Cells Tissues Organs 2001;168:105–112

Carbohydrate-Mediated Formationof the Oviductal Sperm Reservoir inMammals

Susan S. Suarez

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, N.Y., USA

Susan S. SuarezDepartment of Biomedical Sciences, College of Veterinary MedicineCornell University, Ithaca, NY 14853 (USA)Tel. +1 607 253 3589, Fax +1 607 253 3541E-Mail sss7@cornell.edu

ABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com

© 2001 S. Karger AG, Basel1422–6405/01/1682–0105$17.50/0

Accessible online at:www.karger.com/journals/cto

Abbreviation used in this paper

fuc-BSA-FITC bovine serum albumin conjugated to fluoresceinand fucose

Key WordsSpermatozoa W Oviduct W Fallopian tube W Spermreservoir

AbstractMammalian sperm are trapped in a reservoir in the ovi-duct until ovulation is imminent. Then, they are gradual-ly released, such that a few meet the oocytes as they en-ter the ampulla of the oviduct. In the three eutherian spe-cies studied to date, sperm are trapped in the reservoirby carbohydrate-mediated binding to the oviductal mu-cosa. Evidence indicates that a molecule on the surfaceof the plasma membrane overlying the acrosome bindsto a carbohydrate moiety on the surface of the oviduct.While sperm remain bound, they appear to be protectedfrom degradation. When sperm become capacitated,they lose binding affinity for the oviductal mucosa. Themechanism initiating capacitation in the reservoir is un-known; however, it must be tied to the hormonal signal-ling of ovulation. Hyperactivated motility may assistsperm in pulling off from the mucosal surface as binding

affinity declines. The function of the reservoir appears tobe to prevent polyspermy and ensure fertilization by pro-viding a small number of sperm in the proper physiologi-cal condition for fertilization at the time the oocytes enterthe oviduct.

Copyright © 2001 S. Karger AG, Basel

Function of the Sperm Reservoir

In most species of eutherian mammals, millions ofsperm are inseminated in order to fertilize only one or afew oocytes [Hunter, 1988]. Of the millions inseminated,only hundreds reach the Fallopian tube in women [Barrattand Cooke, 1991; Williams et al., 1993] and thousands inrabbits [Overstreet et al., 1978] and large farm animals[Hawk, 1983, 1987]. Evidence acquired for a number ofmammalian species demonstrates that sperm which enterthe oviduct are held in a reservoir until the time of ovula-tion nears [reviewed by Harper, 1994; Suarez, 1998]. Thissperm reservoir may serve a number of functions. First, itmay prevent polyspermic fertilization by allowing only afew sperm at a time to reach the oocyte(s) in the ampulla.Sperm numbers have been artificially increased at the siteof fertilization in the pig by surgical insemination directlyinto the oviduct [Polge et al., 1970; Hunter, 1973], byresecting the oviduct to bypass the reservoir [Hunter andLeglise, 1971], and by administering progesterone intothe tunica muscularis [Day and Polge, 1968; Hunter,

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106 Cells Tissues Organs 2001;168:105–112 Suarez

Fig. 1. A Hamster sperm bound to cilia on an explant of hamster oviductal mucosa. !1,000 (micrograph by Lo andSuarez). B Black labelling over the head of a hamster sperm by fetuin conjugated with colloidal gold enhanced withsilver. !2,000. C Fetuin labelling is decreased on a hamster sperm incubated under capacitating conditions. D Con-trol for fetuin labelling: silver enhancement alone [data from DeMott et al., 1995].

1972]. In all of these cases, the incidence of polyspermyincreased. Second, the oviductal mucosa in the reservoirmay maintain the fertility of sperm until the oocyte isreleased into the oviduct. Evidence for this includes theobservation that bovine sperm fertility and motility weremaintained longer in vitro when the sperm were incu-bated with oviductal epithelium, compared with othertypes of epithelium, or in medium alone [Pollard et al.,1991; Chian and Sirard, 1994]. Human sperm retainedmotility longer when incubated with membrane vesiclesextracted from human oviductal epithelium [Murray andSmith, 1997]. Third, the physiological state of the sperm,specifically the processes of capacitation and motilityhyperactivation, may be regulated within the reservoir toensure that sperm are in the proper state when ovulationoccurs. (Note: ‘Capacitation’ is defined herein as a set ofchanges in the sperm plasma membrane that enablessperm to undergo the acrosome reaction. ‘Hyperactiva-tion’ is a change in sperm flagellar beating pattern ob-served in vivo prior to fertilization that involves a signifi-cant increase in flagellar bend amplitude and usually alsoin beat asymmetry.) Capacitation was delayed in humanand equine sperm when they were incubated with mem-brane vesicles prepared from oviduct epithelium [Do-brinski et al., 1997; Murray and Smith, 1997].

