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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Fucose, Mannose, and β-<italic>N</italic>-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm
Acrosome Reaction through Convergent Signaling Pathways</title><author><name sortKey="Wu, Linghui" sort="Wu, Linghui" uniqKey="Wu L" first="Linghui" last="Wu">Linghui Wu</name></author><author><name sortKey="Sampson, Nicole S" sort="Sampson, Nicole S" uniqKey="Sampson N" first="Nicole S." last="Sampson">Nicole S. Sampson</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24252131</idno><idno type="pmc">4049243</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049243</idno><idno type="RBID">PMC:4049243</idno><idno type="doi">10.1021/cb400550j</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000124</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000124</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Fucose, Mannose, and β-<italic>N</italic>-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm
Acrosome Reaction through Convergent Signaling Pathways</title><author><name sortKey="Wu, Linghui" sort="Wu, Linghui" uniqKey="Wu L" first="Linghui" last="Wu">Linghui Wu</name></author><author><name sortKey="Sampson, Nicole S" sort="Sampson, Nicole S" uniqKey="Sampson N" first="Nicole S." last="Sampson">Nicole S. Sampson</name></author></analytic><series><title level="j">ACS Chemical Biology</title><idno type="ISSN">1554-8929</idno><idno type="eISSN">1554-8937</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="cb-2013-00550j_0008" id="ab-tgr1"></graphic></p><p>The sperm acrosome reaction (AR),
an essential exocytosis step
in mammalian fertilization, is mediated by a species-specific interaction
of sperm surface molecules with glycans on the egg. Previous studies
indicate that a subset of terminal carbohydrates on the mouse egg
zona pellucida (ZP) trigger the AR by cross-linking or aggregating
receptors on the sperm membrane. However, the exact role of those
carbohydrates in AR has not been identified and the mechanism underlying
the AR still needs further investigation. To study this process, a
series of glycopolymers was synthesized. The glycopolymers are composed
of a multivalent scaffold (norbornene), a functional ligand (previously
identified ZP terminal monosaccharides), and a linker connecting the
ligand and the scaffold. The polymers were tested for their ability
to initiate AR and through which signaling pathways AR induction occurred.
Our data demonstrate that mannose, fucose, and β-<italic>N</italic>-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent
manner, and the 100-mers are more potent on a per monomer basis than
the 10-mers. Although nearly equipotent in inducing the AR at the
optimal concentrations, their AR activation kinetics are not identical.
Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding
regulatory proteins (G-proteins), tyrosine kinase, protein kinase
A, protein kinase C, and Ca<sup>2+</sup>-related antagonists. Thus,
the chemotypes of synthetic glycopolymers imitate the physiologic
AR-activation agents and provide evidence that occupation of one of
at least three different receptor binding sites is sufficient to initiate
the AR.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Wassarman, P M" uniqKey="Wassarman P">P. M. Wassarman</name></author><author><name sortKey="Jovine, L" uniqKey="Jovine L">L. Jovine</name></author><author><name sortKey="Litscher, E S" uniqKey="Litscher E">E. S. Litscher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wassarman, P M" uniqKey="Wassarman P">P. M. Wassarman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Benoff, S" uniqKey="Benoff S">S. Benoff</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Clark, G F" uniqKey="Clark G">G. F. Clark</name></author><author><name sortKey="Dell, A" uniqKey="Dell A">A. Dell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bedford, J M" uniqKey="Bedford J">J. M Bedford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Miller, D J" uniqKey="Miller D">D. J. Miller</name></author><author><name sortKey="Gong, X" uniqKey="Gong X">X. Gong</name></author><author><name sortKey="Shur, D B" uniqKey="Shur D">D. B. Shur</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nagdas, S K" uniqKey="Nagdas S">S. K. Nagdas</name></author><author><name sortKey="Araki, Y" uniqKey="Araki Y">Y. Araki</name></author><author><name sortKey="Chayko, C A" uniqKey="Chayko C">C. A. Chayko</name></author><author><name sortKey="Orgebin Crist, M C" uniqKey="Orgebin Crist M">M. C. Orgebin-Crist</name></author><author><name sortKey="Tulsiani, D R" uniqKey="Tulsiani D">D. R. Tulsiani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cornwell, G X0a A" uniqKey="Cornwell G">G.
