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First published online February 9, 2007
doi: 10.1242/10.1242/dev.02752
1 Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes
of Health, Bethesda, MD 20892, USA.
2 Department of Cell Biology and Neuroscience, University of California,
Riverside, CA 92521, USA.
* Author for correspondence (e-mail: borisb{at}mail.nih.gov)
Accepted 21 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Molecular mechanisms of mouse fertilization, Sperm acrosome exocytosis, Mechanosensory signal transduction, Zona pellucida matrix, Sperm-egg recognition, Postfertilization block to polyspermy
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It has been reported that only acrosome-intact mouse sperm
penetrate the cumulus oophorus (a hyaluronic acid matrix with interspersed
somatic cells) surrounding ovulated eggs and bind to the zona pellucida, an
extracellular matrix that encircles the egg
(Storey et al., 1984;
Cherr et al., 1986). However,
only acrosome-reacted sperm are present in the perivitelline space (between
the plasma membrane of the egg and the inner aspect of the zona pellucida),
and only acrosome-reacted sperm fuse with the plasma membrane of the egg to
effect fertilization (Austin,
1975; Saling et al.,
1979). The presence of residual acrosomal shrouds on the zona
pellucida (Yanagimachi and Phillips,
1984; Cherr et al.,
1986; VandeVoort et al.,
1997) suggests that induction of acrosome exocytosis (acrosome
reaction) occurs on the surface of the zona matrix.
The induction of sperm acrosome exocytosis in mouse fertilization has long
engaged investigative interest, but remains incompletely understood. Unlike
recyclable exocytosis in somatic cells, the acrosome reaction occurs once and
is biologically relevant only if it takes place in conjunction with sperm
penetration of the zona pellucida. Exocytosis results from fusion between the
outer acrosomal membrane of the sperm and the overlying plasma membrane,
causing multiple membrane fenestrations and release of acrosomal contents
(Barros et al., 1967). Although
initially considered a binary event (acrosome-intact or acrosome-reacted),
more recent data suggest that the acrosome reaction is a continuum, beginning
with capacitation of sperm, and is dramatically accelerated by interactions
with the zona pellucida (Lee and Storey,
1985; Kim and Gerton,
2003). The acrosome possesses a calcium store that is sufficient
to trigger exocytosis (Herrick et al.,
2005). Whether physiologic mobilization is initiated by
Gi-protein mediated ligand-receptor interactions
(Ward et al., 1994),
extracellular Ca2+ influxes
(Jungnickel et al., 2001),
activation of internal inositol 1,4,5-triphosphate receptors
(Herrick et al., 2005) or
other mechanisms remains unclear. Although multiple agonists induce the
acrosome reaction two- to threefold over background in vitro
(Florman and Storey, 1982;
Bleil and Wassarman, 1983;
Meizel, 1985;
Roldan et al., 1994;
Shi and Roldan, 1995;
Murase and Roldan, 1995;
Sato et al., 2000;
Son and Meizel, 2003), only
binding or penetration of the zona pellucida during fertilization is 100%
effective in triggering acrosome exocytosis
(Austin, 1975;
Saling et al., 1979;
Yanagimachi, 1994).
The mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2 and
ZP3 (Bleil and Wassarman,
1980b; Boja et al.,
2003). Following fertilization, egg cortical granules exocytose
their contents, which modify the zona matrix so that additional sperm do not
bind or penetrate (Austin and Braden,
1956; Ducibella,
1996). Although other biochemical changes have been inferred, only
the proteolytic cleavage of ZP2 has been experimentally observed
(Moller and Wassarman, 1989).
The human homologs to the three mouse zona proteins are 62-71% identical
(Hoodbhoy et al., 2005), and
genetically altered mice in which human ZP2 replaces endogenous mouse ZP2
(huZP2 rescue mice) form a zona pellucida and are fertile.
Unexpectedly, sperm continue to bind to two-cell embryos after in vitro
fertilization of eggs derived from huZP2 rescue mice and this
observation correlates with intact huZP2, which is not cleaved in the chimeric
mouse-human zona pellucida. Nevertheless, the normal postfertilization block
to zona penetration is observed and there is no increased incidence of
polyspermy (Rankin et al.,
2003). These observations form the basis of the `zona scaffold'
model of sperm binding, in which the cleavage status of ZP2 regulates the
three-dimensional structure of the zona matrix, rendering it permissive (ZP2
intact) or non-permissive (ZP2 cleaved) for sperm binding, independent of
fertilization and cortical granule exocytosis
(Dean, 2004).
