N.L. Shaper, J.H.

Shaper, B.D. Shur, J. Cell Biol. 101 (1985) 1501^1510.[247] S.A. Coonrod, J.C. Herr, M.E. Westhusin, J. Reprod. Fer-

til. 107 (1996) 287^297.[248] W.J. Snell, J.M. White, Cell 85 (1996) 629^637.[249] L. Leyton, P.M. Saling, Cell 57 (1989) 1123^1130.[250] R.K. Naz, K. Ahmad, R. Kumar, J. Cell Sci. 99 (1991)

157^165.[251] P. Fenichel, A. Gharib, C. Emiliozzi, M. Donzeau, Y. Me-

nezo, Biol. Reprod. 54 (1996) 1405^1411.[252] I.A. Brewis, R. Clayton, C.E. Browes, M. Martin, C.R.

Barratt, D.P. Hornby, H.D.M. Moore, Mol. Hum. Reprod.4 (1998) 1136^1144.

[253] K. Uma-Devi, M. Basheer-Ahmad, S. Shivaji, Mol. Re-prod. Dev. 47 (1997) 341^350.

[254] B.S. Pukazhenthi, D.E. Wildt, M.A. Ottinger, J. Howard,J. Androl. 17 (1996) 409^419.

[255] B.S. Pukazhenthi, J.A. Long, D.E. Wildt, M.A. Ottinger,D.L. Armstrong, J. Howard, J. Androl. 19 (1998) 675^685.

[256] A.E. Duncan, L.R. Fraser, J. Reprod. Fertil. 97 (1993)287^299.

[257] R.K. Naz, Arch. Androl. 37 (1996) 47^55.[258] L. Leyton, P. LeGuen, D. Bunch, P.M. Saling, Proc. Natl.

Acad. Sci. USA 89 (1992) 11692^11695.[259] P. Kalab, P.E. Visconti, P. Leclerc, G.S. Kopf, J. Biol.

Chem. 269 (1994) 3810^3817.[260] P.E. Visconti, P.J. Olds-Clarke, S.B. Moss, P. Kalab, A.J.

Travis, M.A. De las Heras, G.S. Kopf, Mol. Reprod. Dev.43 (1996) 82^93.



[261] L. Leyton, C. Tomes, P.M. Saling, Mol. Reprod. Dev. 42(1995) 347^358.

[262] D.J. Miller, M.B. Macek, B.D. Shur, Nature 357 (1992)589^593.

[263] R. Shalgi, T. Raz, Histol. Histopathol. 12 (1997) 813^822.[264] Q.X. Lu, B.D. Shur, Development 124 (1997) 4121^4131.[265] E. To«pfer-Petersen, J.J. Calvete, Int. J. Androl. 18 (1995)

20^26.[266] S.C. Kwok, M.J. Soares, J.P. McMurtry, E.C. Yurewicz,

Mol. Reprod. Dev. 35 (1993) 244^250.[267] S.S. Suarez, Biol. Reprod. 36 (1987) 203^210.[268] S.B. McLeskey, C. Dowds, R. Carballada, R.R. White,

P.M. Saling, Int. Rev. Cytol. 177 (1998) 57^113.[269] J.D. Bleil, J.M. Greve, P.M. Wassarman, Dev. Biol. 128

(1988) 376^385.[270] G.R. Hunnicutt, P. Primako¡, D.G. Myles, Biol. Reprod.

55 (1996) 80^86.[271] Y. Lin, L.H. Kimmel, D.G. Myles, P. Primako¡, Proc.

Natl. Acad. Sci. USA 90 (1993) 10071^10075.[272] E. Mori, T. Baba, A. Iwamatsu, T. Mori, Biochem. Bio-

phys. Res. Commun. 196 (1993) 196^202.[273] E. Mori, S.I. Kashiwabara, T. Baba, Y. Inagaki, T. Mori,

Dev. Biol. 168 (1995) 575^583.[274] K. Sabeur, G.N. Cherr, A.I. Yudin, P. Primako¡, M.W. Li,

J.W. Overstreet, J. Androl. 18 (1997) 151^158.[275] R.T. Richardson, M.G. Orand, J. Biol. Chem. 271 (1996)

24069^24074.[276] J.G. Geng, T.J. Raub, C.A. Baker, G.A. Sawada, L. Ma,

A.P. Elhammer, J. Cell Biol. 137 (1997) 743^754.[277] S. Oehninger, M. Patankar, M. Seppala, G.F. Clark, An-

drologia 30 (1998) 269^274.[278] K.L. Polakoski, R.A. McRorie, W.L. Williams, J. Biol.