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<front><journal-meta><journal-id journal-id-type="nlm-ta">ACS Chem Biol</journal-id><journal-id journal-id-type="iso-abbrev">ACS Chem. Biol</journal-id><journal-id journal-id-type="publisher-id">cb</journal-id><journal-id journal-id-type="coden">acbcct</journal-id><journal-title-group><journal-title>ACS Chemical Biology</journal-title></journal-title-group><issn pub-type="ppub">1554-8929</issn><issn pub-type="epub">1554-8937</issn><publisher><publisher-name>American
Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24252131</article-id><article-id pub-id-type="pmc">4049243</article-id><article-id pub-id-type="doi">10.1021/cb400550j</article-id><article-categories><subj-group><subject>Articles</subject></subj-group></article-categories><title-group><article-title>Fucose, Mannose, and β-<italic>N</italic>-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm
Acrosome Reaction through Convergent Signaling Pathways</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Wu</surname><given-names>Linghui</given-names></name></contrib><contrib contrib-type="author" corresp="yes" id="ath2"><name><surname>Sampson</surname><given-names>Nicole S.</given-names></name><xref rid="cor1" ref-type="other">*</xref></contrib><aff id="aff1">Department of Chemistry,<institution>Stony Brook University</institution>, Stony Brook, New York 11794-3400,<country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>nicole.sampson@stonybrook.edu</email>.</corresp></author-notes><pub-date pub-type="pmc-release"><day>19</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>19</day><month>11</month><year>2013</year></pub-date><pub-date pub-type="ppub"><day>21</day><month>02</month><year>2014</year></pub-date><volume>9</volume><issue>2</issue><fpage>468</fpage><lpage>475</lpage><history><date date-type="received"><day>21</day><month>07</month><year>2013</year></date><date date-type="accepted"><day>19</day><month>11</month><year>2013</year></date></history><permissions><copyright-statement>Copyright © 2013 American Chemical
Society</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>American Chemical
Society</copyright-holder></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="cb-2013-00550j_0008" id="ab-tgr1"></graphic></p><p>The sperm acrosome reaction (AR),
an essential exocytosis step
in mammalian fertilization, is mediated by a species-specific interaction
of sperm surface molecules with glycans on the egg. Previous studies
indicate that a subset of terminal carbohydrates on the mouse egg
zona pellucida (ZP) trigger the AR by cross-linking or aggregating
receptors on the sperm membrane. However, the exact role of those
carbohydrates in AR has not been identified and the mechanism underlying
the AR still needs further investigation. To study this process, a
series of glycopolymers was synthesized. The glycopolymers are composed
of a multivalent scaffold (norbornene), a functional ligand (previously
identified ZP terminal monosaccharides), and a linker connecting the
ligand and the scaffold. The polymers were tested for their ability
to initiate AR and through which signaling pathways AR induction occurred.
Our data demonstrate that mannose, fucose, and β-<italic>N</italic>-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent
manner, and the 100-mers are more potent on a per monomer basis than
the 10-mers. Although nearly equipotent in inducing the AR at the
optimal concentrations, their AR activation kinetics are not identical.
Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding
regulatory proteins (G-proteins), tyrosine kinase, protein kinase
A, protein kinase C, and Ca<sup>2+</sup>-related antagonists. Thus,
the chemotypes of synthetic glycopolymers imitate the physiologic
AR-activation agents and provide evidence that occupation of one of
at least three different receptor binding sites is sufficient to initiate
the AR.</p></abstract><funding-group><funding-statement><funding-source>National Institutes of Health, United States</funding-source></funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>cb400550j</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>cb-2013-00550j</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id="sec1">Mammalian
fertilization involves
a series of sophisticated steps. It starts with sperm–egg binding,
sperm penetration of the egg outer membrane, the zona pellucida (ZP),
and finally sperm–egg fusion to form an embryo.<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> In the mouse, the ZP consists of three glycoproteins (ZP1,
ZP2, and ZP3) that form a cross-linked extracellular matrix surrounding
the egg. Each glycoprotein contains glycans covalently linked to asparagine
(N-linked) and/or serine and threonine (O-linked).<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> Previous studies indicated that the adhesion of sperm to
the egg is mainly mediated by a specific interaction of sperm surface
molecules with the glycan moieties on the ZP.<sup><xref ref-type="bibr" rid="ref3">3</xref>−<xref ref-type="bibr" rid="ref5">5</xref></sup> Glycosidase
digestion and mass spectrometric analyses have indicated that several
mouse ZP3 terminal monosaccharide residues, including β-<italic>N</italic>-acetylgalactosamine (GalNAc),<sup><xref ref-type="bibr" rid="ref6">6</xref></sup> β-<italic>N</italic>-acetylglucosamine (GlcNAc),<sup><xref ref-type="bibr" rid="ref7">7</xref></sup> mannose,<sup><xref ref-type="bibr" rid="ref8">8</xref></sup> galactose,<sup><xref ref-type="bibr" rid="ref9">9</xref></sup> and sialyl,<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> may
be critical for sperm binding to the ZP. Although a fucosylated glycan
was identified on human ZP<sup><xref ref-type="bibr" rid="ref11">11</xref></sup> and the addition
of a fucose residue to the GlcNAc and galactose trisaccharides enhances
mouse sperm–egg binding affinity,<sup><xref ref-type="bibr" rid="ref12">12</xref></sup> the participation of fucose in mouse gamete binding is not definitive.
The complex heterogeneity of zona glycosides makes definition of the
exact function of these sugars a challenge.</p><p>To penetrate the
thick ZP layer, sperm need to undergo a cellular
exocytosis known as the acrosome exocytosis or acrosome reaction (AR).
Structurally, the acrosome is composed of a membrane vesicle filled
with soluble components and acrosomal matrix proteins.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> When the AR occurs (Figure <xref rid="fig1" ref-type="fig">1</xref>), the
sperm plasma membrane fuses with the outer acrosomal membrane; the
soluble contents in the vesicle, e.g., hydrolases and proteases, are
sequentially released.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> The acrosome reaction
plays an essential role in fertilization, and only acrosome-reacted
sperm can participate in the following fertilization steps that lead
to sperm–egg fusion.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup></p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Overview of
mouse sperm acrosome reaction.</p></caption><graphic xlink:href="cb-2013-00550j_0001" id="gr1" position="float"></graphic></fig><p>A number of physiological and nonphysiological agonists can
activate
the AR. Though controversial, ZP3 was widely accepted as the primary
AR activator. Multiple sperm–carbohydrate binding events occur
to stimulate the AR by cross-linking or aggregating receptors on the
sperm plasma membrane.<sup><xref ref-type="bibr" rid="ref15">15</xref>,<xref ref-type="bibr" rid="ref16">16</xref></sup> This hypothesis is based on inhibition
and activation studies with deglycosylated ZP and fragmented ZP.</p><p>To elucidate the role of individual carbohydrates in initiating
the AR, activity studies of neoglycoconjugates have been undertaken.
