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

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    Fucose, Mannose and -N-Acetylglucosamine Glycopolymers

    Initiate the Mouse Sperm Acrosome Reaction ThroughConvergent Signaling ath!ays

    Linghui Wu and Nicole S. Sampson*

    Department of Chemistry, Stony Brook Uniersity, Stony Brook, N! ""#$%&'%((

    KEYWORDS: sperm acrosome exocytosis, sperm acrosome reaction, glycopolymer, ROMP

    A"STRACT#)he sperm acrosome reaction +-, an essential eocytosis step in mammalian fertili/ation, ismediated 0y a species&specific interaction of sperm surface molecules 1ith glycans on the egg. 2reious

    studies indicate that a su0set of terminal car0ohydrates on the mouse egg /ona pellucida 32- triggerthe + 0y cross&linking or aggregating receptors on the sperm mem0rane. 4o1eer, the eact role ofthose car0ohydrates in + has not 0een identified and the mechanism underlying the + still needsfurther inestigation. )o study this process, a series of glycopolymers 1as synthesi/ed. )heglycopolymers are composed of a multialent scaffold nor0ornene-, a functional ligand preiouslyidentified 32 terminal monosaccharides-, and a linker connecting the ligand and the scaffold. )hepolymers 1ere tested for their a0ility to initiate + and through 1hich signaling path1ays + inductionoccurred. 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 monomer0asis than the "(&mers. +lthough nearly e7uipotent in inducing the + at the optimal concentrations,their + actiation kinetics are not identical. Similar to mouse 32', all "((&mer&actiated + aresensitie to guanine&0inding regulatory proteins 8&proteins-, tyrosine kinase, protein kinase +, proteinkinase C and Ca9: related antagonists. )hus, the chemotypes of synthetic glycopolymers imitate thephysiologic +&actiation agents and proide eidence that occupation of one of at least three different

    receptor 0inding sites is sufficient to initiate the +.

    ;ammalian fertili/ation inoles a series ofsophisticated steps.

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    + num0er of physiological and non&physiologicalagonists can actiate the +. )houghcontroersial, 32' 1as 1idely accepted as theprimary + actiator. ;ultiple sperm&&car0ohydrate 0inding eents occur to stimulatethe + 0y cross&linking or aggregating receptorson the sperm plasma mem0rane. )his hypothesisis 0ased on inhi0ition and actiation studies 1ithdeglycosylated 32 and fragmented 32.

    )o elucidate the role of indiidual car0ohydrates

    in initiating the +, actiity studies ofneoglycoconEugates hae 0een undertaken.

    Loeser et al."#eamined the properties of seeralneoglycoproteins that are 0oine serum al0uminBS+- conEugates 1ith an aerage of copies ofthe glycan of interest per protein molecule. )heyconcluded that mannose&BS+, 8lcN+c&BS+, and8alN+c&BS+ could initiate the + 1hile glucose&BS+ and galactose&BS+ had no effect. ;oreoer,unconEugated sugars failed to 0lockneoglycoprotein&induced + suggesting that ascaffold to display the sugars is necessary. Later,

    4anna et al." found that Le1is F

    8al6%GucH'I8lcN+c- and Le1is +8al6'GucH%I8lcN+c- 1hen conEugated to BS+could initiate the +, and Le1isF&BS+ 1as morepotent than Le1is+&BS+. )his finding indicatedthat fucose may 0e another glycan ligandinoled in + initiation in addition to the three

    monosaccharides identified 0y Loeser et al.17

    4o1eer, 4anna et al." did not o0sere +actiation 1ith 8lcN+c&BS+ in contrast to the

    results of Loeser et al."#

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    scaffold. +ll of the 32 monosaccharides proposedto 0e inoled in the +J mannose, fucose,8lcN+c and 8alN+c, 1ere chosen as ligands.

    +lthough Loeser et al.17 demonstrated thatgalactose&BS+ and glucose&BS+ did not induce +and glucose is not present on the 32,'" theproperties of nor0ornene&deried galactose andglucose polymers 1ere still tested to confirmthese results. 2reiously utili/ed linkers are ' or"% atoms long, ho1eer their structures are

    proprietary.

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    + significantly greater num0er of spermundergo the + 1hen treated 1ith "(( ;poly;an-"(, polyuc-"( or poly8lcN+c-"( than1ith the other three glycopolymers igure 'a-.