Trapping Sperm

There is strong evidence in multiple species that theoviductal reservoir is created by the binding of sperm tooviductal epithelium. Motile sperm have been observedto bind to the apical surface of the oviductal epithelium in

humans [Baillie et al., 1997], cattle [Suarez et al., 1990],mice [Suarez, 1987], hamsters [Smith and Yanagimachi,1991], pigs [Suarez et al., 1991] and horses [Thomas et al.,1994]. As mentioned above, this contact appears to ex-tend the fertile life span of sperm. The trapping action ofthe sperm-binding moieties is undoubtedly enhanced bythe narrow, tortuous lumen of the uterotubal junction andisthmus, as well as the presence of a sticky mucus [Jansen,1978, 1980; Suarez et al., 1997]. Thus, as sperm pass intothe uterotubal junction, they are forced to swim slowlyagainst the mucosal surface of the oviduct, thereby in-creasing their contact with potential binding sites.

Carbohydrate Mediation of Sperm Binding toOviductal Epithelium

Sperm binding to oviductal epithelium involves carbo-hydrate recognition. The first evidence for this came froma study of sperm transport in the hamster. Hamster spermwere mixed with various glycoproteins and polysaccha-rides and then infused into oviducts set in chambers on amicroscope stage. Transillumination allowed the viewingand videorecording of the behavior of the sperm withinthe oviducts. The percentage of sperm bound to the epi-thelium was ascertained for the various macromoleculestested, and fetuin was discovered to significantly inhibitbinding [DeMott et al., 1995]. Asialofetuin, which isfetuin with its terminal sialic acids removed, did notinhibit binding, while sialic acid alone did. Fetuin taggedwith colloidal gold was then found to label the heads ofhamster sperm (fig. 1), as well as certain glycoproteinbands on Western blots of extracts of sperm membrane

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Oviductal Sperm Reservoir Cells Tissues Organs 2001;168:105–112 107

Fig. 2. The role of fucose in bull sperm binding to explants of oviduc-tal epithelium from the isthmuses of estrous heifers (cows). A Com-petitive inhibition of binding by various monosaccharides. c = Nomonosaccharides added; fuc = fucose; gal = galactose; glc = glucose;gln = N-acetylglucosamine; man = mannose; sia = sialic acid, specifi-cally, N-acetylneuraminic acid. Mean B SEM for five replicates;* p ! 0.05 indicates significant reduction of binding compared with

control. B Effect on sperm binding of decreasing amounts of fucose.Mean B SEM for five replicates. C Pretreatment of explants withenzymes. c = No pretreatment; f = 0.1 U/ml bovine epididymal fuco-sidase; f + i = fucosidase plus its specific inhibitor deoxyfuconojiri-mycin; g = 5 U/ml ß-galactosidase. Mean B SEM for three replicates;** p ! 0.01 indicates significant reduction of sperm binding com-pared with no pretreatment [data from Lefebvre et al., 1997].

[DeMott et al., 1995]. These data indicate that there is amolecule on the head of hamster sperm that binds sialicacid and is responsible for attachment of the sperm to theepithelium.

For other species, in which transillumination of theoviduct was not possible, explants were prepared of ovi-ductal mucosa and tested for carbohydrate specificity ofsperm binding. Binding of equine sperm to explants ofoviductal epithelium in vitro was most effectively inhibit-ed by asialofetuin and its terminal sugar, galactose [Le-febvre et al., 1995a; Dobrinski et al., 1996a]. Bovinesperm binding to explants of oviductal epithelium wasdetermined to be specifically blocked by fucoidan and itscomponent fucose [Lefebvre et al., 1997] (fig. 2). Lectinhistochemistry [Danguy et al., 1998], employing the fu-cose-specific lectins UEA-I and LTL, demonstrated thatthe mucosal surface of the bovine oviduct is covered withmolecules containing fucose (fig. 3). Pretreatment of bo-vine epithelium with fucosidase, but not galactosidase,reduced sperm binding [Lefebvre et al., 1997] (fig. 2).Fucosylated bovine serum albumin, tagged with fluores-cein, specifically labelled the rostral head regions of mo-tile bovine sperm [Revah et al., 2000].

