Article

Acrosomal Exocytosis of Mouse Sperm Progresses in a Consistent Direction in Response to Zona Pellucida

Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.
Journal of Cellular Physiology (Impact Factor: 3.84). 09/2009; 220(3):611-20. DOI: 10.1002/jcp.21781
Source: PubMed
ABSTRACT
Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor-mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model.

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Available from: George L Gerton, Mar 23, 2015
Acrosomal Exocytosis of
Mouse Sperm Progresses in a
Consistent Direction in Response
to Zona Pellucida
MARIANO G. BUFFONE,
1
ESMERALDA RODRIGUEZ-MIRANDA,
1,2
BAYARD T. STOREY,
1
AND GEORGE L. GERTON
1
*
1
Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women’s Health,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
2
Instituto Tecnolo
´
gico Sup erior de Irapuato (ITESI), Colonia El Copal, Irapuato, Guanajuato, Mexico
Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the
primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction
paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome.
It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a
live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We
compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by
the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used,
exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment
with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an
anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other
reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor-mediated
event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model.
J. Cell. Physiol. 220: 611–620, 2009. ! 2009 Wiley-Liss, Inc.
For a mammalian egg to be fertilized, a sperm must penetrate
the egg’s extracellular coat, the zona pellucida (ZP), to reach
the perivitelline space before the plasma membranes of both
gametes can come in contact and fuse (Glassner et al., 1991;
Yanagimachi, 1994; Storey, 1995; Florman and Ducibella, 2006).
A specialized organelle on the apical aspect of the head of the
mammalian sperm, the acrosome, has long been recognized as
playing a key role in this process (reviewed by Buffone et al.,
2008a). Subsequent studies of Bleil and Wassarman (1983)
showed that one of the three glycoproteins making up the
mouse egg zona pellucida, ZP3, served as both the binding site
for the sperm and inducer/stimulator of acrosomal exocytosis.
The acrosome lies directly under the plasma membrane
(PM). The acrosomal contents are enclosed by the outer
acrosomal membrane (OAM), which is in direct apposition to
the PM, and the inner acrosomal membrane (IAM), which is
closely associated with the anterior region of the nuclear
membrane. Although in early studies the role of the acrosome
was portrayed as a modified lysosome that could release lytic
enzymes active in mediating sperm penetration through the ZP,
recent investigations have demonstrated that there is also a
particulate component of the acrosome called the acrosomal
matrix that is composed of proteins, such as the mouse sperm
protein sp56, that are capable of mediating sperm binding to the
ZP (Allison and Hartree, 1970; Bleil and Wassarman, 1990;
Cohen and Wassarman, 2001; Kim et al., 2001; Buffone et al.,
2008b).
The process of releasing acrosomal components has
historically been called the ‘acrosome reaction’, for which
extracellular Ca
2þ
was found to be essential (Stambaugh and
Buckley, 1968; Allison and Hartree, 1970; Bedford, 1974;
Yanagimachi, 1994; Florman and Ducibella, 2006). The
physiological agonist that acts as a trigger of the mouse sperm
acrosome reaction was first demonstrated to be the mouse egg
zona pellucida (Florman and Storey, 1982). The acrosome
reaction was regarded as occurring through formation of
fenestration pores, opened by punctate fusion of the OAM and
PM, followed by widening of the pores to a continuum. The
hybrid membrane vesicles then disperse, leaving the IAM as the
sperm cell plasma membrane over the anterior head (Bedford
and Cooper, 1978; Yanagimachi, 1994). This view led to
formulation of the Acrosome Reaction Model, which depicts
the acrosome proceeding in a single step from an intact
membrane-bounded vesicle whose contents are sealed-in to a
dispersed organelle with fragmented membranes and scattered
contents. In essence, this is a ‘‘binary’’ model, in analogy to the
‘‘on–off’’ states underlying the operation of computer binary
digital code (Buffone et al., 2008a). This model gained
Additional Supporting Information may be found in the online
version of this article.
Contract grant sponsor: National Institutes of Health;
Contract grant numbers: R01 HD-41552, Fogarty
5D43TW000671.
*Correspondence to: George L. Gerton, Department of
Obstetrics and Gynecology, Center for Research on Reproduction
and Women’s Health, University of Pennsylvania Medical Center,
421 Curie Blvd., 1311 BRB II/III, Philadelphia, PA 19104-6160.
E-mail: gerton@mail.med.upenn.edu
Received 19 December 2008; Accepted 3 March 2009
Published online in Wiley InterScience
(www.interscience.wiley.com.), 16 April 2009.