    )he actiation of the + 0y these threeglycopolymers is steeply dose dependent at a 9&fold lo1er concentration, the + is not actiated.Sperm samples treated 1ith a 9&fold higherconcentration 9(( ;- 1ere + actiated 1ith alo1er efficiency or no efficacy. )hese datasuggest that at high polymer concentrations,multialent 0inding and thus the clustering effect

    Figure *' +ctiation of sperm acrosome reaction 0y homoglycopolymers.Capacitated sperm 1ere incu0ated 1ith glycopolymers at differentconcentrations sho1n as polymer concentration-. a- "(&mers. 0- "((&mers. )heaerage acrosome reaction percentage +K- of glycopolymer treated sperm1ere normali/ed using G+Kglycopolymers- = +Knegatie control-I?G+Kpositie control- = +Knegatie control-I. )he aerage +K for the positiecontrol, +9'"#=treated @ M;- sperm, 1as 9%K and for the negatie control,2BS=treated sperm, 1as "(K. Data represent mean S>; of at least threeindependent eperiments. * pO (.(@ 1hen compared to the negatie control.

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    is not faored.'% Neither poly8lc-"(, poly8al-"(nor poly8alN+c-"( triggered the + atconcentrations of "(( or 9(( ;.

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    )%%ect o% pairs o% (++-mers on AR'Net, 1e pairedthe actie "((&mers poly;an-"((, polyuc-"((,and poly8lcN+c-"((at their optimal "( ;- andat much lo1er concentrations 9.@ ;- toeamine the effect of inducing the +simultaneously 1ith t1o different ligands. +sglucose has not 0een identified on 32 andpoly8lc-"(( had no + actiation a0ility,poly8lc-"((1as used as a negatie control. Wesa1 no further increase in the amount of sperm+ comparing the polymer pairs and singleglycopolymers at their optimal concentrations

    Figure Comparison of mied "((&mers and the corresponding single "((&mers. a-"((&mers paired at "( ; each. 0- "((&mers paired at 9.@ ; each. )heconcentration sho1n in the chart is polymer concentration. )he aerage +K ofglycopolymer treated sperm 1ere normali/ed using G+Kglycopolymers- = +Knegatie control-I?G+Kpositie control- = +Knegatie control-I. )he aerage+K for the positie control, +9'"#=treated @ M;- sperm, 1as 9%K and for thenegatie control, 2oly8lc-"((=treated "( M;- sperm, 1as ""K. Data representmean S>; of at least three independent eperiments. * pO (.(@ 1hencompared to the corresponding single "((&mers.

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    the efficacy remained at "((K of the positie

    control igure %a-. ;itures of threeglycopolymers at their optimal concentrations1ere also tested, 0ut no significant differences in+ percentage 0et1een a pair and a miture ofthree 1ere o0sered.

    +lthough poly8lcN+c-"(( and poly;an-"(( hadsimilar dose&dependent + actiating patternsigure '0- poly8lcN+c-"(( 1as more effectiethan poly;an-"(( at 9.@ ; igure %0-.2oly8lcN+c-"(( and poly;an-"(( paired at 9.@ ;each sho1ed a slight enhancement in +actiation compared to poly8lcN+c-"((at 9.@ ;,yet the miture did not actiate + to the sameleel as @ ; of a poly8lcN+c-"((or poly;an-"((

    igure %0-. )his result suggests that the t1ocar0ohydrate ligands 0ind to different receptorson the sperm.

    Sperm samples treated 1ith the other t1ocom0inations of actiating polymer 9.@ ; each-sho1ed efficacies e7ual to treatment 1ith asingle polymer at 9.@ ;. ; of at least threeindependent eperiments. * pO (.(@1hen compared to the +K ofpoly8lc-"((at each time point .

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    .inetics o% AR induced /y (++-mers' We furtherstudied the effects of the three actie "((&merson the + as they 1ere more potent than the "(&mers. )he time courses for the three "((&mers1ere monitored in parallel for %@ minutes igure@-. Data for longer incu0ation periods 1ere notincluded due to high + in the negatie controland reduced sperm ia0ility.

    +t the three time points, the etent of + in thepositie control and the poly;an-"(( andpoly8lcN+c-"(( treated samples is the same.