Using Acr3-EGFP sperm that accumulate soluble enhanced green fluorescent protein within the acrosome, we examined the acrosome status and binding of sperm to eggs and embryos derived from wild-type, huZP2 transgenic and huZP2 rescue mice. We observed persistent sperm binding to huZP2 transgenic and huZP2 rescue eggs and two-cell embryos as predicted by the `zona scaffold' model. Unexpectedly, sperm acrosomes remained intact when bound to wild-type and huZP2 rescue eggs or embryos for greater than 2 and 24 hours, respectively. These data have implications for the mechanisms by which fertilizing sperm bind, acrosome react and penetrate the zona pellucida.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), two
transgenic mouse lines, TgN(Acr3-EGFP)1NIH and
TgN(Acr3EGFP)2NIH, were established. Each accumulated EGFP in their
acrosomes (see Fig. S1A in the supplementary material) and were fertile in
vivo. Sperm were isolated from the caudal epididymis and incubated under
capacitating conditions (see below). After treatment with the calcium
ionophore A23187 (3 µmol/l), individual sperm underwent the acrosome
reaction exposing the inner acrosomal membrane to which Alexa 568-conjugated
soybean trypsin inhibitor (SBTI) bound (see Fig. S1B in the supplementary
material). Motile Acr3-EGFP sperm bound to ovulated eggs as
effectively as wild-type sperm (see Fig. S1C in the supplementary material)
and eggs fertilized in vitro underwent the cortical granule exocytosis (see
Fig. S1D in the supplementary material)
(Rankin et al., 2003).
TgN(Acr3-EGFP)1NIH was used in these studies.
Zp3-EGFP transgenic mice were established using
BglII-AceI and AceI-HindIII fragments of
the mouse Zp3 promoter (1.5 kbp) isolated from pXPZP3WT
(Millar et al., 1991) and
cloned into the BglII-HindIII sites of pEGFP1 (Clontech,
Palo Alto, CA). A purified BglII-AflII DNA fragment was
injected into the male pronucleus of fertilized FVB/N eggs
(Rankin et al., 2003) to
establish two transgenic lines that accumulate EGFP in the cytoplasm of
growing oocytes and preimplantation embryos (to be fully described elsewhere).
TgN(Zp3-EGFP)1NIH was used in these studies. All experiments were
conducted in compliance with the guidelines of the Animal Care and Use
Committee of the National Institutes of Health under a Division of Intramural
Research, NIDDK approved animal study protocol.
Sperm capacitation
Unless noted otherwise, caudal epididymal sperm isolated from wild-type and
Acr3-EGFP mice were placed under oil (Irvine Scientific, Santa Ana,
CA) in M16 media (Specialty Media, Chemicon International, Phillipsburg, NJ)
and incubated under capacitating conditions (1 hour, 37°C, 5%
CO2) before use in sperm binding and in vitro fertilization
assays.
Confocal microscopy
Confocal laser scanning images were obtained on a Zeiss LSM 510 confocal
microscope (Rankin et al.,
2003). To detect acrosome-reacted sperm, samples were incubated
for 15 minutes before fixation with soybean trypsin inhibitor (SBTI)
(Worthington, Lakewood, NJ) conjugated with Alexa 568 (Invitrogen Molecular
Probes, Carlsbad, CA) according to the manufacturer's protocol. Samples were
fixed in 2% paraformaldehyde and stained with Hoechst 33342 before
imaging.
In vitro fertilization
Wild-type or Acr3-EGFP sperm were incubated (1, 2, 4 or 24 hours)
with ovulated eggs obtained from gonadotropin-stimulated mice
(Rankin et al., 1998). Embryos
were then isolated, fixed, stained with Hoechst 33342 and imaged by confocal
microscopy. Fertilization was confirmed by the presence of Hoechst-positive
sperm nuclei within the egg cytoplasm. To count sperm bound to the zona
pellucida, consecutive 6 µm optical sections were collected through
individual embryos and projected onto a single-plane image. Acrosome-intact
(EGFP-positive), acrosome-reacted (Alexa 568-SBTI-positive) and the total
number (Hoechst positive nuclei) of sperm were determined.
lmmunoblot analyses
Eggs or two-cell embryos were isolated from 4-week-old wild-type,
huZP2 transgenic and huZP2 rescue mice after stimulation
with gonadotropins with or without in vivo mating
(Rankin et al., 1998) and were
prepared for immunoblot (Rankin et al.,
2003). Chemiluminescence signals were acquired by Luminescent
Image Analyzer LAS-3000 (Fuji Film Medical Systems, Stamford, CT). To present
mouse and human ZP2 results on the same blot, digital images were displayed in
green or red channels, respectively, using Adobe Photoshop (Adobe Systems, San
Jose, CA) RGB color mode.
