Fucose, Mannose and -N-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm Acrosome Reaction Through Convergent Signaling ath!

ays
Linghui Wu and Nicole S. Sampson*
Department of Chemistry, Stony Brook Uni ersity, Stony Brook, N! ""#$%&'%(( KEYWORDS: sperm acrosome exocytosis, sperm acrosome reaction, glycopolymer, ROMP
A"STRACT# )he sperm acrosome reaction *+,-, an essential e.ocytosis step in mammalian fertili/ation, is mediated 0y a species&specific interaction of sperm surface molecules 1ith glycans on the egg. 2re ious studies indicate that a su0set of terminal car0ohydrates on the mouse egg /ona pellucida *32- trigger the +, 0y cross&linking or aggregating receptors on the sperm mem0rane. 4o1e er, the e.act role of those car0ohydrates in +, has not 0een identified and the mechanism underlying the +, still needs further in estigation. )o study this process, a series of glycopolymers 1as synthesi/ed. )he glycopolymers are composed of a multi alent scaffold *nor0ornene-, a functional ligand *pre iously identified 32 terminal monosaccharides-, and a linker connecting the ligand and the scaffold. )he polymers 1ere tested for their a0ility to initiate +, and through 1hich signaling path1ays +, induction occurred. 5ur data demonstrate that mannose, fucose, and 6&N&acetylglucosamine "(&mers and "((& mers initiate +, in a dose&dependent manner, and the "((&mers are more potent on a per monomer 0asis than the "(&mers. +lthough nearly e7uipotent in inducing the +, at the optimal concentrations, their +, acti ation kinetics are not identical. Similar to mouse 32', all "((&mer&acti ated +, are sensiti e to guanine&0inding regulatory proteins *8&proteins-, tyrosine kinase, protein kinase +, protein kinase C and Ca9: related antagonists. )hus, the chemotypes of synthetic glycopolymers imitate the physiologic +,&acti ation agents and pro ide e idence that occupation of one of at least three different receptor 0inding sites is sufficient to initiate the +,.

;ammalian fertili/ation in ol es a series of sophisticated steps. <t starts 1ith sperm&egg 0inding, sperm penetration of egg outer mem0rane=/ona pellucida *32-, and finally sperm&egg fusion to form an em0ryo." <n the mouse, the 32 consists of three glycoproteins *32", 329, and 32'- that form a cross&linked e.tracellular matri. surrounding the egg. >ach glycoprotein contains glycans co alently linked to asparagine *N&linked- and?or serine and threonine *5&linked-.9 2re ious studies indicated that the adhesion of sperm to the egg is mainly mediated 0y a specific interaction of sperm surface molecules 1ith the glycan moieties on the 32. '&@ 8lycosidase digestion and mass spectrometric analyses ha e indicated that se eral mouse 32' terminal monosaccharide residues, including 6&N& acetylgalactosamine *8alN+c-,A 6&N& acetylglucosamine *8lcN+c-,# mannose,B galactose,$ and sialyl"( may 0e critical for sperm 0inding to the 32. +lthough a fucosylated glycan 1as identified on human 32"" and the addition of

a fucose residue to the 8lcN+c and galactose trisaccharides enhances mouse sperm&egg 0inding affinity"9, the participation of fucose in mouse gamete 0inding is not definiti e. )he comple. heterogeneity of /ona glycosides makes definition of the e.act function of these sugars a challenge. )o penetrate the thick 32 layer, sperm need to undergo a cellular e.ocytosis kno1n as the acrosome e.ocytosis or acrosome reaction *+,-. Structurally, the acrosome is composed of a mem0rane esicle filled 1ith solu0le components and acrosomal matri. proteins.9 When the +, occurs *Cigure "-, the sperm plasma mem0rane fuses 1ith the outer acrosomal mem0raneD the solu0le contents in the esicle e.g., hydrolases and proteases, are se7uentially released."' )he acrosome reaction plays an essential role in fertili/ation and only acrosome&reacted sperm can participate in the follo1ing fertili/ation steps 1hich lead to sperm&egg fusion."%