In vitro, bull sperm bound equally well to explantsoriginating from both the isthmus and the ampulla of the

oviduct. Lectin histochemistry of sections of whole ovi-duct indicated that fucose is present on the mucosal sur-face in both the isthmus and the ampulla. In vivo, thesperm reservoir does not extend beyond the first few cen-timeters of isthmus proximal to the uterotubal junction[Hunter and Wilmut, 1984; Hunter et al., 1991]. To shedlight on this apparent discrepancy, bull sperm were surgi-cally infused into the isthmuses and ampullas of cowsunder local anesthesia. After 10 min, the oviducts wereremoved, opened longitudinally, and rinsed to removeunbound sperm. Sperm binding was assessed by scanningelectron microscopy. Equal numbers of sperm were seenon the mucosal surface in both segments. It was concludedthat sperm may bind anywhere in the bovine oviduct, butthe reservoir is confined to the initial segment because thenarrow lumen and mucus present in the initial segmentforce enough contact between the sperm and oviduct wallto enable all sperm to become bound to the mucosa [Le-febvre et al., 1995b]. The function of the fucosylated mol-ecules on the mucosal surface of the ampulla is notknown.

From these studies, carbohydrate recognition has beenimplicated in the binding of three different species ofsperm to oviductal epithelium [see also Töpfer-Petersen,1999]. In each of these species, a different monosaccha-

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108 Cells Tissues Organs 2001;168:105–112 Suarez

Fig. 3. A Scanning electron micrograph of bull sperm in the isthmic reservoir. The inset illustrates the contactbetween the plasma membrane of the acrosomal region of the sperm with ciliated epithelial cells (micrograph byLefebvre et al.). B A section of the isthmus taken from an estrous heifer (cow), labelled with the fucose lectin UEA-I,conjugated with peroxidase. The arrows indicate the dark precipitate labelling the mucosal surface and cilia. !1,000.C Control, in which labelling was blocked by preincubation of UEA-I with fucose [data from Lefebvre et al., 1997].

ride was most effective at inhibiting binding. These spe-cies differences may not seem so unusual when one con-siders that changing a single amino acid residue in a lectincan alter its carbohydrate ligand-binding specificity [Ko-gan et al., 1995; Revelle et al., 1996; Solis et al., 2001], andthat closely related animal lectins have different carbohy-drate specificities [Weiss, 1994; Gabius 1997; von derLieth et al., 1998].

In the studies described above, the focus was on identi-fying a key, specific monosaccharide involved in spermbinding. The next step to characterizing the binding sitein cattle was to determine how the monosaccharide islinked to the carbohydrate portion of a macromolecule inthe oviduct, whether it be a glycoprotein or glycolipid.Because fucose had been determined to be a key monosac-charide for sperm binding in cattle, the next step was todetermine which linkage of fucose to other sugars is mosteffective at blocking bovine sperm binding to oviductalepithelium. Oligosaccharides containing fucose in variouslinkages were tested for capacity to inhibit sperm bindingto explants in vitro. Fucose in an ·1-4 linkage to N-acetyl-glucosamine, as represented by the trisaccharide Lewis-a,inhibited binding more efficiently than fucose in otherlinkages [Suarez et al., 1998]. It was also more effectivethan fucose alone. Furthermore, Lewis-· tagged by conju-gation to fluorescein-labelled polyacrylamide could be

seen attaching to the heads of motile bovine sperm [Sua-rez et al., 1998]. At this point, it can be concluded thatthere is a carbohydrate-binding molecule on the surface ofbovine sperm that binds fucose, preferentially in an ·1-4linkage to N-acetylglucosamine. The exact identities ofthe carbohydrate-binding molecule on sperm and the cor-responding glycoprotein or glycolipid ligand on the ovi-duct epithelium are yet to be determined.

Binding of bovine sperm to oviductal epithelium isdependent on the presence of Ca2+. In the absence ofextracellular Ca2+, bovine sperm failed to bind to oviduc-tal explants in vitro and they did not bind the Lewis-·label [Suarez et al., 1998]. Other animal cell lectins areknown to be dependent on Ca2+ for binding activity[Weiss, 1994; Gabius, 1997; Kaltner and Stierstorfer,1998], so this sperm lectin may be related to the animal‘C’ or calcium-dependent lectins.