Electron microscopy
After washing three times with a wide bore pipette, embryos from in vitro
fertilization with wild-type, huZP2 transgenic and huZP2
rescue eggs were embedded in Spurr's plastic (SPI-Chem Low Viscosity `Spurr'
Kit, SPI Supplies, West Chester, PA) and thin sections were cut using a
diamond knife on a RMC MTX ultramicrotome (Boeckler Instruments, Tucson, AZ).
Thin sections were stained with uranium and lead salts and examined on a
Tecnai 12 transmission electron microscope (FEI, Hillsborrow, OR)
(Rankin et al., 1999).
Sperm binding assay
Ovulated eggs and two-cell embryos were isolated from wild-type (FVB
strain), huZP2 rescue and huZP2 transgenic mice for sperm
binding assays (Rankin et al.,
1998). As a wash control in these assays, two-cell embryos were
isolated from TgN(Zp3-EGFP)1NIH mice. Wild-type and/or
Acr3-EGFP sperm binding to the zona pellucida was assessed after 1 or
24 hours by confocal microscopy (Rankin et
al., 1999).
Filter penetration assay
To enrich for acrosome-intact wild-type and Acr3-EGFP sperm
(Bangham and Hancock, 1955),
450 µl epididymal sperm was applied to a column (55x8 mm) packed with
the 250-320 µm glass beads and equilibrated with M16 media (1 hour,
37°C, 5% CO2). After washing the column with M16 media (2 ml),
sperm were collected in a second fraction (4 ml) and counted on a cellometer
(Nexcelom Bioscience, Lawrence, MA).
The sperm penetration assay chamber was a 25 mm Swinnex Filter (Millipore, Bedford, MA) holder from which the syringe connector tip was removed to establish a 10 mm opening into the upper chamber. In the center of the filter support, a 10 mm opening was drilled and the holder outlet was blocked with a piece of plastic. Epididymal or glass-bead-washed sperm (2.1x104 sperm, 850 µl) were placed in the lower chamber through the opening in the filter support. Polycarbonate filters (1.2, 3, 5 µm pores, Millipore, Bedford, MA; 12 µm pores, Whatman, Florham Park, NJ) were degassed in PBS under vacuum and placed, glossy side up, over the opening in the filter support, ensuring the exclusion of trapped air. After assembly of the filter holder, 500 µl preincubated (1 hour, 37°C, 5% CO2) M16 media was added on top of the filter in the upper chamber. Following a 30 minute incubation (37°C, 5% CO2), 400 µl was removed sequentially from the upper and lower chambers, fixed with an equal amount of 4% paraformaldehyde in PBS, and stained with propidium iodide to visualize nuclei.
Aliquots (400 µl) of sperm from the upper and lower chambers were analyzed by fluorescence activated cell sorting (FACS) (Calibur, Becton Dickinson BD, San Jose, CA). Using forward or side scatter density data, a discrete population of cells was selected that excluded clumps as well as subcellular debris; events below cellular auto fluorescence on EGFP or forward scatter plots were not included. Regions used to determine acrosome-intact or -reacted sperm on PI/EGFP dot plot were selected from analysis of glass-bead washed sperm and confirmed morphologically by confocal microscopy.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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120 kDa into 30 kDa and 90
kDa) following fertilization. The human ZP2 transgenic zona contains
the same three mouse proteins, plus human ZP2 (huZP2), and huZP2 entirely
replaces endogenous moZP2 in human ZP2 rescue zonae
(Rankin et al., 2003). After
treatment with gonadotropins to stimulate ovulation, eggs were collected in
cumulus from each of three genotypes (three independently obtained biological
samples, 8-18 eggs each) and inseminated with 5x105
epididymal Acr3-EGFP sperm that had been incubated under capacitating
conditions. During in vitro fertilization, a cohort of sperm approach the egg,
and acrosome exocytosis of the `fertilizing' sperm occurs shortly after sperm
bind to the zona pellucida of normal, huZP2 transgenic, and huZP2
rescue mice.