Chem. 248 (1973) 8178^8182.[279] P.M. Wassarman, E.S. Litscher, Curr. Top. Dev. Biol. 30

(1995) 1^19.[280] P.M. Wassarman, Biol. Reprod. 46 (1992) 186^191.[281] P.M. Saling, J. Sowinski, B.T. Storey, J. Exp. Zool. 209

(1979) 229^238.[282] M.N. Llanos, P. Vigil, A.M. Salgado, P. Morales, J. Re-

prod. Fertil. 97 (1993) 173^178.[283] K. Toshimori, Biol. Reprod. 26 (1982) 475^481.[284] J.M. Cummins, W.R. Edirisinghe, Y. Odawara, R.G.

Wales, J.L. Yovich, Gamete Res. 24 (1989) 461^469.[285] F.P. Cheng, A.R. Fazeli, W.F. Voorhout, A. Marks, M.M.

Bevers, B. Colenbrander, J. Androl. 17 (1996) 674^682.[286] Y. Endo, P. Mattei, G.S. Kopf, R.M. Schultz, Dev. Biol.

123 (1987) 574^577.[287] L. Leyton, A. Robinson, P.M. Saling, Dev. Biol. 132 (1989)

174^178.[288] J.M. Cummins, S.M. Pember, A.M. Jequier, J.L. Yovich,

P.E. Hartmann, J. Androl. 12 (1991) 98^103.[289] J.D. Bleil, P.M. Wassarman, Dev. Biol. 95 (1983) 317^324.[290] L. Leyton, P.M. Saling, J. Cell Biol. 108 (1989) 2163^

2168.[291] M.B. Macek, L.C. Lopez, B.D. Shur, Dev. Biol. 147 (1991)

440^444.

[292] D. Aarons, H. Boettger-Tong, G. Holt, G.R. Poirier, Mol.Reprod. Dev. 30 (1991) 258^264.

[293] L.S. Argetsinger, C. Carter-Su, Physiol. Rev. 76 (1996)1089^1107.

[294] P.R. Gouldson, C.R. Snell, R.P. Bywater, C. Higgs, C.A.Reynolds, Protein Eng. 11 (1998) 1181^1193.

[295] M.W. Wilde, C.R. Ward, G.S. Kopf, Mol. Reprod. Dev. 31(1992) 297^306.

[296] H. Bastiaan, D. Franken, P. Wranz, J. Assist. Reprod.Genet. 16 (1999) 332^336.

[297] Y. Lax, S. Rubinstein, H. Breitbart, FEBS Lett. 339 (1994)234^238.

[298] C.M. Doherty, S.M. Tarchala, E. Radwanska, C.J. DeJonge, J. Androl. 16 (1995) 36^46.

[299] K. Sengoku, K. Tamate, N. Takuma, Y. Takaoka, T.Yoshida, K. Nishiwaki, M. Ishikawa, Mol. Hum. Reprod.2 (1996) 401^404.

[300] Y. Sistina, J.C. Rodger, Reprod. Fertil. Dev. 9 (1997) 803^809.

[301] J. Wess, FASEB J. 11 (1997) 346^354.[302] N.B. Garty, D. Galiani, A. Aharonheim, Y.K. Ho, D.M.

Phillips, N. Dekel, Y. Salomon, J. Cell Sci. 91 (1988) 21^31.[303] M. Glassner, J. Jones, I. Kligman, M.J. Woolkalis, G.L.

Gerton, G.S. Kopf, Dev. Biol. 146 (1991) 438^450.[304] F. Merlet, L.S. Weinstein, P.K. Goldsmith, T. Rarick, J.L.

Hall, J.P. Bisson, P. De Mazancourt, Mol. Hum. Reprod. 5(1999) 38^45.