+ num0er of physiological and non&physiological agonists can acti ate the +,. )hough contro ersial, 32' 1as 1idely accepted as the primary +, acti ator. ;ultiple sperm&& car0ohydrate 0inding e ents occur to stimulate the +, 0y cross&linking or aggregating receptors on the sperm plasma mem0rane. )his hypothesis is 0ased on inhi0ition and acti ation studies 1ith deglycosylated 32 and fragmented 32. )o elucidate the role of indi idual car0ohydrates in initiating the +,, acti ity studies of neoglycoconEugates ha e 0een undertaken. Loeser et al. "# e.amined the properties of se eral neoglycoproteins that are 0o ine serum al0umin *BS+- conEugates 1ith an a erage of B copies of the glycan of interest per protein molecule. )hey concluded that mannose&BS+, 8lcN+c&BS+, and 8alN+c&BS+ could initiate the +, 1hile glucose& BS+ and galactose&BS+ had no effect. ;oreo er, unconEugated sugars failed to 0lock neoglycoprotein&induced +, suggesting that a scaffold to display the sugars is necessary. Later, 4anna et al."B found that Le1is F *8al6%GCucH'I8lcN+cand Le1is + *8al6'GCucH%I8lcN+c- 1hen conEugated to BS+ could initiate the +,, and Le1isF&BS+ 1as more potent than Le1is+&BS+. )his finding indicated that fucose may 0e another glycan ligand in ol ed in +, initiation in addition to the three monosaccharides identified 0y Loeser et al.17 4o1e er, 4anna et al."B did not o0ser e +, acti ation 1ith 8lcN+c&BS+ in contrast to the results of Loeser et al."# <nterpretation is difficult as little is kno1n a0out the spatial display of the saccharides on the BS+. ;oreo er, fucosyl 0ioconEugates 1ere not directly studied. ,ecent e.periments ha e further challenged the proposed roles of some glycan residues in the +, and the concept of 32' as the +, stimulus. 5ocyte&specific deletion of 0oth the &",'& galactosyl transferase * !syn- and the mannoside acetylglucosaminyltransferase " *Mgat"- genes generated mouse eggs lacking core&"&deri ed 5& glycans and 1ith modified N&glycans lacking terminal 8al and 8lcN+c residues."$ )hese mice, in 1hich 5&linked or N&linked galactose and 8lcN+c 1ere genetically deleted from the 32 are still fertile, suggesting these t1o monosaccharides are genetically unessential."$ 4o1e er, the knock&out mice display considera0ly lo1er fecundity compared to 1ild& type mice, and the remaining glycans on the 32, for e.ample, mannose or 8alN+c may still contri0ute to fertili/ation. + model through 1hich sperm induce acrosome e.ocytosis 0y mechanosensory signal transduction 1ithout re7uiring sperm 0inding to the 32 1as also reported,9( 0ut it is difficult to imagine ho1 mechanical signals can 0e transmitted 1ithout molecular 0inding interactions 0et1een sperm and the egg surface. <t has also 0een suggested

Figure (' 5 er ie1 of mouse sperm acrosome reaction. Figure &' Structures of glycopolymer pro0es.

that most fertili/ing sperm 0egin acrosomal e.ocytosis 0efore 0inding to the 32 through molecular interactions 0et1een sperm and the female reproducti e tract. <t appears that there are multiple interactions that lead to the +,, most likely in ol ing car0ohydrates. )o 1hat e.tent signaling is initiated through a single type of receptor&ligand interaction ersus multiple types of interactions is poorly understood. +lthough the signaling path1ays initiating the +, are not completely understood, considera0le progress in understanding do1nstream e ents has 0een made. +t minimum, the +, is triggered 0y a cascade of signal e ents including acti ation of 8&proteins, protein phosphorylation, and ele ation of intracellular calcium and p4 le els.9@& 9# 4o1e er, the identity of the cell surface receptor or receptors that initiates these signals is unkno1n, despite the identification of se eral candidates.9A Contro ersy a0out the mechanism of the +, calls for a modified strategy to elucidate the molecular players in this complicated process. We hypothesi/ed that synthetic glycopolymers 1ill pro ide further insights into the molecular comple.ity of sperm +, acti ation.9B )he length, su0stitution and ligand density of glycopolymers can 0e easily controlled. )hus, 1e applied neoglycopolymers prepared 0y ring opening metathesis polymeri/ation *,5;2to in estigation of the +,. We further e.amined the glycopolymer&acti ated +, in the a0sence and presence of esta0lished pharmacological agents kno1n to pre ent the +, 0y 0locking specific signaling path1ays. 5ur data demonstrate that synthetic mannose, fucose and 8lcN+c polymers acti ate mouse acrosome reaction through con ergent signaling path1ays. ,>SUL)S +ND D<SCUSS<5N $esign and preparation o% glycopolymers . Synthetic polymeric pro0es pro ide a ersatile strategy to in estigate ligand&receptor interactions. )heir design, 1hich is simple and fle.i0le, re7uires a multi alent scaffold, a minimal ligand and a spacer to link the ligand to the