Loeser et al. (<xref ref-type="bibr" rid="ref17">17</xref>) examined the properties
of several neoglycoproteins that are bovine serum albumin (BSA) conjugates
with an average of 8 copies of the glycan of interest per protein
molecule. They concluded that mannose-BSA, GlcNAc-BSA, and GalNAc-BSA
could initiate the AR while glucose-BSA and galactose-BSA had no effect.
Moreover, unconjugated sugars failed to block neoglycoprotein-induced
AR, suggesting that a scaffold to display the sugars is necessary.
Later, Hanna et al.<sup><xref ref-type="bibr" rid="ref18">18</xref></sup> found that Lewis
X (Galβ4[Fucα3]GlcNAc) and Lewis A (Galβ3[Fucα4]GlcNAc)
when conjugated to BSA could initiate the AR, and LewisX-BSA was more
potent than LewisA-BSA. This finding indicated that fucose may be
another glycan ligand involved in AR initiation in addition to the
three monosaccharides identified by Loeser et al.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> However, Hanna et al.<sup><xref ref-type="bibr" rid="ref18">18</xref></sup> did
not observe AR activation with GlcNAc-BSA, in contrast to the results
of Loeser et al.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> Interpretation is difficult
as little is known about the spatial display of the saccharides on
the BSA. Moreover, fucosyl bioconjugates were not directly studied.</p><p>Recent experiments have further challenged the proposed roles of
some glycan residues in the AR and the concept of ZP3 as the AR stimulus.
Oocyte-specific deletion of both the β-1,3-galactosyl transferase
(<italic>T-syn</italic>) and the mannoside acetylglucosaminyltransferase
1 (<italic>Mgat</italic>1) genes generated mouse eggs lacking core-1-derived <italic>O</italic>-glycans and with modified <italic>N</italic>-glycans lacking
terminal Gal and GlcNAc residues.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> These
mice, in which <italic>O</italic>-linked or <italic>N</italic>-linked
galactose and GlcNAc were genetically deleted from the ZP are still
fertile, suggesting these two monosaccharides are genetically unessential.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup> However, the knockout mice display considerably
lower fecundity compared to wild-type mice, and the remaining glycans
on the ZP, for example, mannose or GalNAc, may still contribute to
fertilization. A model through which sperm induce acrosome exocytosis
by mechanosensory signal transduction without requiring sperm binding
to the ZP was also reported,<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> but it is
difficult to imagine how mechanical signals can be transmitted without
molecular binding interactions between sperm and the egg surface.
It has also been suggested that most fertilizing sperm begin acrosomal
exocytosis before binding to the ZP through molecular interactions
between sperm and the female reproductive tract.<sup><xref ref-type="bibr" rid="ref21">21</xref>,<xref ref-type="bibr" rid="ref22">22</xref></sup> It appears that there are multiple interactions that lead to the
AR, most likely involving carbohydrates. To what extent signaling
is initiated through a single type of receptor–ligand interaction
versus multiple types of interactions is poorly understood.</p><p>Although the signaling pathways initiating the AR are not completely
understood, considerable progress in understanding downstream events
has been made.<sup><xref ref-type="bibr" rid="ref23">23</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> At minimum, the AR is triggered
by a cascade of signal events including activation of G-proteins,
protein phosphorylation, and elevation of intracellular calcium and
pH levels.<sup><xref ref-type="bibr" rid="ref25">25</xref>−<xref ref-type="bibr" rid="ref27">27</xref></sup> However, the identity of the cell surface receptor
or receptors that initiates these signals is unknown, despite the
identification of several candidates.<sup><xref ref-type="bibr" rid="ref26">26</xref></sup></p><p>Controversy about the mechanism of the AR calls for a modified
strategy to elucidate the molecular players in this complicated process.
We hypothesized that synthetic glycopolymers will provide further
insights into the molecular complexity of sperm AR activation.<sup><xref ref-type="bibr" rid="ref28">28</xref></sup> The length, substitution, and ligand density
of glycopolymers can be easily controlled. Thus, we applied neoglycopolymers
prepared by ring-opening metathesis polymerization (ROMP) to investigation
of the AR. We further examined the glycopolymer-activated AR in the
absence and presence of established pharmacological agents known to
prevent the AR by blocking specific signaling pathways. Our data demonstrate
that synthetic mannose, fucose, and GlcNAc polymers activate mouse
acrosome reaction through convergent signaling pathways.</p><sec id="sec2"><title>Results and Discussion</title><sec id="sec2.1"><title>Design
and Preparation of Glycopolymers</title><p>Synthetic polymeric
probes provide a versatile strategy to investigate ligand–receptor
interactions.<sup><xref ref-type="bibr" rid="ref29">29</xref>,<xref ref-type="bibr" rid="ref30">30</xref></sup> Their design, which is simple
and flexible, requires a multivalent scaffold, a minimal ligand, and
a spacer to link the ligand to the scaffold. All of the ZP monosaccharides
proposed to be involved in the AR, i.e., mannose, fucose, GlcNAc,