[305] Y. Endo, M.A. Lee, G.S. Kopf, Dev. Biol. 119 (1987) 210^216.

[306] M.A. Lee, J.H. Check, G.S. Kopf, Mol. Reprod. Dev. 31(1992) 78^86.

[307] H.M. Florman, R.M. Tombes, N.L. First, D.F. Babcock,Dev. Biol. 135 (1989) 133^146.

[308] C.R. Ward, B.T. Storey, G.S. Kopf, J. Biol. Chem. 267(1992) 14061^14067.

[309] C.R. Ward, B.T. Storey, G.S. Kopf, J. Biol. Chem. 269(1994) 13254^13258.

[310] X. Ning, C.R. Ward, G.S. Kopf, Mol. Reprod. Dev. 40(1995) 355^363.

[311] A. Brandelli, Biochem. Mol. Biol. Int. 41 (1997) 1217^1225.[312] X. Gong, D.H. Dubois, D.J. Miller, B.D. Shur, Science 269

(1995) 1718^1721.[313] P. Leclerc, G.S. Kopf, J. Androl. 20 (1999) 126^134.[314] C. Arnoult, Y. Zeng, H.M. Florman, J. Cell Biol. 134

(1996) 637^645.[315] L. Linares-Hernandez, A.M. Guzman-Grenfell, J.J. Hicks-

Gomez, M.T. Gonzalez-Mart|nez, Biochim. Biophys. Acta1372 (1998) 1^12.

[316] R.E. Westenbroek, D.F. Babcock, Dev. Biol. 207 (1999)457^469.

[317] S.J. Publicover, C.R. Barratt, Hum. Reprod. 14 (1999) 873^879.

[318] C. Foresta, M. Rossato, F. Di Virgilio, Biochem. J. 294(1993) 279^283.

[319] A. Ruknudin, Gamete Res. 22 (1989) 375^384.[320] M.N. Llanos, J. Exp. Zool. 269 (1994) 484^488.



[321] C. Meyer, R. Schmid, K. Schmieding, E. Falkenstein, M.Wehling, Steroids 63 (1998) 111^116.

[322] C. Mendoza, A. Soler, J. Tesarik, Biochem. Biophys. Res.Commun. 210 (1995) 518^523.

[323] P.F. Blackmore, F.A. Lattanzio, Biochem. Biophys. Res.Commun. 181 (1991) 331^336.

[324] R. Kumar, E.B. Thompson, Steroids 64 (1999) 310^319.[325] K. Sabeur, D.P. Edwards, S. Meizel, Biol. Reprod. 54

(1996) 993^1001.[326] E.A. Libersky, D.E. Boatman, Biol. Reprod. 53 (1995) 477^

482.[327] R.A. Osman, M.L. Andria, A.D. Jones, S. Meizel, Bio-

chem. Biophys. Res. Commun. 160 (1989) 828^833.[328] C.S. Melendrez, S. Meizel, T. Berger, Mol. Reprod. Dev. 39

(1994) 433^438.[329] E. Baldi, R. Casano, C. Falsetti, C. Krausz, M. Maggi, G.

Forti, J. Androl. 12 (1991) 323^330.[330] M.D. Majewska, N.L. Harrison, R.D. Schwartz, J.L.

Barker, S.M. Paul, Science 232 (1986) 1004^1007.[331] C.S. Melendrez, S. Meizel, Biol. Reprod. 53 (1995) 676^

683.[332] C. Emiliozzi, H. Cordonier, J.F. Guerin, B. Ciapa, M.

Benchaib, P. Fenichel, Int. J. Androl. 19 (1996) 39^47.[333] T. Murase, E.R.S. Roldan, Biochem. J. 320 (1996) 1017^

1023.[334] S. Meizel, Biol. Reprod. 56 (1997) 569^574.[335] K.O. Turner, S. Meizel, Biochem. Biophys. Res. Commun.

213 (1995) 774^780.[336] H. Breitbart, B. Spungin, Mol. Hum. Reprod. 3 (1997)

195^202.[337] F. Espinosa, J.L. DelaVega-Beltran, I. Lopez-Gonzalez, R.

Delgado, P. Labarca, A. Darszon, FEBS Lett. 426 (1998)47^51.