scaffold. +ll of the 32 monosaccharides proposed to 0e in ol ed in the +,J mannose, fucose, 8lcN+c and 8alN+c, 1ere chosen as ligands. +lthough Loeser et al.17 demonstrated that galactose&BS+ and glucose&BS+ did not induce +, and glucose is not present on the 32,'" the properties of nor0ornene&deri ed galactose and glucose polymers 1ere still tested to confirm these results. 2re iously utili/ed linkers are ' or "% atoms long, ho1e er their structures are proprietary. <n this 1ork, 1e designed a simple ethyl amide linker to connect the nor0ornene& deri ed 0ack0one and the monosaccharide ligand. Nor0ornene *NB- ser ed as the scaffold in our 1ork due to its high rigidity and 1idespread adoption. )he desired polymers 1ere generated 0y ,5;2 that affords polymers 1ith defined lengths and narro1 molecular mass distri0utions. ;oreo er, the density of ligands on nor0ornyl ,5;2 polymers fa ors clustering receptors and acti ating signaling transduction.'% 4ence, a series of homogenous glycopolymers 1ith t1o different a erage lengths *"(&mer and "((&mer*Cigure 9- 1ere synthesi/ed *Supplementary Schemes "&#- and tested to determine the 0etter scaffold length for acti ating the +, 1ith monosaccharide ligands. )%%ect o% homoglycopolymers on acrosome reaction' We e.amined the effect of homoglycopolymers on the sperm +, 0y sperm immunofluorescence assay *Supplementary Cigure "-. <n the assay, sperm 1ere capacitated 0y treatment 1ith (.'K 0o ine serum al0umin *BS+-, 1hich is kno1n to facilitate sperm capacitation 0y altering fatty acids and?or cholesterol on the sperm plasma mem0rane. Calcium ionophore +9'"B#, a kno1n sperm +, stimulus, 1as used as a positi e control instead of 32, 0ecause the role of 32 in +, acti ation is contro ersial and the calcium concentration has 0een demonstrated to 0e essential to acti ate the +,. Since all polymer samples 1ere prepared in phosphate 0uffered saline *2BS-, sperm treated 1ith 2BS 1as used as a negati e control. Sperm samples 1ere incu0ated 1ith controls and glycopolymers at arying concentrations for '( minutes and then stained 1ith rhodamine=la0eled peanut agglutinin *2N+-. 2N+, 1hich has 0een identified to 0ind 0oth the outer acrosomal mem0rane and certain components of the acrosomal matri., is 1idely used to discriminate 0et1een acrosome&intact and acrosome&reacted sperm.'@

Figure *' +cti ation of sperm acrosome reaction 0y homoglycopolymers. Capacitated sperm 1ere incu0ated 1ith glycopolymers at different concentrations *sho1n as polymer concentration-. a- "(&mers. 0- "((&mers. )he a erage acrosome reaction percentage *+,K- of glycopolymer treated sperm 1ere normali/ed using G+,K*glycopolymers- = +,K*negati e control-I?G+,K *positi e control- = +,K*negati e control-I. )he a erage +,K for the positi e control, +9'"B#=treated *@ M;- sperm, 1as 9%K and for the negati e control, 2BS=treated sperm, 1as "(K. Data represent mean N S>; of at least three independent e.periments. * pO (.(@ 1hen compared to the negati e control.