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More strikingly, sperm bound for 
24 hours to fertilized eggs and early
embryos derived from huZP2 transgenic and huZP2 rescue
females (Fig. 1A) and by
regression analysis, changes in sperm numbers were modest or non-existent
(Fig. 1B). Sperm associated
with huZP2 rescue-derived embryos
(Fig. 1A, 17-24) maintained an
intact acrosome (EGFP-positive) for up to 24 hours after in vitro
fertilization. Thus, the presence of intact human ZP2 provides a zona
structure to which sperm will bind 24 hours despite fertilization, and
sperm binding to normal and huZP2 rescue eggs or embryos was not
sufficient to induce acrosome exocytosis. Sperm bound to huZP2
transgenic eggs or embryos, however, underwent acrosome exocytosis beginning 4
hours after insemination, with all becoming EGFP-negative and SBTI-positive by
24 hours (Fig. 1A, 9-16 and
Fig. 1B). The late acrosome
reaction of these `non-fertilizing' sperm may provide mechanistic insights,
but is distinct from acrosome exocytosis induced by the initial interactions
of `fertilizing' sperm with ZP2-intact zonae pellucidae.
Electron microscopy of sperm bound to the zona pellucida
To better define the acrosome status of EGFP- and SBTI-positive sperm,
embryos (3-5) derived from in vitro fertilization of normal (moZP1, moZP2,
moZP3), huZP2 transgenic (moZP1, moZP2, huZP2, moZP3) and
huZP2 rescue eggs (moZP1, huZP2, moZP3) were imaged by transmission
electron microscopy (Fig. 2).
Sperm bound to normal fertilized eggs (2 hours post-insemination), adhering to
the zona pellucida with intact acrosomes contained within the inner and outer
acrosomal membranes immediately apposed to the plasma membrane overlying the
sperm head (15 of 15 sperm, Fig.
2A). Sperm also bound to huZP2 transgenic embryos (12
hours post-insemination), but for the most part (31 of 34 sperm, 91%) their
plasma membrane and outer acrosomal membranes had fused
(Fig. 2B), presumably leading
to loss of EGFP through the resulting fenestrations and consistent with the
similarly bound SBTI-positive sperm observed by confocal microscopy
(Fig. 1A, 16). By contrast,
sperm that bound to huZP2 rescue embryos (12 hours post-insemination)
were mostly acrosome-intact (42 of 51 sperm, 82%), and bound by intact plasma
membrane (Fig. 2C), much like
normal controls. Thus, confocal images of EGFP- and SBTI-positive sperm
provide realistic proxies of the ultrastructure of the mouse acrosome, and
both acrosome-intact and acrosome-reacted sperm can bind to early embryos
containing human ZP2 in the zona pellucida.
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). To
further examine this model, two-cell embryos derived from normal,
huZP2 transgenic and huZP2 rescue mice were flushed from
oviducts after in vivo mating. Additional Acr3-EGFP sperm
(5x105) were added to these in vitro fertilized two-cell
embryos (three independently obtained biological samples, ten embryos each) in
a sperm binding assay using Zp3-EGFP two-cell embryos as wash
controls. After washing with a wide-bore pipette to remove loosely adherent sperm (Fig. 3A, 1,4,7), no sperm bound to normal two-cell embryos and comparable numbers bound to embryos derived from huZP2 transgenic and huZP2 rescue eggs (49±8 and 73±15, respectively). Virtually all of the sperm associated with embryos derived from both the huZP2 transgenic and huZP2 rescue eggs remained acrosome-intact (EGFP-positive), with few showing evidence of having undergone the acrosome reaction (SBTI-positive) (Fig. 3A, 4-9). If the embryos were incubated for an additional 24 hours, almost all of the sperm bound to huZP2 rescue zonae remained acrosome-intact (Fig. 3B, 4-6), whereas all of the sperm bound to the huZP2 transgenic zonae pellucidae underwent the acrosome reaction (Fig. 3B, 1-3). Thus, persistent sperm binding (up to 24 hours) to the zona pellucida is not sufficient to induce the acrosome reaction when human ZP2 replaces endogenous mouse ZP2, but does occur, albeit delayed, if both mouse and human ZP2 are present in the zona matrix.