[338] E. Baldi, C. Krausz, M. Luconi, L. Bonaccorsi, M. Maggi,G. Forti, J. Steroid Biochem. Mol. Biol. 53 (1995) 199^203.

[339] M. Luconi, T. Barni, G.B. Vannelli, C. Krausz, F. Marra,P.A. Benedetti, V. Evangelista, S. Francavilla, G. Properzi,G. Forti, E. Baldi, Biol. Reprod. 58 (1998) 1476^1489.

[340] D.A. Harrison, D.W. Carr, S. Meizel, Biol. Reprod. 62(2000) 811^820.

[341] E. Baldi, M. Luconi, L. Bonaccorsi, M. Maggi, S. Franca-villa, A. Gabriele, G. Properzi, G. Forti, Steroids 64 (1999)143^148.

[342] J. Aitken, Fertil. Steril. 62 (1994) 17^19.[343] L. Grunfeld, J. Reprod. Med. 34 (1989) 143^149.[344] J.P. Evans, R.M. Schultz, G.S. Kopf, Biol. Reprod. 59

(1998) 145^152.[345] D.G. Myles, C.H. Cho, P. Primako¡, in: C. Gagnon (Ed.),

The Male Gamete, Cache River Press, Vienna, IL, 1999,pp. 249^257.

[346] P. Gaddum-Rosse, Am. J. Anat. 174 (1985) 347^356.[347] C.M. Yoshihara, Z.W. Hall, J. Cell Biol. 122 (1993) 169^

179.[348] H. Takano, R. Yanagimachi, U.A. Urch, Zygote 1 (1993)

79^91.

[349] R. Yanagimachi, Curr. Top. Membr. Trans. 32 (1988) 3^43.

[350] J.M. Clark, J.K. Koehler, Mol. Reprod. Dev. 27 (1990)351^365.

[351] T.G. Wolfsberg, P. Primako¡, D.G. Myles, J.M. White,J. Cell Biol. 131 (1995) 275^278.

[352] T.G. Wolfsberg, J.M. White, Dev. Biol. 180 (1996) 389^401.

[353] B. Linder, S. Bammer, U.A.O. Heinlein, Exp. Cell Res. 221(1995) 66^72.

[354] A. Forsbach, U.A.O. Heinlein, J. Exp. Biol. 201 (1998)861^867.

[355] U.A.O. Heinlein, S. Wallat, A. Senftleben, L. Lemaire,Dev. Growth Di¡er. 36 (1994) 49^58.

[356] B. Linder, U.A.O. Heinlein, Dev. Growth Di¡er. 39 (1997)243^247.

[357] R.Y. Yuan, P. Primako¡, D.G. Myles, J. Cell Biol. 137(1997) 105^112.

[358] R. Shalgi, D.M. Phillips, Biol. Reprod. 23 (1980) 433^444.[359] P. Primako¡, H. Hyatt, J. Tredick-Kline, J. Cell Biol. 104

(1987) 141^149.[360] C.P. Blobel, T.G. Wolfsberg, C.W. Turck, D.G. Myles, P.

Primako¡, J.M. White, Nature 356 (1992) 248^252.[361] T.G. Wolfsberg, P.D. Straight, R.L. Gerena, A.P.J. Huo-

vila, P. Primako¡, D.G. Myles, J.M. White, Dev. Biol. 169(1995) 378^383.

[362] D.G. Myles, L.H. Kimmel, C.P. Blobel, J.M. White, P.Primako¡, Proc. Natl. Acad. Sci. USA 91 (1994) 4195^4198.

[363] C.H. Cho, D.O. Bunch, J.E. Faure, E.H. Goulding, E.M.Eddy, P. Primako¡, D.G. Myles, Science 281 (1998) 1857^1859.

[364] P. Fenichel, M. Durand-Clement, Hum. Reprod. 13((Suppl. 4)) (1998) 31^46.

[365] E.A.C. Almeida, A.P.J. Huovila, A.E. Sutherland, L.E. Ste-phens, P.G. Calarco, L.M. Shaw, A.M. Mercurio, A. Son-nenberg, P. Primako¡, D.G. Myles, Cell 81 (1995) 1095^1104.