+ significantly greater num0er of sperm undergo the +, 1hen treated 1ith "(( L; poly*;an-"(, poly*Cuc-"( or poly*8lcN+c-"( than 1ith the other three glycopolymers *Cigure 'a-. )he acti ation of the +, 0y these three glycopolymers is steeply dose dependentD at a 9& fold lo1er concentration, the +, is not acti ated. Sperm samples treated 1ith a 9&fold higher concentration *9(( L;- 1ere +, acti ated 1ith a lo1er efficiency or no efficacy. )hese data suggest that at high polymer concentrations, multi alent 0inding and thus the clustering effect

is not fa ored.'% Neither poly*8lc-"(, poly*8al-"( nor poly*8alN+c-"( triggered the +, at concentrations of "(( or 9(( L;. <nduction of the +, 0y poly*Cuc-"( is not as effecti e as 1ith poly*;an-"( or poly*8lcN+c-"(. Similarly, dose& dependent +, initiation 1as o0ser ed 1hen sperm 1ere incu0ated 1ith poly*;an-"((, poly*Cuc-"(( and poly*8lcN+c-"((, 0ut not poly*8lc-"(( and poly*8al-"(( and poly*8alN+c-"(( *Cigure '0-. )here is no statistically significant difference 0et1een the efficacy of mannose polymers and 8lcN+c polymers. +gain, the +, initiation efficacy of poly*Cuc-"(( is lo1er than for poly*;an-"((, and poly*8lcN+c-"((, consistent 1ith the lo1ered efficacy of poly*Cuc-"(. )aken together, these results indicate that galactose and 8alN+c may play a role in sperm&egg 0inding 0ut not in acti ation of the +,. ;annose, 8lcN+c and fucose function as sperm +, acti ators, 0ut fucose is less efficient at +, acti ation. )he concentrations reported here are in polymer concentrations. <f the 0ulk concentration of glycan ligand utili/ed is considered in comparing the efficacies of "(&mers ersus "((&mers, 1e o0ser e that the "((&mers are more potent. Sperm did not undergo the +, 1hen incu0ated 1ith the "(&mers at @(( L; ligand concentration*@( L; polymer- *Cigure 'a-, 1hereas @(( L; poly*;an-"((, poly*Cuc-"(( and poly*8lcN+c-"(( *@ L; polymer- *Cigure '0successfully initiated the +,. )he higher potency of the "((&mers further confirms that the polymers stimulate the +, through a multi alent interaction 1ith sperm.

Figure , Comparison of mi.ed "((&mers and the corresponding single "((&mers. a"((&mers paired at "( L; each. 0- "((&mers paired at 9.@ L; each. )he concentration sho1n in the chart is polymer concentration. )he a erage +,K of glycopolymer treated sperm 1ere normali/ed using G+,K*glycopolymers- = +,K *negati e control-I?G+,K*positi e control- = +,K*negati e control-I. )he a erage +,K for the positi e control, +9'"B#=treated *@ M;- sperm, 1as 9%K and for the negati e control, 2oly*8lc-"((=treated *"( M;- sperm, 1as ""K. Data represent mean N S>; of at least three independent e.periments. * pO (.(@ 1hen compared to the corresponding single "((&mers.

)%%ect o% pairs o% (++-mers on AR' Ne.t, 1e paired the acti e "((&mers poly*;an-"((, poly*Cuc-"((, and poly*8lcN+c-"(( at their optimal *"( L;- and at much lo1er concentrations *9.@ L;- to e.amine the effect of inducing the +, simultaneously 1ith t1o different ligands. +s glucose has not 0een identified on 32 and poly*8lc-"(( had no +, acti ation a0ility, poly*8lc-"(( 1as used as a negati e control. We sa1 no further increase in the amount of sperm +, comparing the polymer pairs and single glycopolymers at their optimal concentrationsD

glycopolymer&receptor 0inding and signal transduction, and that the three sugars act independently to acti ate the +,. We also used dynamic light scattering to in estigate 1hether polymer aggregation, 1hich could interfere 1ith sperm acti ation, had occurred. No aggregation 1as o0ser ed *data not sho1n-. )hese results together suggest that ma.imal sperm +, is achie ed upon treatment 1ith a single homopolymer at its optimal concentration *Cigure %a-, and that the car0ohydrate ligands 0ind to different receptors on the sperm *Cigure %0-.