Cleavage status of mouse and human ZP2 proteins
To investigate the mechanism of this dichotomy, the cleavage status of
mouse and human ZP2 of eggs and two-cell embryos derived from normal,
huZP2 transgenic and huZP2 rescue female mice was examined
(Fig. 4). The primary
structures of secreted mouse ZP2 (599 amino acids) and human ZP2 (602 amino
acids) are 62% identical, but their molecular masses differ (120-140 kDa and
90-110 kDa, respectively) due to differences in post-translational
modifications. Each ZP2 protein is normally cleaved following fertilization
resulting in two fragments (30 kDa, 90 kDa for mouse; 35 kDa, 65 kDa
for human) that remain linked by disulfide bands
(Moller and Wassarman, 1989;
Bauskin et al., 1999).
Eggs and two-cell embryos (10-15) were isolated from normal, huZP2
transgenic and huZP2 rescue mice. As determined by immunoblots
(Fig. 4) using monoclonal
antibodies specific to the carboxyl terminus of mouse (green) or human ZP2
(red), mouse ZP2 was intact in normal and huZP2 transgenic eggs and
cleaved in normal and huZP2 transgenic two-cell embryos
(Fig. 4, lanes 1,2,5,6). By
contrast, human ZP2 remained uncleaved in both eggs and two-cell embryos
derived from huZP2 transgenic and huZP2 rescue female mice
(Fig. 4, lanes 3-6). Neither
the mouse-nor the human-specific antibodies crossreacted with ZP2 from the
other species (Fig. 4, lanes
1-4). Normally, ZP2 is cleaved by a cortical granule protease released by the
egg following fertilization. The ability of the protease to cleave mouse, but
not human, ZP2 in huZP2 transgenic mice indicates access within the
`humanized' zona architecture and suggests that intrinsic differences in the
two homologs prevent cleavage of human ZP2
(Rankin et al., 2003). Thus,
uncleaved human ZP2 in the absence of mouse ZP2 is sufficient to support mouse
sperm binding for 24 hours without inducing the acrosome reaction.
Reversibility of sperm binding to ovulated eggs and two-cell embryos
The number of sperm binding to normal, huZP2 transgenic and
huZP2 rescue eggs (three independently obtained biological samples,
ten eggs each) was similar (100±10, 112±8, 103±5,
respectively) in a 1 hour binding assay in which sperm were labeled with
Hoechst dye (Fig. 5,
1,2,5,6,9,10). The fertilized eggs were rinsed by serial passage through three
drops of media (30 µl) and challenged with Acr3-EGFP sperm (1 hour
incubation) followed by washing with 0.2 mm pipettes using Zp3-EGFP
two-cell embryos as controls (Fig.
5, 3,4,7,8,11,12). Although comparable numbers of total sperm
bound normal, huZP2 transgenic and huZP2 rescue eggs
(92±12, 115±17, 104±22, respectively), 84-88% of the
initially bound sperm had been replaced with Acr3-EGFP sperm
(Fig. 5, 4,8,12). Thus, under
these assay conditions, a fairly constant number of sperm bound to the zona
pellucida of normal, huZP2 transgenic and huZP2 rescue eggs,
and the binding was almost completely reversible 1 hour after
insemination.
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Mechanical induction of the acrosome reaction
The ability of Acr3-EGFP sperm to bind and remain acrosome-intact
for 2-3 hours with normal or 24 hours with huZP2 rescue eggs or embryos
appears inconsistent with the induction of acrosomal exocytosis by a signal
transduction pathway predicated on a zona ligand binding to a sperm surface
receptor. However, initial penetration into the zona matrix might trigger a
mechanosensory signal transduction, leading to exocytosis, and account for the
presence of only acrosome-reacted sperm in the perivitelline space. This
possibility has been investigated using a filter penetration assay with inert
polycarbonate filters unadorned with zona pellucida ligands
(Fig. 6). Normally, after 1
hour of incubation in capacitating conditions, 70% of epididymal
Acr3-EGFP sperm were acrosomeintact. However, passage through a
column of glass beads, to which acrosome-reacted, non-viable sperm
preferentially bind (Bangham and Hancock,
1955), resulted in a population that was 90-95% acrosome-intact.
These glass-bead-treated sperm (three independently obtained biological
samples) were used in a filterpenetration assay in which the progress of sperm
was impeded by inert filters with pore sizes ranging from 1.2 to 12 µm
(Fig. 6A). After incubation (30
minutes), aliquots of sperm from each side of the filters were obtained,
fixed, treated with propidium iodide (nuclear stain) and analyzed by FACS. Two
populations of sperm were observed according to the presence of EGFP and DNA
or DNA alone and confirmed morphologically as acrosome-intact and
acrosome-reacted, respectively (Fig.