[366] T. Kellom, A. Vick, J. Boldt, Mol. Reprod. Dev. 33 (1992)46^52.

[367] N. Tanphaichitr, J. Smith, S. Mongkolsirikieart, C. Gradil,C.A. Lingwood, Dev. Biol. 156 (1993) 164^175.

[368] V. Ahnonkitpanit, D. White, S. Suwajanakorn, F.K. Kan,M. Namking, G. Wells, N. Kamolvarin, N. Tanphaichitr,Biol. Reprod. 61 (1999) 749^756.

[369] R.P. Amann, R.H. Hammerstedt, R.B. Shabanowitz,J. Androl. 20 (1999) 34^41.

[370] D.J. Cohen, M.J. Munuce, P.S. Cuasnicu, Biol. Reprod. 55(1996) 200^206.

[371] W. Xu, D.W. Hamilton, Mol. Reprod. Dev. 43 (1996) 347^357.

[372] T. Weber, B.V. Zemelman, J.A. McNew, B. Westermann,M. Gmachl, F. Parlati, T.H. Sollner, J.E. Rothman, Cell 92(1998) 759^772.

[373] A. Muga, W. Neugebauer, T. Hirama, W.K. Surewicz, Bio-chem. 33 (1994) 4444^4448.



[374] S.I. Waters, J.M. White, Biol. Reprod. 56 (1997) 1245^1254.

[375] C.M. Hardy, M.K. Holland, Mol. Reprod. Dev. 45 (1996)107^116.

[376] A.C.F. Perry, R. Jones, L. Hall, Biochem. J. 312 (1995)239^244.

[377] K. Ash, T. Berger, C.M. Horner, C.C. Calvert, Zygote 3(1995) 163^170.

[378] M.M. Noor, H.D.M. Moore, J. Reprod. Fertil. 115 (1999)215^224.

[379] T. Gougoulidis, A.O. Trounson, A. Dowsing, Mol. Re-prod. Dev. 54 (1999) 173^178.

[380] E.G.J.M. Arts, S. Jager, D. Hoekstra, Biochem. J. 304(1994) 211^218.

[381] E.G.J.M. Arts, J. Kuiken, S. Jager, D. Hoekstra, Eur. J.Biochem. 217 (1993) 1001^1009.

[382] E.G.J.M. Arts, J.G. Wijchman, S. Jager, D. Hoekstra, Bio-chem. J. 325 (1997) 191^198.

[383] J.P. Evans, G.S. Kopf, Andrologia 30 (1998) 297^307.[384] D.J. Miller, R.L. Ax, Mol. Reprod. Dev. 26 (1990) 184^

198.

[385] R. Reyes, A. Carranco, O. Hernandez, A. Rosado, H. Mer-chant, N.M. Delgado, Arch. Androl. 12 (1984) 203^209.

[386] Y. Gur, H. Breitbart, Y. Lax, S. Rubinstein, N. Zamir,Am. J. Physiol. 275 (1998) E87^E93.

[387] N. Zamir, D. Barkan, N. Keynan, Z. Naor, H. Breitbart,Am. J. Physiol. Endocrinol. Met. 32 (1995) E216^E221.

[388] H. Boettger-Tong, D. Aarons, B. Biegler, T. Lee, G.R.Poirier, Biol. Reprod. 47 (1992) 716^722.

[389] M. Banerjee, M. Chowdhury, Hum. Reprod. 10 (1995)3147^3153.

[390] H. Breitbart, Y. Shalev, S. Marcus, M. Shemesh, Hum.Reprod. 10 (1995) 2079^2084.

[391] M.J. Angle, R. Tom, K. Jarvi, R.D. McClure, J. Reprod.Fertil. 98 (1993) 541^548.

[392] G. Oliphant, Fertil. Steril. 27 (1976) 28^38.[393] C.W. Lui, S. Meizel, J. Exp. Zool. 207 (1979) 173^185.[394] C.J. De Jonge, H.L. Han, H. Lawrie, S.R. Mack, L.J.D.

Zaneveld, J. Exp. Zool. 258 (1991) 113^125.[395] J. Santos-Sacchi, M. Gordon, J. Cell Biol. 85 (1980) 798^

803.



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