Figure -' poly*Cuc-"((, poly*;an-"(( and poly*8lcN+c-"(( ha e different +, acti ation rates. )he concentration sho1n in the chart is polymer concentration. )he a erage +,K of glycopolymer treated sperm 1ere normali/ed using G+,K*glycopolymers= +,K*negati e control-I?G+,K*positi e control- = +,K*negati e control-I. )he a erage +,K for the positi e control, +9'"B#&treated *@ M;- sperm at %@ min 1as ''K and for the negati e control, poly*8lc-"((&treated *"( M;sperm at "@ min, 1as $K. Data represent mean N S>; of at least three independent e.periments. * pO (.(@ 1hen compared to the +,K of poly*8lc-"(( at each time point .

the efficacy remained at "((K of the positi e control *Cigure %a-. ;i.tures of three glycopolymers at their optimal concentrations 1ere also tested, 0ut no significant differences in +, percentage 0et1een a pair and a mi.ture of three 1ere o0ser ed. +lthough poly*8lcN+c-"(( and poly*;an-"(( had similar dose&dependent +, acti ating patterns *Cigure '0-D poly*8lcN+c-"(( 1as more effecti e than poly*;an-"(( at 9.@ L; *Cigure %0-. 2oly*8lcN+c-"(( and poly*;an-"(( paired at 9.@ L; each sho1ed a slight enhancement in +, acti ation compared to poly*8lcN+c-"(( at 9.@ L;, yet the mi.ture did not acti ate +, to the same le el as @ L; of a poly*8lcN+c- "(( or poly*;an-"(( *Cigure %0-. )his result suggests that the t1o car0ohydrate ligands 0ind to different receptors on the sperm. Sperm samples treated 1ith the other t1o com0inations of acti ating polymer *9.@ L; eachsho1ed efficacies e7ual to treatment 1ith a single polymer at 9.@ L;. <n addition, the pair1ise mi.tures *9.@ L; each- 1ere less effecti e acti ators of +, than a single polymer at @ L;, 1hich is e7ual to the total concentration of polymer in the paired mi.ture. )he data suggest that there is a concentration threshold for

Figure 0' )he signaling path1ays of +, acti ation 0y the three effecti e glycopolymers are similar. )GTAJ ethylene glycol tetraacetic acid, e.tracellular Ca9: inhi0itor. erJ pertussis to.in, 8&protein inhi0itor. AmiJ amiloride hydrochloride, )&type Ca9: channel inhi0itor. 123J protein kinase + inhi0itor. GenJ genistein, protein tyrosine kinase inhi0itor. Ni%J nifedipine, L&type Ca9: channel inhi0itor. CheJ chelerylthrine, protein kinase C inhi0itor. )he a erage inhi0ition percentage of inhi0itor and glycopolymer treated sperm 1ere normali/ed using G+,K*positi e control- = +,K*glycopolymers-I ?G+,K *positi e control- = +,K*negati e control-I. )he a erage +,K for the positi e control, +9'"B#&treated *@ M;sperm, 1as 9%K, and for the negati e control, inhi0itor&treated sperm, 1as $& "'K. Data represent mean N S>; of three independent e.periments.