    4o1eer, polyuc-"((induced lo1er leels of the+ than poly;an-"((and poly8lcN+c-"((at '( or%@ minutes, and there 1as no initiation after "@minutes. )hese data indicate that the induction ofthe + 0y poly;an-"(( and poly8lcN+c-"(( ismore rapid than 0y polyuc-"((, 1hich is inagreement 1ith the preious o0seration thatpoly;an-"((and poly8lcN+c-"((is more effectiethan polyuc-"((. )he reason for the slo1er +actiation kinetics of polyuc-"((compared to theother t1o "((&mers is unclear. 5ne possi0ility isthat differences in glycopolymer conformations

    Figure 0')he signaling path1ays of +actiation 0y the three effectieglycopolymers are similar. )GTAJethylene glycol tetraacetic acid,etracellular Ca9: inhi0itor. erJpertussis toin, 8&protein inhi0itor. AmiJamiloride hydrochloride, )&type Ca9:

    channel inhi0itor. 123J protein kinase +inhi0itor. GenJ genistein, proteintyrosine kinase inhi0itor. Ni%J

    nifedipine, L&type Ca9: channelinhi0itor. CheJ chelerylthrine, proteinkinase C inhi0itor. )he aerageinhi0ition percentage of inhi0itor andglycopolymer treated sperm 1erenormali/ed using G+Kpositiecontrol- = +Kglycopolymers-I ?G+Kpositie control- = +Knegatiecontrol-I. )he aerage +K for thepositie control, +9'"#&treated @ M;-sperm, 1as 9%K, and for the negatiecontrol, inhi0itor&treated sperm, 1as $&"'K. Data represent mean S>; ofthree independent eperiments.

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    arising from su0stitution 1ith differentmonosaccharides affect efficacy of signaltransduction. Note that the + in the negatiecontrol increased sharply at %@ minutes due tospontaneous +. We selected '( minutes for all ofour endpoint assays 0ecause the leel ofspontaneous + 1as much lo1er.

    Signaling ath!ay o% Glycopolymers Induced AR'We eamined 1hich signaling transduction eentsare actiated 0y +&actie glycopolymers using

    1ell characteri/ed inhi0itors for protein kinase +2P+-, protein kinase C 2PC-, protein tyrosinekinase 2)P-, 8&protein, )&type?L&type Ca9:

    channels and etracellular Ca9:. )hese signalingmolecules and channels hae 0een detected in0oth mouse and human sperm and hae 0eensuggested to play an important role in 0oth themouse and the human 32&induced acrosomereaction. )he inhi0itors do not hae a0solutespecificity they 0lock + actiation non&selectiely at high concentrations. )he doses ofthe inhi0itors 1ere carefully chosen in order tonot affect sperm ia0ility and motility. Conersely,inhi0itors do not completely a0olish thespontaneous +.

    +ll seen inhi0itors significantly suppressedpoly;an-"((&, poly8lcN+c-"((& and polyuc-"((&actiated + 1ith at least A(K inhi0ition igureA-. +s the three glycopolymers all actiate +, itis not surprising to find that they share signalingpath1ays in common. )hese results demonstratethat poly;an-"((, poly8lcN+c-"((, and polyuc-"((actiate the + though conergent signalingpath1ays. Sperm treated 1ith inhi0itors alonealso sho1 a lo1 + percentage consistent 1ith alo1 leel of spontaneous + that is independentof these path1ays.

    )he precise sperm + signaling path1ays arenot completely elucidated, though seeraltentatie signaling path1ay mechanisms of 32&initiated + hae 0een proposed.

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    +ll of the a0oe&descri0ed signaling factors areinoled in actiation of the + 0y poly;an- "((,poly8lcN+c-"((, and polyuc-"((. ;oreoer, thesehomopolymers actiate through the samepath1ays as mouse 32 )a0le "-. 5ur datastrongly support that glycopolymer&actiated +is analogous to 32&actiated + and that theseglycopolymers are suita0le mimics of the 32and?or other physiologic ligands for actiatingsperm +. 5ur 1ork emphasi/es the highredundancy of car0ohydrate ligands that can 0eused to actiate the +.

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    used in su0se7uent eperiments. )heconcentration of sperm 1as assessed 0yhemocytometer. +li7uots 9( L- containinga0out @ R "(@ capacitated sperm 1eretransferred to microcentrifuge tu0es andincu0ated 1ith glycopolymers, negatie, andpositie controls at '# C under @K C59 for thespecified time "@, '( or %@ min-. )he positiecontrol used in our assay is a 1ell&studied Ca9:

    transporting ionophore +9'"# Sigma&+ldrich-.