and GalNAc, were chosen as ligands. Although Loeser et al.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> demonstrated that galactose-BSA and glucose-BSA
did not induce AR and glucose is not present on the ZP,<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> the properties of norbornene-derived galactose
and glucose polymers were still tested to confirm these results. Previously
utilized linkers are 3 or 14 atoms long; however, their structures
are proprietary. In this work, we designed a simple ethyl amide linker
to connect the norbornene-derived backbone and the monosaccharide
ligand. Norbornene (NB) served as the scaffold in our work due to
its high rigidity and widespread adoption. The desired polymers were
generated by ROMP that affords polymers with defined lengths and narrow
molecular mass distributions.<sup><xref ref-type="bibr" rid="ref32">32</xref>,<xref ref-type="bibr" rid="ref33">33</xref></sup> Moreover, the density
of ligands on norbornyl ROMP polymers favors clustering receptors
and activating signaling transduction.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> Hence, a series of homogeneous glycopolymers with two different
average lengths (10-mer and 100-mer) (Figure <xref rid="fig2" ref-type="fig">2</xref>) were synthesized (<xref rid="notes-1" ref-type="notes">Supplementary Schemes 1–7</xref>) and tested to determine the better scaffold length for activating
the AR with monosaccharide ligands.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Structures of glycopolymer probes.</p></caption><graphic xlink:href="cb-2013-00550j_0002" id="gr2" position="float"></graphic></fig></sec><sec id="sec2.2"><title>Effect of Homoglycopolymers
on Acrosome Reaction</title><p>We
examined the effect of homoglycopolymers on the sperm AR by sperm
immunofluorescence assay (<xref rid="notes-1" ref-type="notes">Supplementary Figure
1</xref>). In the assay, sperm were capacitated by treatment with
0.3% bovine serum albumin (BSA), which is known to facilitate sperm
capacitation by altering fatty acids and/or cholesterol on the sperm
plasma membrane. Calcium ionophore A23187, a known sperm AR stimulus,
was used as a positive control instead of ZP, because the role of
ZP in AR activation is controversial and the calcium concentration
has been demonstrated to be essential to activate the AR. Since all
polymer samples were prepared in phosphate buffered saline (PBS),
sperm treated with PBS was used as a negative control. Sperm samples
were incubated with controls and glycopolymers at varying concentrations
for 30 min and then stained with rhodamine-labeled peanut agglutinin
(PNA). PNA, which has been identified to bind both the outer acrosomal
membrane and certain components of the acrosomal matrix, is widely
used to discriminate between acrosome-intact and acrosome-reacted
sperm.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup></p><p>A significantly greater
number of sperm undergo the AR when treated with 100 μM poly(Man)<sub>10</sub>, poly(Fuc)<sub>10</sub>, or poly(GlcNAc)<sub>10</sub> than
with the other three glycopolymers (Figure <xref rid="fig3" ref-type="fig">3</xref>a). The activation of the AR by these three glycopolymers is steeply
dose-dependent; at a 2-fold lower concentration, the AR is not activated.
Sperm samples treated with a 2-fold higher concentration (200 μM)
were AR activated with a lower efficiency or no efficacy. These data
suggest that at high polymer concentrations, multivalent binding and
thus the clustering effect are not favored.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> Neither poly(Glc)<sub>10</sub>, poly(Gal)<sub>10</sub>, nor poly(GalNAc)<sub>10</sub> triggered the AR at concentrations of 100 or 200 μM.
Induction of the AR by poly(Fuc)<sub>10</sub> is not as effective
as with poly(Man)<sub>10</sub> or poly(GlcNAc)<sub>10</sub>. Similarly,
dose-dependent AR initiation was observed when sperm were incubated
with poly(Man)<sub>100</sub>, poly(Fuc)<sub>100</sub>, and poly(GlcNAc)<sub>100</sub>, but not poly(Glc)<sub>100</sub>, poly(Gal)<sub>100</sub>, and poly(GalNAc)<sub>100</sub> (Figure <xref rid="fig3" ref-type="fig">3</xref>b). There is no statistically significant difference between the
efficacy of mannose polymers and GlcNAc polymers. Again, the AR initiation
efficacy of poly(Fuc)<sub>100</sub> is lower than for poly(Man)<sub>100</sub> and poly(GlcNAc)<sub>100</sub>, consistent with the lowered
efficacy of poly(Fuc)<sub>10</sub>. Taken together, these results
indicate that galactose and GalNAc may play a role in sperm–egg
binding but not in activation of the AR. Mannose, GlcNAc, and fucose
function as sperm AR activators, but fucose is less efficient at AR
activation. The concentrations reported here are polymer concentrations.
If the bulk concentration of glycan ligand utilized is considered
in comparing the efficacies of 10-mers versus 100-mers, we observe
that the 100-mers are more potent. Sperm did not undergo the AR when
incubated with the 10-mers at 500 μM ligand concentration (50
μM polymer) (Figure <xref rid="fig3" ref-type="fig">3</xref>a), whereas 500
μM poly(Man)<sub>100</sub>, poly(Fuc)<sub>100</sub>, and poly(GlcNAc)<sub>100</sub> (5 μM polymer) (Figure <xref rid="fig3" ref-type="fig">3</xref>b)
successfully initiated the AR. The higher potency of the 100-mers
further confirms that the polymers stimulate the AR through a multivalent
interaction with sperm.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Activation of sperm acrosome reaction by homoglycopolymers.