.inetics o% AR induced /y (++-mers' We further studied the effects of the three acti e "((&mers on the +, as they 1ere more potent than the "(& mers. )he time courses for the three "((&mers 1ere monitored in parallel for %@ minutes *Cigure @-. Data for longer incu0ation periods 1ere not included due to high +, in the negati e control and reduced sperm ia0ility. +t the three time points, the e.tent of +, in the positi e control and the poly*;an-"(( and poly*8lcN+c-"(( treated samples is the same. 4o1e er, poly*Cuc-"(( induced lo1er le els of the +, than poly*;an-"(( and poly*8lcN+c-"(( at '( or %@ minutes, and there 1as no initiation after "@ minutes. )hese data indicate that the induction of the +, 0y poly*;an-"(( and poly*8lcN+c-"(( is more rapid than 0y poly*Cuc-"((, 1hich is in agreement 1ith the pre ious o0ser ation that poly*;an-"(( and poly*8lcN+c-"(( is more effecti e than poly*Cuc-"((. )he reason for the slo1er +, acti ation kinetics of poly*Cuc-"(( compared to the other t1o "((&mers is unclear. 5ne possi0ility is that differences in glycopolymer conformations

arising from su0stitution 1ith different monosaccharides affect efficacy of signal transduction. Note that the +, in the negati e control increased sharply at %@ minutes due to spontaneous +,. We selected '( minutes for all of our endpoint assays 0ecause the le el of spontaneous +, 1as much lo1er. Signaling ath!ay o% Glycopolymers Induced AR' We e.amined 1hich signaling transduction e ents are acti ated 0y +,&acti e glycopolymers using 1ell characteri/ed inhi0itors for protein kinase + *2P+-, protein kinase C *2PC-, protein tyrosine kinase *2)P-, 8&protein, )&type?L&type Ca 9: channels and e.tracellular Ca9:. )hese signaling molecules and channels ha e 0een detected in 0oth mouse and human sperm and ha e 0een suggested to play an important role in 0oth the mouse and the human 32&induced acrosome reaction. )he inhi0itors do not ha e a0solute specificityD they 0lock +, acti ation non& selecti ely at high concentrations. )he doses of the inhi0itors 1ere carefully chosen in order to not affect sperm ia0ility and motility. Con ersely, inhi0itors do not completely a0olish the spontaneous +,. +ll se en inhi0itors significantly suppressed poly*;an-"((&, poly*8lcN+c-"((& and poly*Cuc-"((& acti ated +, 1ith at least A(K inhi0ition *Cigure A-. +s the three glycopolymers all acti ate +,, it is not surprising to find that they share signaling path1ays in common. )hese results demonstrate that poly*;an-"((, poly*8lcN+c-"((, and poly*Cuc-"(( acti ate the +, though con ergent signaling path1ays. Sperm treated 1ith inhi0itors alone also sho1 a lo1 +, percentage consistent 1ith a lo1 le el of spontaneous +, that is independent of these path1ays. )he precise sperm +, signaling path1ays are not completely elucidated, though se eral tentati e signaling path1ay mechanisms of 32& initiated +, ha e 0een proposed. <n these mechanisms, 32 is thought to 0ind to at least t1o receptors on the sperm mem0rane. 5ne is a 8& protein coupled receptor, 1hich is reported to regulate adenylyl cyclase and acti ate 2P+, phospholipase C *2LC- 6" and 4: efflu.. Upon acti ation, 2P+ phosphorylates and further triggers do1nstream factors. )he other is a 2)P receptor, 1hich is suggested to trigger a sperm Na:?4: e.changer promoting cell alkalini/ation, mem0rane depolari/ation, and )&type and L&type calcium channels acti ation on the sperm plasma mem0rane. )he calcium channels play ital roles in ele ating intracellular Ca9: and p4 preceding the +,. 8&protein and 2)P can also acti ate 2PC, 1hich mediates calcium entry into the sperm cytosol from intracellular stores. )hese signaling factors all lead to an increase in cytosolic calcium, resulting in the fusion of sperm plasma mem0rane and the outer acrosomal mem0rane, and e entually the +,.

Ta/le (' Comparison of signaling path1ays initiated 0y different acti ators.