Survival of Sperm during Storage

The oviductal mucosa apparently protects spermagainst aging damage during storage. Such damage is asignificant problem for sperm compared with many othertypes of cells, because sperm lack the machinery for recy-cling plasma membrane lipids. Sperm incubated with ovi-

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Oviductal Sperm Reservoir Cells Tissues Organs 2001;168:105–112 109

ductal epithelium remain viable longer in vitro than whenthey are incubated in medium alone [porcine: Suarez etal., 1991; equine: Ellington et al., 1993; human: Kervan-cioglu et al., 1994] or with tracheal epithelium [bovine:Pollard et al., 1991]. Viability can be extended by incubat-ing sperm with membrane vesicles prepared from the api-cal membranes of oviductal epithelium [rabbit: Smithand Nothnick, 1997; equine: Dobrinski et al., 1997;human: Murray and Smith, 1997], indicating that the epi-thelium can produce at least some of the protective effectby direct contact rather than by products secreted into thelumen. It was reported that equine sperm binding to epi-thelium or membrane vesicles maintain low levels of cyto-plasmic Ca2+, compared to free-swimming sperm, spermattached to Matrigel, or sperm incubated with vesiclesmade from kidney membranes [Dobrinski et al., 1996b,1997]. Equine and human sperm incubated with oviductmembrane vesicles also capacitated more slowly thansperm incubated in capacitating medium alone, whencapacitation was assayed by chlortetracycline fluores-cence patterns [Dobrinski et al., 1997; Murray and Smith,1997]. Possibly, viability is maintained by preventingcapacitation and its concomitant rise in cytoplasmic Ca2+.Identification of the carbohydrate-binding molecule onsperm and its oviductal ligand may provide insight intothe mechanism responsible for prolonging the fertile lifeof sperm in the reservoir.

The Reservoir in Marsupials and PrimitiveEutherians

In marsupial mammals studied so far [Bedford, 1991;Taggart, 1994], sperm are stored in mucosal crypts in theoviduct called ‘sperm storage tubules’. However, in atleast some marsupial species, the sperm do not attach tothe epithelium in the tubules. Many of the sperm in thetubules of the marsupial Sminthopsis crassicaudata wereobserved to be immotile [Bedford and Breed, 1994].Hence, motility suppression may serve to keep sperm inthe tubules until ovulation, particularly when sperm donot bind to the epithelium. In the primitive eutherianmammals, the shrews, some species have been reported topossess distinctive bubble-like outpocketings of the ovi-duct wall in the caudal ampulla. Sperm enter these struc-tures and do not adhere to the epithelium [Bedford et al.,1997a, b]. In more advanced eutherian mammals, thestorage structures are less tubular and less distinctive,being organized as grooves created by folds of mucosa.Adhesion may be more effective at trapping sperm in

these structures. Motility suppression has been observedin the isthmus of rabbits and has been proposed as amechanism of storage [Overstreet and Cooper, 1975;Overstreet et al., 1980; Burkman et al., 1984]; however,sticking of rabbit sperm to oviductal mucosa has also beenobserved [Smith and Nothnick, 1997]. In eutherian spe-cies of hamsters [Smith and Yanagimachi, 1990] andmice [Suarez, 1987], immotile sperm have been observedin the central part of the isthmic lumen; however, in thiscase, it is thought that these sperm are damaged and maynot fertilize [Smith and Yanagimachi, 1990]. Motilitysuppression may or may not be operative in sperm storagein species where sperm binding occurs. It is interesting toconsider that, during evolution of modern eutherianmammals, distinctive storage structures were lost andsperm binding evolved to replace them.

Release from the Reservoir

Little is known about sperm release from the epithe-lium for fertilization. At this point, the available evidenceindicates that the hormonal state of oviductal epitheliumdoes not affect the number of binding sites for sperm onthe epithelium [Suarez et al., 1991; Thomas et al., 1994;Lefebvre et al., 1995b; Baillie et al.,1997]. The density ofbull sperm bound to oviductal explants was similar whenthe explants were taken from preovulatory, postovulatoryor diestrous cows [Lefebvre et al., 1995b]. Instead, it is thestate of the sperm that affects binding. Capacitationinvolves changes in the plasma membrane over the spermhead and, therefore, may release sperm by eliminating ormodifying binding molecules on the head. Hyperactiva-tion may provide the force necessary for overcoming theattraction between the sperm and oviductal epithelium.Smith and Yanagimachi [1991] reported that hamstersperm which had undergone both capacitation and hyper-activation in vitro did not bind to epithelium wheninfused into hamster oviducts. Studying motile spermwithin oviducts of mated mice, we noted that only hyper-activated mouse sperm detached from epithelium [De-Mott and Suarez, 1992]. Pacey et al. [1995] observed thathuman sperm detaching from cultured oviductal epithe-lium were hyperactivated. We found that capacitatingbull sperm in vitro with heparin reduced binding to ovi-ductal epithelium [Lefebvre and Suarez, 1996]. Altogeth-er, these observations indicate that changes in the spermhead surface which occur during the development ofcapacitation and hyperactivation include a loss or inacti-vation of the carbohydrate-binding molecule on the head