6B). As the filter pore size decreased, the number of sperm in the
lower chamber gradually increased from 1.5 to 1.9x104
(Fig. 7A), but spontaneous
acrosome exocytosis remained comparable, ranging between 13-15%
(Fig. 6C,
Fig. 7B). As expected, the
number of sperm recovered in the upper chamber after passage through filters
decreased with smaller pore size (Fig.
6C, Fig. 7A), but
the proportion of acrosome-reacted sperm increased dramatically to
85±5.3% in filters with 3 µm and 93±5.8% with 1.2 µm pores
(Fig. 6C,
Fig. 7B).
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In addition, two capacitated Acr3-EGFP sperm aliquots from the same epididymal sperm sample were compared. One was pretreated with glass beads, the other was not, and each was observed after 30 minutes in the filter penetration assay. In three independently obtained biological samples, 21-31% of the untreated and 5-7% of the glass-bead-treated sperm in the lower chamber were acrosome-reacted, whereas more than 90% of sperm from each sample were acrosome-reacted in the upper chamber (Fig. 6D). If the presence of acrosome-reacted sperm in the upper chamber merely reflected `preferential passage' of spontaneously acrosome-reacted sperm, an increased number of sperm would be anticipated in the untreated, compared with the glass-bead-treated, Acr3-EGFP sperm samples. However, no such increase was observed, and the number of acrosome-reacted sperm was similar (318.3±202.6, 141.7±81.0, respectively) between the glass-bead-treated and untreated samples, despite fourfold more untreated than treated sperm in the starting samples. Taken together, these observations suggest that the passage of motile sperm through the inert matrix is sufficient to induce the acrosome reaction and raise the possibility that similar mechanical forces may have physiological import for induction of the acrosome reaction during normal fertilization (Fig. 8).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm-egg recognition
The zona pellucida surrounding ovulated eggs, but not two-cell embryos, is
permissive for sperm binding. To account for this dichotomy, two models are
considered. The `glycan release' model postulates that sperm bind to a
carbohydrate ligand, which is removed by a glycosidase released
postfertilization by egg cortical granules, to account for the absence of
sperm binding to the early embryo
(Wassarman et al., 2005;
Shur et al., 2006). The `zona
scaffold' model hypothesizes a three-dimensional zona matrix that is initially
permissive for sperm binding and is rendered nonpermissive by the
postfertilization cleavage of ZP2
(Hoodbhoy and Dean, 2004).
These two models make disparate predictions concerning de novo sperm binding
to two-cell embryos derived from huZP2 rescue mice in which ZP2
remains intact despite cortical granule exocytosis
(Jungnickel et al., 2003). The
first predicts no sperm binding because of postfertilization release of the
glycan ligand, whereas the second predicts sperm binding to a zona matrix
because of intact ZP2. The experimental observation of de novo
Acr3-EGFP sperm binding to embryos derived from huZP2
transgenic and huZP2 rescue mice
(Fig. 3) is not consistent with
the `glycan release' model and supports the `zona scaffold' model by
confirming earlier observations of persistent, postfertilization sperm binding
to huZP2 rescue eggs (Rankin et
al., 2003).
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;
Liu et al., 1995;
Thall et al., 1995;
Lu and Shur, 1997;
Ellies et al., 1998;
Easton et al., 2000;
Boja et al., 2003;
Lowe and Marth, 2003;
Shi et al., 2004), and genetic
studies in which individual zona proteins are absent or replaced with human
homologs do not support a single zona protein as a sperm adhesion molecule
(Rankin et al., 1998;
Rankin et al., 1999;
Rankin et al., 2003). Thus,
the three-dimensional structure of the zona pellucida probably plays a crucial
role in forming the cognate binding site, and further high resolution
structural definition of individual zona pellucida proteins and their
relationship within the supramolecular architecture of the zona matrix should
provide insights into the molecular basis of sperm-egg recognition.