Signaling molecule AR activator BSA-manno e! Gal"Ac!Glc"Aca BSA-Le#i $d Extracellular Ca2+ T-type Ca2+ L-type Ca2+ PKC PKA PTK G protein

+ + + + + +

"A "A

"A "A

"A "A

+ + + +

+ + + + + +

"Ae "A

BSA-Le#i Af

%ou e &Pg

+ + + +

+ + + +

+ + + +

+ + + +

poly'%an()**

poly'Glc"Ac()**

poly'+uc()**

Data from ref.'# "=, Signaling path1ay is not acti ated. c:, Signaling path1ay is acti ated. #Data from ref."B eN+, not a aila0le, no signaling path1ay e.periment 1as performed. $Data from ref."B gData from ref.'A +ll of the a0o e&descri0ed signaling factors are in ol ed in acti ation of the +, 0y poly*;an- "((, poly*8lcN+c-"((, and poly*Cuc-"((. ;oreo er, these homopolymers acti ate through the same path1ays as mouse 32 *)a0le "-. 5ur data strongly support that glycopolymer&acti ated +, is analogous to 32&acti ated +, and that these glycopolymers are suita0le mimics of the 32 and?or other physiologic ligands for acti ating sperm +,. 5ur 1ork emphasi/es the high redundancy of car0ohydrate ligands that can 0e used to acti ate the +,. <n contrast to conditional genetic knockouts for 1hich no +, phenotypes 1ere o0ser ed, the use of glycopolymers has ena0led the identification of 1hich terminal car0ohydrates are important for the +,. )he glycopolymer chemotypes o0ser ed in this 1ork suggest that at least three different sperm receptor 0inding sites can 0e utili/ed to initiate the +, in mouse. +fter receptor acti ation 0y glycopolymer, signaling con erges onto the same path1ays intracellularly. )he receptors acti ated 0y poly*;an- "((, poly*8lcN+c-"((, and poly*Cuc-"(( ha e not 0een definiti ely identified. None of the large num0er of egg 0inding receptors proposed and characteri/ed has 0een demonstrated to 0e essential.%(&%9 2re ious results also suggested a high le el of redundancy, 0ut 1hether multiple egg 0inding receptors acted indi idually or as a multi&protein comple. 1as unclear. 5ur results fa or the single ligand&receptor interaction model and pro ide further e idence that induction of the sperm acrosome reaction proceeds through duplicati e sperm&egg interactions. C5NCLUS<5N + multi alent display of mannose, 8lcN+c, or fucose triggers the sperm acrosome reaction in a concentration&dependent manner, and high alency polymers *"(( ligands- are more effecti e than lo1 alency polymers *"( ligands-. +lthough fucose sho1ed lo1er +, acti ation potency and the kinetics of fucose&induced +, are distinct from those of mannose or 8lcN+c&induced +,, all the glycopolymers rely on 8&proteins, protein kinase C, protein kinase +, e.tracellular Ca9:, L& and )&type Ca9: channels, and a protein tyrosine kinase. )he three single ligand&receptor interactions all induce the acrosome&reaction and are functionally e7ui alent, 0ut they are redundant. )hus, the chemotypes of ,5;2& deri ed glycopolymers mimic the 0iological function of physiologic +,&acti ation agents and pro ide e idence that occupation of one of at least three different receptor 0inding sites is sufficient to initiate the +,. ;>)45DS General Methods and Materials' +ll e.periments performed 1ith mice 1ere in accordance 1ith the National <nstitute of 4ealth and United States Department of +griculture guidelines, and the specific procedures performed 1ere appro ed 0y the Stony Brook Uni ersity <+CUC *protocol (A"A-. Chemicals for assay 0uffers 1ere purchased from Sigma&+ldrich, Cisher Scientific, and QW,. Sperm Treatment' Sperm 1ere isolated from the cauda epididymis of t1o "( to "9&1eek&old <C, male 0reeders *)aconic- in ;"A medium *A mLsupplemented 1ith (.'K BS+ *1? -. )he sperm suspension 1as then gently pipetted into a polypropylene culture tu0e *"9 R #@ mm- and incu0ated at '# SC for '( min under @K C5 9 * ? in air. 5nce the incu0ation 1as complete, the sperm motility 1as e.amined 0y phase&contrast microscopy *9( R ? (.@ air-. 5nly samples of capacitated sperm displaying TB(K motility 1ere