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110 Cells Tissues Organs 2001;168:105–112 Suarez

Fig. 4. Mean fluorescence signals from live bull sperm labelled withfuc-BSA-FITC. Live sperm are those which excluded ethidiumhomodimer dye. Means B SD for three replicates, with approximate-ly 10,000 sperm analyzed per sample by flow cytometry. Barslabelled with different letters indicate significant differences betweenthe treatments (p ! 0.05). Inset indicates percentage of live sperm pertreatment. Semen = Unwashed sperm, labelled in whole semen;Wash = sperm washed in TALP medium; Wash + s = washed spermwith seminal plasma added back; capac = washed sperm incubatedunder capacitating conditions; fuc-con = labelling blocked with freefucose; con = labelling of washed sperm with BSA-FITC; cap-con =sperm incubated under capacitating conditions and then labelledwith BSA-FITC.

of the sperm. As binding affinity for the oviductal epithe-lium decreases, hyperactivation can provide sperm withincreased tugging force to aid in detachment.

There is evidence that this loss of binding affinity ofsperm for oviductal epithelium can be traced to a loss ofcarbohydrate-binding affinity. Fresh hamster sperm, re-trieved from the caudal epididymis, labelled over theacrosomal region with fetuin conjugated to colloidal gold(fig. 1). When the sperm were incubated under capacitat-ing conditions until hyperactivation was observed invitro, they no longer labelled over the acrosomal cap withthe fetuin probe. Labelling of a few bands of protein bythe fetuin gold was significantly more intense in mem-brane extracts of uncapacitated sperm than in extracts ofcapacitated sperm [DeMott et al., 1995]. Revah et al.[2000] used bovine serum albumin conjugated to fluores-cein and fucose (fuc-BSA-FITC) as a fluorescent markerfor the fucose-binding capacity of bull sperm. Freshlyejaculated sperm, when washed free of seminal plasmaand incubated with the probe, quickly showed fluores-

cence over the acrosomal region. This is the region bywhich bull sperm attach to oviductal epithelium. Whenflow cytometry was used to monitor binding of fuc-BSA-FITC in samples of sperm, the fluorescence signals weresignificantly reduced in samples of bull sperm that hadbeen incubated under capacitating conditions (fig. 4), i.e.in TALP medium containing 10 Ìg/ml of heparin [Parrishet al., 1988]. During this study, it was also observed thatseminal plasma blocked binding of fuc-BSA-FITC tosperm. It may be that components of seminal plasma con-tain fucose and thereby compete for the binding site withfuc-BSA-FITC, or that the fucose-binding molecule isexpressed in a soluble form in seminal plasma that com-petes with the membrane-bound form for the fuc-BSA-FITC.

Although the role of the epithelium in sperm releasedoes not appear to involve reduction of expression of car-bohydrate ligand, epithelium could instigate sperm re-lease by secreting initiators of capacitation and/or hyper-activation. Soluble oviductal factors have been shown toenhance capacitation of bull sperm [Chian et al., 1995;Mahmoud and Parrish, 1996]. Thus, hormones whoselevels rise before ovulation could stimulate secretion ofinitiators of capacitation and hyperactivation.

Conclusion

Current evidence supports the following scenario forfunctioning of the sperm reservoir in the oviduct. There isa specific interaction between sperm and epithelium, inwhich a carbohydrate-binding molecule on sperm bindsto a specific carbohydrate moiety on the epithelium.While bound, the viability of sperm is maintained andcapacitation is suppressed. Signals associated with im-pending ovulation could release sperm for fertilization byinitiating capacitation, which brings about removal orinactivation of the binding molecule on the sperm plasmamembrane. Hyperactivation could provide extra pullingforce for detachment. Identifying the molecules on spermand epithelium that are involved in carbohydrate-me-diated sperm binding would elucidate how sperm aremaintained in a fertile state in the reservoir.

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