Acrosome reaction
Acr3-EGFP sperm bind reversibly to normal eggs and embryos after
in vitro fertilization, but binding is absent after 4 hours and correlates
with partial proteolytic cleavage of moZP2. Those sperm initially bound to the
surface of the zona pellucida remain acrosome-intact for several hours
(Fig. 1A, 1-8). In eggs and
embryos derived from huZP2 rescue mice in which huZP2 is not cleaved,
reversible Acr3-EGFP sperm binding persists for at least 24 hours
after insemination, and again, sperm remain acrosome-intact
(Fig. 1A, 17-24). The long
dwell time of acrosome-intact sperm on the surface on the zona pellucida of
normal (2-3 hours) and huZP2 rescue (24 hours) mice is seemingly
inconsistent with a zona ligand interacting with a sperm receptor to induce
acrosomal exocytosis via a classic ligand-receptor signal transduction
pathway.
O-glycan ligands in the zona pellucida have been implicated as inducers of
the acrosome reaction (Bleil and
Wassarman, 1983; Leyton and
Saling, 1989). However, ZP1 is not required for fertility
(Rankin et al., 1999), and
O-linked glycosylation of ZP2 and ZP3 is surprisingly sparse
(Nagdas et al., 1994;
Boja et al., 2003). Confidence
in the role of O-glycans in acrosome exocytosis has been eroded by the
inability to define definitively either the zona ligand or the sperm surface
receptor as well as the observation that genetic ablation of leading
candidates does not prevent in vivo fertility
(Thall et al., 1995;
Lu and Shur, 1997;
Asano et al., 1997;
Liu et al., 1997). Moreover,
occupancy of putative attachments sites by O-glycans implicated as zona
ligands (Chen et al., 1998) is
not detected by microscale mass spectrometry of native ZP3
(Boja et al., 2003) and
mutation of the sites to preclude glycosylation does not affect in vivo
fertility in transgenic mice (Liu et al.,
1995). The observed heterogeneity of O-glycan side chains (1-7
monosaccharide residues) in the zona pellucida
(Easton et al., 2000;
Chalabi et al., 2006) further
complicates these models by suggesting that either not all glycan ligands at a
particular attachment site are utilized or multiple sperm surface receptors
are required to accommodate differing glycan isoforms.
Because sperm binding to normal or huZP2 rescue zonae pellucidae
is seemingly not sufficient to induce acrosome exocytosis, initial penetration
into the zona pellucida matrix was explored as a possible alternative. In the
filter penetration assay, Acr3-EGFP sperm were induced to undergo the
acrosome reaction by passage through inert polycarbonate filters. In the
absence of any zona ligand, 93±5.8% of the sperm underwent acrosome
exocytosis if pore sizes were 1.2 µm, whereas only 10-11% were
acrosome-reacted if the pore sizes were 5 µm. A simple explanation of
these data is that passage through filter pores comparable in size to the zona
pellucida matrix mechanically `induces' acrosome exocytosis in virtually all
sperm. This `induction' model predicts that there would be little or no
physical constraint until the pore size (3 µm) approaches the size of the
acrosome-intact sperm (3.5x8 µm)
(Wyrobek et al., 1976;
Cummins and Woodall, 1985). At
that point, contact between motile sperm and filter would generate sufficient
shear force to elicit a mechanosensory signal and acrosome exocytosis. The
dramatic threshold increase in acrosome-reacted sperm in the upper chamber
(Fig. 7B) as the pore size
decreased (2 µm) from 5 µm (11% acrosome-reacted) to 3 µm (85%
acrosome-reacted) is consistent with the `induction' model of acrosome
exocytosis.
|
) and induction of the acrosome reaction
(Fig. 8). Mechanosensory
signaling is widespread among plants and animals, although molecular details
have been more extensively described in bacteria
(Kung, 2005). Unlike the lock
and key binding of ligand to receptor transducing a signal, mechanically gated
channels can respond to forces imposed on the lipid bilayer to regulate ion
fluxes. This effect can be direct, as in stretch-activated channels, or
indirect, whereby cytoskeletal or extracellular matrices augment minor
perturbations in force. Multiple mechanotransducers have been described in
animals (Orr et al., 2006),
including polycystins (Delmas,
2004) and members of the transient receptor potential (TRP) family
(Barritt and Rychkov, 2005).
Sea urchin polycystin-2 has been implicated in the induction of the acrosome
reaction of Strongylocentrotus purpuratus sperm
(Neill et al., 2004) and
TRPCl, 2, 3 and 5 are present in mouse sperm
(Jungnickel et al., 2001;
Trevino et al., 2001).