used in su0se7uent e.periments. )he concentration of sperm 1as assessed 0y hemocytometer. +li7uots *9( LL- containing a0out @ R "(@ capacitated sperm 1ere transferred to microcentrifuge tu0es and incu0ated 1ith glycopolymers, negati e, and positi e controls at '# SC under @K C59 for the specified time *"@, '( or %@ min-. )he positi e control used in our assay is a 1ell&studied Ca 9: transporting ionophore +9'"B# *Sigma&+ldrich-. +fter incu0ation, the sperm 1ere pelleted 0y centrifugation at @(( g for A min. )he supernatant 1as remo ed and the pelleted sperm 1ere 1ashed once 1ith %( LL 2BS and fi.ed 1ith %( LL #(K ethanol. +fter fi.ing at % SC for '( min, the sperm 1ere pelleted and 1ashed t1ice 1ith 2BS. )he final pellet 1as resuspended in %( LL of DD< 1ater. +li7uots *"( LL- of each sample 1ere transferred to co er slips and air&dried. Assessment o% Sperm Acrosome Reaction' "( LL ,hodamine la0eled peanut agglutinin *2N+*Qector la0s- at a concentration of 9( Lg mL &" 1as incu0ated 1ith fi.ed sperm *from 1hich polymers had 0een remo ed 0y e.tensi e 1ashing- on co er slips for "( min at rt. +fter 1ashing 1ith 9 mL DD< 1ater *t1o times "( min-, the co er slips 1ere mounted on SuperCrost 2lus microscope slides *Cisher Scientific- o er a drop *A LL- of mounting medium Qectashield *Qector la0s-, sealed 1ith nail polish, and the acrosomal status 1as assessed 0y in erse fluorescence microscopy *3eiss-. Sperm that displayed continuous red fluorescence along their acrosomal arcs 1ere scored as acrosome&intactD those that displayed no red or punctuate fluorescence 1ere scored as acrosome&reacted. )he slides 1ere coded and counted 0lindlyD all e.periments 1ere conducted at least three times. >ach time, three independent replicates of each test group 1ere analy/ed, and 9(( sperm from each replicate 1ere counted. Signaling ath!ay Inhi/ition Assay' <nhi0itor stock solutions 1ere prepared 0y dissol ing the reagents either in distilled 1ater *pertussis to.inor in D;S5. +li7uots of the stock solutions 1ere mi.ed 1ith the capacitated sperm solution to achie e the desired concentration, and pre& incu0ated for @ min 0efore treatment 1ith polymers or +9'"B#. )he concentrations of inhi0itors 1ere chosen 0ased on references and to.icity tests. )he acrosomal status of sperm 1as e aluated as descri0ed a0o e. Statistical Analysis. Comparisons of the a erage alues for the control and e.perimental groups 1ere carried out 0y a paired t1o&tailed t&test to determine statistically significant differences *p O(.(@-. )he results are presented as mean N S>;. Supporting in%ormation

Supporting information including all synthesis scheme and e.perimental procedures is a aila0le free of charge ia the <nternet at httpJ??pu0s.acs.org.

A4T15R INF5RMATI5N
Corresponding Author 6 nicole'sampson7stony/roo8'edu Author Contri/utions
)he manuscript 1as 1ritten through contri0utions of all authors. +ll authors ha e gi en appro al to the final ersion of the manuscript.

Funding Sources
N<4 & ,("8;($#$#"?,("4D'B@"$ *N.S.S.-.

AC.N59:)$GM)NT
We thank Brookha en <nstruments *Brookha en, N!for the dynamic light scattering soft1are.

A""R);IATI5NS
+,, acrosome reactionD 32, /ona pellucidaD 32', /ona pelucida protein 'D NB, nor0orneneD 8alN+c, 6&N&acetylgalactosamine, 8lcN+c, 6&N& acetylglucosamineD ;an, mannoseD 8lc, glucose, 8al, galactoseD Cuc, fucoseD BS+, 0o ine serum al0uminD 2P+, protein kinase +D 2PC, protein kinase CD 2)P, protein tyrosine kinase. ,>C>,>NC>SJ
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<nsert )a0le of Contents art1ork here