Capacitated
sperm were incubated with glycopolymers at different concentrations
(shown as polymer concentration). (a) 10-mers. (b) 100-mers. The average
acrosome reaction percentages (AR%) of glycopolymer-treated sperm
were normalized using [AR%(glycopolymers) – AR%(negative control)]/[AR%(positive
control) – AR%(negative control)]. The average AR% for the
positive control, A23187-treated (5 μM) sperm, was 24% and for
the negative control, PBS-treated sperm, was 10%. Data represent mean ±
SEM of at least three independent experiments. * <italic>p</italic> < 0.05 when compared to the negative control.</p></caption><graphic xlink:href="cb-2013-00550j_0003" id="gr3" position="float"></graphic></fig></sec><sec id="sec2.3"><title>Effect of Pairs of 100-mers on AR</title><p>Next, we paired the
active 100-mers poly(Man)<sub>100</sub>, poly(Fuc)<sub>100</sub>,
and poly(GlcNAc)<sub>100</sub> at their optimal (10 μM) and
at much lower concentrations (2.5 μM) to examine the effect
of inducing the AR simultaneously with two different ligands. As glucose
has not been identified on ZP and poly(Glc)<sub>100</sub> had no AR
activation ability, poly(Glc)<sub>100</sub> was used as a negative
control. We saw no further increase in the amount of sperm AR comparing
the polymer pairs and single glycopolymers at their optimal concentrations;
the efficacy remained at 100% of the positive control (Figure <xref rid="fig4" ref-type="fig">4</xref>a). Mixtures of three glycopolymers at their optimal
concentrations were also tested, but no significant differences in
AR percentage between a pair and a mixture of three were observed.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Comparison
of mixed 100-mers and the corresponding single 100-mers.
(a) 100-mers paired at 10 μM each. (b) 100-mers paired at 2.5
μM each. The concentration shown in the chart is polymer concentration.
The average AR% of glycopolymer-treated sperm were normalized using
[AR%(glycopolymers) – AR%(negative control)]/[AR%(positive
control) – AR%(negative control)]. The average AR% for the
positive control, A23187-treated (5 μM) sperm, was 24% and for
the negative control, poly(Glc)<sub>100</sub>-treated (10 μM)
sperm, was 11%. Data represent mean ± SEM of at least three independent
experiments. * <italic>p</italic> < 0.05 when compared to the corresponding
single 100-mers.</p></caption><graphic xlink:href="cb-2013-00550j_0004" id="gr4" position="float"></graphic></fig><p>Although poly(GlcNAc)<sub>100</sub> and poly(Man)<sub>100</sub> had similar dose-dependent
AR activating patterns (Figure <xref rid="fig3" ref-type="fig">3</xref>b), poly(GlcNAc)<sub>100</sub> was more effective
than poly(Man)<sub>100</sub> at 2.5 μM (Figure <xref rid="fig4" ref-type="fig">4</xref>b). Poly(GlcNAc)<sub>100</sub> and poly(Man)<sub>100</sub> paired at 2.5 μM each showed a slight enhancement in AR activation
compared to poly(GlcNAc)<sub>100</sub> at 2.5 μM, yet the mixture
did not activate AR to the same level as 5 μM of a poly(GlcNAc)<sub>100</sub> or poly(Man)<sub>100</sub> (Figure <xref rid="fig4" ref-type="fig">4</xref>b). This result suggests that the two carbohydrate ligands bind to
different receptors on the sperm.</p><p>Sperm samples treated with
the other two combinations of activating
polymer (2.5 μM each) showed efficacies equal to treatment with
a single polymer at 2.5 μM. In addition, the pairwise mixtures
(2.5 μM each) were less effective activators of AR than a single
polymer at 5 μM, which is equal to the total concentration of
polymer in the paired mixture. The data suggest that there is a concentration
threshold for glycopolymer-receptor binding and signal transduction
and that the three sugars act independently to activate the AR.</p><p>We also used dynamic light scattering to investigate whether polymer
aggregation, which could interfere with sperm activation, had occurred.
No aggregation was observed (data not shown). These results together
suggest that maximal sperm AR is achieved upon treatment with a single
homopolymer at its optimal concentration (Figure <xref rid="fig4" ref-type="fig">4</xref>a) and that the carbohydrate ligands bind to different receptors
on the sperm (Figure <xref rid="fig4" ref-type="fig">4</xref>b).</p></sec><sec id="sec2.4"><title>Kinetics of
AR Induced by 100-mers</title><p>We further studied
the effects of the three active 100-mers on the AR as they were more
potent than the 10-mers. The time courses for the three 100-mers were
monitored in parallel for 45 min (Figure <xref rid="fig5" ref-type="fig">5</xref>).
Data for longer incubation periods were not included due to high AR
in the negative control and reduced sperm viability.</p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Poly(Fuc)<sub>100</sub>, poly(Man)<sub>100</sub>, and poly(GlcNAc)<sub>100</sub> have different
AR activation rates. The concentration shown
in the chart is polymer concentration. The average AR% of glycopolymer-treated
sperm were normalized using [AR%(glycopolymers) – AR%(negative
control)]/[AR%(positive control) – AR%(negative control)].
The average AR% for the positive control, A23187-treated (5 μM)
sperm at 45 min, was 33% and for the negative control, poly(Glc)<sub>100</sub>-treated (10 μM) sperm at 15 min, was 9%. Data represent
mean ± SEM of at least three independent experiments. * <italic>p</italic> < 0.05 when compared to the AR% of poly(Glc)<sub>100</sub> at each time point.</p></caption><graphic xlink:href="cb-2013-00550j_0005" id="gr5" position="float"></graphic></fig><p>At the three time points, the extent of AR in the positive
control
and the poly(Man)<sub>100</sub>- and poly(GlcNAc)<sub>100</sub>-treated
samples is the same. However, poly(Fuc)<sub>100</sub> induced lower
levels of the AR than poly(Man)<sub>100</sub> and poly(GlcNAc)<sub>100</sub> at 30 or 45 min, and there was no initiation after 15 min.