    +fter incu0ation, the sperm 1ere pelleted 0ycentrifugation at @(( g for A min. )he supernatant1as remoed and the pelleted sperm 1ere1ashed once 1ith %( L 2BS and fied 1ith %( L#(K ethanol. +fter fiing at % C for '( min, thesperm 1ere pelleted and 1ashed t1ice 1ith 2BS.

    )he final pellet 1as resuspended in %( L of DDidence for intermediate states ofsecretion during spontaneous acrosomal eocytosis

    in mouse sperm, De2. 'iol./*4, "%"&"@9."%- Wassarman, 2. ;., oine, L., Vi, 4., Williams,

    3., Darie, C., and Litscher, >. S. 9((%- ecent

    aspects of mammalian fertili/ation research, Mol.

    &ell. En#ocrinol./(4, $@&"('."@- )ulsiani, D. . 2. 9(((- Car0ohydrates

    mediate sperm&oum adhesion and triggering of the

    acrosome reaction sian -. n#rol. /, #&$#."A- Wassarman, 2. ;. 9((@- Contri0ution of

    mouse egg /ona pellucida glycoproteins to gamete

    recognition during fertili/ation, -. &ell. Pysiol./54,'&'$".

    "#- Loeser, C. ., and )ulsiani, D. . 2. "$$$- )herole of car0ohydrates in the induction of the

    acrosome reaction in mouse spermato/oa, 'iol.

    Repro#.*5, $%&"("."- 4anna, W. ., Perr, C. L., Shaper, . 4., and

    Wright, W. W. 9((%- Le1is &containingneoglycoproteins mimic the intrinsic a0ility of /onapellucida glycoprotein /p' to induce the acrosome

    reaction in capacitated mouse sperm, 'iol. Repro#.

    71, ##$."$- Williams, S. +., Fia, L., Cummings, . D.,

    ;c>er, . 2., and Stanley, 2. 9((#- ertili/ation inmouse does not re7uire terminal galactose or n&

    acetylglucosamine on the /ona pellucida glycans, -.

    &ell Sci.1/5, "'%"&"'%$.9(- Bai0ako, B., 8authier, L., )al0ot, 2., ankin, ).

    L., and Dean, . 9((#- Sperm 0inding to the /onapellucida is not sufficient to induce acrosome

    eocytosis, De2elopment1(4, $''&$%'.

    9"- in, ;., uEi1ara, >., Pakiuchi, !., 5ka0e, ;.,Satouh, !., Ba0a, S. +., Chi0a, P., and 4irohashi, N.9(""- ;ost fertili/ing mouse spermato/oa 0egintheir acrosome reaction 0efore contact 1ith the /ona

    pellucida during in itro fertili/ation, Proc. %atl.

    ca#. Sci. S150, %$9&%$A.99- !anagimachi, . 9(""- ;ammalian sperm

    acrosome reactionJ Where does it 0egin 0efore

    fertili/ation, 'iol. Repro#.03, %&@.9'- +ella, ;. +., and Dean, . 9(""- ertili/ation

    1ith acrosome&reacted mouse spermJ aston, . L., 2atankar, ;. S., Lattan/io, . +.,

    Leaen, ). 4., ;orris, 4. ., Clark, 8. ., and Dell, +.9(((- Structural analysis of murine /ona pellucidaglycans. >idence for the epression of core 9&type

    o&glycans and the sda- antigen, -. 'iol. &em./73,##'"#%9.

    '9- Lee, !., and Sampson, N. S. 9((A- ompingthe cellular landscapeJ Linear scaffolds for molecular

    recognition, &rr. Opin. Strct. 'iol.1*, @%%&@@(.''- Biela1ski, C. W., and 8ru00s, . 4. 9((#-

    Liing ring&opening metathesis polymeri/ation, Prog.

    Polym. Sci.(/, "&9$.'%- 8est1icki, . >., Cairo, C. W., Strong, L. >.,

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    human spermato/oa, 'iol. Repro#.7), A$#.'#- Loeser, C. ., Lynch, C., and )ulsiani, D. . 2.

    "$$$- Characteri/ation of the pharmacological&

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    sensitiity profile of neoglycoprotein induced

    acrosome reaction in mouse spermato/oa, 'iol.

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    fertili/ation, ynecol. O"stet. /, doiJ"(.%"#9?9"A"&($'9."(((e%"(#.

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