Although TRPC2 has been incorporated into a ligand-receptor model of acrosome
exocytosis (Arnoult et al.,
1999; Jungnickel et al.,
2001), mice lacking TRPC2 remain fertile
(Stowers et al., 2002;
Leypold et al., 2002) and the
human homolog is a pseudo gene (Wes et
al., 1995). Whether the other TRPC mechanosensory signal
transducers present in mouse sperm play a role in the acrosome reaction
remains to be determined.
Block to zona penetration
The `fertilizing' sperm succeeds by binding to the zona pellucida,
penetrating the matrix and fusing with the plasma membrane of the egg. The
interaction of the `fertilizing' sperm with the zona pellucida is rapid, and
the binding to the zona matrix, induction of the sperm acrosome reaction, zona
penetration and fusion with the plasma membrane of the egg occurs within
minutes (Tollner et al., 2003;
Bedford, 2004). After gamete
fusion, there is a prompt, effective block to polyspermy at the plasma
membrane of the egg (Wolf,
1978) and the observation of few, if any, supernumerary sperm in
the perivitelline space (Sato,
1979) indicates that the postfertilization block to zona
penetration is rapid as well. The observed accumulation of supernumerary sperm
within the perivitelline space of Cd9-null eggs, deficient in
sperm-egg fusion (Le Naour et al.,
2000; Miyado et al.,
2000), indicates that the block to penetration occurs after gamete
fusion. Although this block has been ascribed to cleavage of ZP2 into two
fragments that remain disulfide-bonded
(Bleil et al., 1981), complete
cleavage takes >4 hours (Fig.
1C) and huZP2 rescue mice in which ZP2 is not cleaved
have an effective postfertilization block to zona penetration
(Rankin et al., 2003). Thus,
the block to zona penetration appears to be independent of the relatively
late-occurring cleavage of ZP2, and it seems likely that postfertilization
release of stored materials, presumably small and highly diffusible, from the
egg rapidly modify the zona pellucida to prevent sperm penetration.
The rapid block to zona penetration may account for the accumulation of
reversibly adherent, `non-fertilizing' Acr3-EGFP sperm on the zona
pellucida (Fig. 5). Initial
sperm-egg recognition has been described as `loose' and then `tight' (or
`primary' and `secondary') sperm binding to the surface of the zona matrix
(Hartmann et al., 1972;
Hartmann and Hutchison, 1974;
Bleil and Wassarman, 1980a;
Bleil and Wassarman, 1986).
However, without invoking repeated cycles of attachment and release, binding
to zona glycoprotein(s) seemingly would impede the forward progression of the
`fertilizing' sperm through the zona pellucida. Alternatively, if sperm are
attracted to the egg by chemoattractant guidance systems
(Cohen-Dayag et al., 1995;
Spehr et al., 2003;
Eisenbach and Giojalas, 2006),
then the initial penetration of the zona matrix by the `fertilizing' sperm
could induce acrosome exocytosis by mechanosensory signal transduction without
requiring that sperm bind to the zona pellucida. In this formulation, the
`non-fertilizing' sperm would remain under the influence of the egg's
chemoattractant, but be unable to progress through the zona matrix because of
the rapid, effective postfertilization block to penetration. Thus, the
perceived, reversible binding of `non-fertilizing' sperm on the surface of the
zona pellucida may reflect a conflict between chemoattraction or chemotaxis
and the inability to penetrate the matrix.
Conclusions
These data support a `zona scaffold' model of sperm-egg recognition in
which the cleavage status of ZP2 determines whether the three-dimensional zona
matrix will be permissive (ZP2 intact) or non-permissive (ZP2 cleaved) for
sperm adherence, independent of fertilization and cortical granule exocytosis.
The observed ability of sperm to remain acrosome-intact despite hours-long
adherence to normal and huZP2 rescue zona matrices is seemingly
inconsistent with widely embraced involvement of ligand-receptor signal
transduction in acrosome exocytosis. Thus, the `zona scaffold' model has been
extended to implicate initial penetration into the zona matrix as eliciting
`mechanosensory' signals sufficient for induction of acrosome exocytosis. This
model differs from `glycan release' models, in which ZP3 glycans act as
ligands for sperm binding as well as the induction of acrosome exocytosis,
with the postfertilization loss of the glycan accounting for the inability of
sperm to adhere to embryos. The validity of these disparate models will
require further investigations.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/933/DC1
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ACKNOWLEDGMENTS |
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