These data indicate that the induction of the AR by poly(Man)<sub>100</sub> and poly(GlcNAc)<sub>100</sub> is more rapid than by poly(Fuc)<sub>100</sub>, which is in agreement with the previous observation that
poly(Man)<sub>100</sub> and poly(GlcNAc)<sub>100</sub> are more effective
than poly(Fuc)<sub>100</sub>. The reason for the slower AR activation
kinetics of poly(Fuc)<sub>100</sub> compared to the other two 100-mers
is unclear. One possibility is that differences in glycopolymer conformations
arising from substitution with different monosaccharides affect efficacy
of signal transduction. Note that the AR in the negative control increased
sharply at 45 min due to spontaneous AR. We selected 30 min for all
of our end point assays because the level of spontaneous AR was much
lower.</p></sec><sec id="sec2.5"><title>Signaling Pathway of Glycopolymers-Induced AR</title><p>We examined
which signaling transduction events are activated by AR-active glycopolymers
using well characterized inhibitors for protein kinase A (PKA), protein
kinase C (PKC), protein tyrosine kinase (PTK), G-protein, T-type/L-type
Ca<sup>2+</sup> channels, and extracellular Ca<sup>2+</sup>. These
signaling molecules and channels have been detected in both mouse
and human sperm and have been suggested to play an important role
in both the mouse and the human ZP-induced acrosome reaction.<sup><xref ref-type="bibr" rid="ref36">36</xref>,<xref ref-type="bibr" rid="ref37">37</xref></sup> The inhibitors do not have absolute specificity; they block AR activation
nonselectively at high concentrations. The doses of the inhibitors
were carefully chosen in order to not affect sperm viability and motility.
Conversely, inhibitors do not completely abolish the spontaneous AR.</p><p>All seven inhibitors significantly suppressed poly(Man)<sub>100</sub>-, poly(GlcNAc)<sub>100</sub>-, and poly(Fuc)<sub>100</sub>-activated
AR with at least 60% inhibition (Figure <xref rid="fig6" ref-type="fig">6</xref>).
As the three glycopolymers all activate AR, it is not surprising to
find that they share signaling pathways in common. These results demonstrate
that poly(Man)<sub>100</sub>, poly(GlcNAc)<sub>100</sub>, and poly(Fuc)<sub>100</sub> activate the AR though convergent signaling pathways. Sperm
treated with inhibitors alone also show a low AR percentage, consistent
with a low level of spontaneous AR that is independent of these pathways.</p><fig id="fig6" position="float"><label>Figure 6</label><caption><p>Signaling
pathways of AR activation by the three effective glycopolymers
are similar. EGTA: ethylene glycol tetraacetic acid, extracellular
Ca<sup>2+</sup> inhibitor. Per: pertussis toxin, G-protein inhibitor.
Ami: amiloride hydrochloride, T-type Ca<sup>2+</sup> channel inhibitor.
H89: protein kinase A inhibitor. Gen: genistein, protein tyrosine
kinase inhibitor. Nif: nifedipine, L-type Ca<sup>2+</sup> channel
inhibitor. Che: chelerylthrine, protein kinase C inhibitor. The average
inhibition percentage of inhibitor and glycopolymer treated sperm
were normalized using [AR%(positive control) – AR%(glycopolymers)]
/[AR%(positive control) – AR%(negative control)]. The average
AR% for the positive control, A23187-treated (5 μM) sperm, was
24%, and for the negative control, inhibitor-treated sperm, was 9–13%.
Data represent mean ± SEM of three independent experiments.</p></caption><graphic xlink:href="cb-2013-00550j_0006" id="gr6" position="float"></graphic></fig><p>The precise sperm AR signaling
pathways are not completely elucidated,
though several tentative signaling pathway mechanisms of ZP-initiated
AR have been proposed.<sup><xref ref-type="bibr" rid="ref26">26</xref>,<xref ref-type="bibr" rid="ref38">38</xref>,<xref ref-type="bibr" rid="ref39">39</xref></sup> In these mechanisms, ZP is thought to bind to at least two receptors
on the sperm membrane. One is a G-protein coupled receptor, which
is reported to regulate adenylyl cyclase and activate PKA, phospholipase
C (PLC) β1, and H<sup>+</sup> efflux. Upon activation, PKA phosphorylates
and further triggers downstream factors. The other is a PTK receptor,
which is suggested to trigger a sperm Na<sup>+</sup>/H<sup>+</sup> exchanger promoting cell alkalinization, membrane depolarization,
and T-type and L-type calcium channels activation on the sperm plasma
membrane. The calcium channels play vital roles in elevating intracellular
Ca<sup>2+</sup> and pH preceding the AR. G-protein and PTK can also
activate PKC, which mediates calcium entry into the sperm cytosol
from intracellular stores. These signaling factors all lead to an
increase in cytosolic calcium, resulting in the fusion of sperm plasma
membrane and the outer acrosomal membrane and eventually the AR.</p><p>All of the above-described signaling factors are involved in activation
of the AR by poly(Man)<sub>100</sub>, poly(GlcNAc)<sub>100</sub>,
and poly(Fuc)<sub>100</sub>. Moreover, these homopolymers activate
through the same pathways as mouse ZP (Table <xref rid="tbl1" ref-type="other">1</xref>). Our data strongly support that glycopolymer-activated AR is analogous
to ZP-activated AR and that these glycopolymers are suitable mimics
of the ZP and/or other physiologic ligands for activating sperm AR.
Our work emphasizes the high redundancy of carbohydrate ligands that
can be used to activate the AR. In contrast to conditional genetic
knockouts for which no AR phenotypes were observed, the use of glycopolymers
has enabled the identification of which terminal carbohydrates are
important for the AR. The glycopolymer chemotypes observed in this
work suggest that at least three different sperm receptor binding
sites can be utilized to initiate the AR in mouse. After receptor
activation by glycopolymer, signaling converges onto the same pathways
intracellularly.</p><p>The receptors activated by poly(Man)<sub>100</sub>, poly(GlcNAc)<sub>100</sub>, and poly(Fuc)<sub>100</sub> have not
been definitively
identified. None of the large number of egg binding receptors proposed
and characterized has been demonstrated to be essential.<sup><xref ref-type="bibr" rid="ref40">40</xref>−<xref ref-type="bibr" rid="ref42">42</xref></sup> Previous results also suggested a high level of redundancy, but
whether multiple egg binding receptors acted individually or as a
multiprotein complex was unclear. Our results favor the single ligand–receptor
interaction model and provide further evidence that induction of the
sperm acrosome reaction proceeds through duplicative sperm–egg
interactions.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>Comparison of Signaling Pathways Initiated
by Different Activators</title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead><tr><th style="border:none;" align="center"> </th><th colspan="7" align="center">signaling molecule<hr></hr></th></tr><tr><th style="border:none;" align="center">AR activator</th><th style="border:none;" align="center">extracellular
Ca<sup>2+</sup></th><th style="border:none;" align="center">T-type Ca<sup>2+</sup></th><th style="border:none;" align="center">L-type Ca<sup>2+</sup></th><th style="border:none;" align="center">PKC</th><th style="border:none;" align="center">PKA</th><th style="border:none;" align="center">PTK</th><th style="border:none;" align="center">G protein</th></tr></thead><tbody><tr><td style="border:none;" align="left">BSA-mannose/GalNAc/GlcNAc<xref rid="t1fn1" ref-type="table-fn">a</xref></td><td style="border:none;" align="center">–<xref rid="t1fn2" ref-type="table-fn">b</xref></td><td style="border:none;" align="center">–</td><td style="border:none;" align="center">+<xref rid="t1fn3" ref-type="table-fn">c</xref></td><td style="border:none;" align="center">–</td><td style="border:none;" align="center">–</td><td style="border:none;" align="center">–</td><td style="border:none;" align="center">–</td></tr><tr><td style="border:none;" align="left">BSA-Lewis X<xref rid="t1fn4" ref-type="table-fn">d</xref></td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">NA<xref rid="t1fn5" ref-type="table-fn">e</xref></td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">–</td></tr><tr><td style="border:none;" align="left">BSA-Lewis A<xref rid="t1fn6" ref-type="table-fn">f</xref></td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">NA</td><td style="border:none;" align="center">–</td></tr><tr><td style="border:none;" align="left">Mouses ZP<xref rid="t1fn7" ref-type="table-fn">g</xref></td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td></tr><tr><td style="border:none;" align="left">poly(Man)<sub>100</sub></td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td></tr><tr><td style="border:none;" align="left">poly(GlcNAc)<sub>100</sub></td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td></tr><tr><td style="border:none;" align="left">poly(Fuc)<sub>100</sub></td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td><td style="border:none;" align="center">+</td></tr></tbody></table><table-wrap-foot><fn id="t1fn1"><label>a</label><p>Data from ref (<xref ref-type="bibr" rid="ref37">37</xref>).</p></fn><fn id="t1fn2"><label>b</label><p>–, Signaling pathway is not
activated.</p></fn><fn id="t1fn3"><label>c</label><p>+, Signaling
pathway is activated.</p></fn><fn id="t1fn4"><label>d</label><p>Data
from ref (<xref ref-type="bibr" rid="ref18">18</xref>).</p></fn><fn id="t1fn5"><label>e</label><p>NA, not available, no signaling
pathway experiment was performed.</p></fn><fn id="t1fn6"><label>f</label><p>Data from ref (<xref ref-type="bibr" rid="ref18">18</xref>).</p></fn><fn id="t1fn7"><label>g</label><p>Data from ref (<xref ref-type="bibr" rid="ref36">36</xref>).</p></fn></table-wrap-foot></table-wrap></sec><sec sec-type="conclusions" id="sec3"><title>Conclusion</title><p>A multivalent display
of mannose, GlcNAc,
or fucose triggers the sperm acrosome reaction in a concentration-dependent
manner, and high valency polymers (100 ligands) are more effective
than low valency polymers (10 ligands). Although fucose showed lower
AR activation potency and the kinetics of fucose-induced AR are distinct
from those of mannose or GlcNAc-induced AR, all of the glycopolymers
rely on G-proteins, protein kinase C, protein kinase A, extracellular
Ca<sup>2+</sup>, L- and T-type Ca<sup>2+</sup> channels, and a protein
tyrosine kinase. The three single ligand–receptor interactions
all induce the acrosome reaction and are functionally equivalent but
are redundant. Thus, the chemotypes of ROMP-derived glycopolymers
mimic the biological function of physiologic AR-activation agents
and provide evidence that occupation of one of at least three different
receptor binding sites is sufficient to initiate the AR.</p></sec></sec><sec sec-type="methods" id="sec4"><title>Methods</title><sec id="sec4.1"><title>General Methods and Materials</title><p>All experiments performed
with mice were in accordance with the National Institute of Health
and United States Department of Agriculture guidelines, and the specific
procedures performed were approved by the Stony Brook University IACUC
(protocol 0616). Chemicals for assay buffers were purchased from Sigma-Aldrich,
Fisher Scientific, and VWR.</p></sec><sec id="sec4.2"><title>Sperm Treatment</title><p>Sperm were isolated
from the cauda
epididymis of two 10- to 12-week-old ICR male breeders (Taconic) in
M16 medium (6 mL) supplemented with 0.3% BSA (w/v). The sperm suspension
was then gently pipetted into a polypropylene culture tube (12 mm
× 75 mm) and incubated at 37 °C for 30 min under 5% CO<sub>2</sub> (v/v) in air. Once the incubation was complete, the sperm
motility was examined by phase-contrast microscopy (20×/0.5 air).
Only samples of capacitated sperm displaying >80% motility were
used
in subsequent experiments. The concentration of sperm was assessed
by hemocytometer. Aliquots (20 μL) containing about 5 ×
10<sup>5</sup> capacitated sperm were transferred to microcentrifuge
tubes and incubated with glycopolymers, negative, and positive controls
at 37 °C under 5% CO<sub>2</sub> for the specified time (15,
30, or 45 min). The positive control used in our assay is the well-studied
Ca<sup>2+</sup> transporting ionophore A23187 (Sigma-Aldrich). After
incubation, the sperm were pelleted by centrifugation at 500<italic>g</italic> for 6 min. The supernatant was removed, and the pelleted
sperm were washed once with 40 μL of PBS and fixed with 40 μL
of 70% ethanol. After fixing at 4 °C for 30 min, the sperm were
pelleted and washed twice with PBS. The final pellet was resuspended
in 40 μL of DDI water. Aliquots (10 μL) of each sample
were transferred to coverslips and air-dried.</p></sec><sec id="sec4.3"><title>Assessment of Sperm Acrosome
Reaction</title><p>Ten microliters
of rhodamine-labeled peanut agglutinin (PNA) (Vector laboratories)
at a concentration of 20 μg mL<sup>–1</sup> was incubated
with fixed sperm (from which polymers had been removed by extensive
washing) on coverslips for 10 min at rt. After washing with 2 mL of
DDI water (two × 10 min), the coverslips were mounted on SuperFrost
Plus microscope slides (Fisher Scientific) over a drop (6 μL)
of mounting medium Vectashield (Vector laboratories) and sealed with
nail polish, and the acrosomal status was assessed by inverse fluorescence
microscopy (Zeiss). Sperm that displayed continuous red fluorescence
along their acrosomal arcs were scored as acrosome-intact; those that
displayed no red or punctuate fluorescence were scored as acrosome-reacted.
The slides were coded and counted blindly; all experiments were conducted
at least three times. Each time, three independent replicates of each
test group were analyzed, and 200 sperm from each replicate were counted.</p></sec><sec id="sec4.4"><title>Signaling Pathway Inhibition Assay</title><p>Inhibitor stock
solutions were prepared by dissolving the reagents either in distilled
water (pertussis toxin) or in DMSO. Aliquots of the stock solutions
were mixed with the capacitated sperm solution to achieve the desired
concentration and preincubated for 5 min before treatment with polymers
or A23187. The concentrations of inhibitors were chosen on the basis
of references<sup><xref ref-type="bibr" rid="ref36">36</xref>,<xref ref-type="bibr" rid="ref37">37</xref></sup> and toxicity tests. The acrosomal
status of sperm was evaluated as described above.</p></sec><sec id="sec4.5"><title>Statistical
Analysis</title><p>Comparisons of the average values
for the control and experimental groups were carried out by a paired
two-tailed <italic>t</italic>-test to determine statistically significant
differences (<italic>p</italic> < 0.05). The results are presented
as mean ± SEM.</p></sec></sec></body><back><notes id="notes-1" notes-type="si"><title>Supporting Information Available</title><p>Schemes of all syntheses and
experimental procedures. This material is available free of charge
via the Internet at <uri xlink:href="http://pubs.acs.org">http://pubs.acs.org</uri>.</p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="cb400550j_si_001.pdf"><caption><p>cb400550j_si_001.pdf</p></caption></media></supplementary-material></sec><notes id="NOTES-d1143e1047-autogenerated" notes-type="conflict-of-interest"><p>The authors
declare no
competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>We thank Brookhaven Instruments
(Brookhaven, NY) for the dynamic
light scattering software. Funding was providing by the National Institutes
of Health (R01GM097971/R01HD38519 to N.S.S.)</p></ack><glossary id="dl1"><def-list><title>Abbreviations</title><def-item><term>AR</term><def><p>acrosome reaction</p></def></def-item><def-item><term>ZP</term><def><p>zona pellucida</p></def></def-item><def-item><term>ZP3</term><def><p>zona pelucida protein 3</p></def></def-item><def-item><term>NB</term><def><p>norbornene</p></def></def-item><def-item><term>GalNAc</term><def><p>β-<italic>N</italic>-acetylgalactosamine</p></def></def-item><def-item><term>GlcNAc</term><def><p>β-<italic>N</italic>-acetylglucosamine</p></def></def-item><def-item><term>Man</term><def><p>mannose</p></def></def-item><def-item><term>Glc</term><def><p>glucose</p></def></def-item><def-item><term>Gal</term><def><p>galactose</p></def></def-item><def-item><term>Fuc</term><def><p>fucose</p></def></def-item><def-item><term>BSA</term><def><p>bovine serum albumin</p></def></def-item><def-item><term>PKA</term><def><p>protein kinase A</p></def></def-item><def-item><term>PKC</term><def><p>protein kinase C</p></def></def-item><def-item><term>PTK</term><def><p>protein tyrosine kinase</p></def></def-item></def-list></glossary><ref-list><title>References</title><ref id="ref1"><mixed-citation publication-type="journal" id="cit1"><name><surname>Wassarman</surname><given-names>P. M.</given-names></name>; <name><surname>Jovine</surname><given-names>L.</given-names></name>; <name><surname>Litscher</surname><given-names>E. S.</given-names></name> (<year>2001</year>) <article-title>A profile
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