DOI: 10.1002/jcp.21781
ORIGINAL ARTICLE
611
J o u r n a l o f
J o u r n a l o f
Cellular
Physiology
Cellular
Physiology
! 2 0 0 9 W I L E Y - L I S S , I N C .
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acceptance in large part because of relative ease in assaying the
‘‘intact’’ and ‘‘reacted’’ states, as opposed to difficulties in
assaying intermediate stages (Kligman et al., 1991). The best
assay available in the earlier studies for studying such stages
was the fluorescent probe chlortetracycline, which revealed
intermediate stages that have not been fully characterized
(Ward and Storey, 1984).
Recent studies challenged the concept of the binary reaction,
as it became ever more evident that the acrosomal lumen
contained an array of soluble components and protein
structures that make up the acrosomal matrix. We have begun
to conceptualize the loss of acrosomal components as an
‘‘analog’’ (i.e., continuously variable) process (Buffone et al.,
2008a). To distinguish this model from the Acrosome Reaction
Model, we have introduced the term ‘‘Acrosomal Exocytosis
Model’’ that incorporates a role for the acrosomal matrix
proteins in ZP binding and differential release of acrosomal
components (Kim et al., 2001; Kim and Gerton, 2003). Entry of
extracellular Ca
2þ
into the cell during capacitation was
postulated to first reach a ‘‘capacitation threshold’’ (Kim and
Gerton, 2003), at which point the membrane modifications
and phosphorylation on tyrosines of a select cohort of sperm
proteins have occurred, and the acrosomal lumen pH has
increased from 5.3 to 6.2 (Nakanishi et al., 2001). Continued
entry of Ca
2þ
allows the cell to reach an ‘‘exocytosis
threshold,’’ at which point punctate pore formation between
outer acrosomal and plasma membranes could occur (Kim and
Gerton, 2003). Once the cell reaches this threshold, further
stimulus of Ca
2þ
entry by intact zonae pellucidae, solubilized
zona pellucida protein (ZP), or calcium ionophore accelerates
the rate of pore formation to the point of rapid exocytosis
(Kim and Gerton, 2003). In the absence of stimulus by ZP or
ionophore, pore formation occurs far more slowly but does
allow matrix protein to be exposed at the sperm cell surface.
This proposed pathway offers an explanation for what has
long been called the ‘‘spontaneous acrosome reaction,’’
a process that has been ignored with regard to mechanism or
physiological significance. It also provides a rationale for the
role of acrosomal matrix protein sp56 in binding mouse sperm
to the zona pellucida.
The two different models for acrosomal dynamics raise
several questions concerning the exposure or release of
acrosomal components. Does the fusion of the OAM and PM
occur randomly, initiating at various points in different sperm?
Once started, does the fusion of the membranes and the
subsequent loss of acrosomal proteins exhibit any directionality
as the process progresses? Are the fusion patterns and kinetics
of ionophore-stimulated and ZP-promoted acrosomal changes
comparable? Experiments to address these questions became
feasible following the original demonstration by Okabe and
coworkers (Ikawa et al., 1995; Okabe et al., 1997) that a
transgene encoding green fluorescent protein (GFP) (Prasher
et al., 1992; Chalfie et al., 1994) could be incorporated into mice
and expressed in most tissues. Nakanishi et al. (1999)
developed a transgenic mouse that strictly localizes a mutant
form of GFP, enhanced green fluorescent protein (EGFP),
to the mouse sperm acrosome and allows the continuous
monitoring of acrosomal status of live sperm. EGFP is not
bound to any of the matrix proteins and so is released rapidly
from the acrosomal lumen upon pore formation at the onset
of acrosomal exocytosis, resulting in the loss of acrosomal
fluorescence. EGFP thus provides a probe for two aspects of
the initial stage of acrosomal exocytosis stimulated by either ZP
or by Ca
2þ
ionophore. The first is regional: where on the sperm
acrosome does the loss of EGFP fluorescence start? The second
is temporal: what is the time course of the fluorescence loss?
The studies of Nakanishi et al. (1999) and Yamashita et al.
(2007) did not address the regional aspect but showed that the
time course is on the order of seconds. In this study we
determined that EGFP fluorescence was lost rapidly in a
random pattern from the acrosomes of mouse sperm treated
with the calcium ionophore ionomycin. In contrast, the
fluorescence was lost in a slower and virtually non-variant
pattern from the acrosomes of sperm treated with soluble ZP
proteins. These findings are incorporated into a model
presented in this report.
Materials and Methods
Reagents and culture media
Inorganic chemicals were reagent grade from J.T. Baker Chemical
(Phillipsburg, NJ) and from Fisher Scientific (Pittsburgh, PA).
Aprotinin, DNAase, and Complete Protease Inhibitor tablets
were from Roche (Indianapolis, IN). All other biochemicals were
obtained from Sigma Chemical Co. (St. Louis, MO). The basic
medium used throughout these studies for the preparation and
culture of mouse sperm was the bicarbonate–HEPES buffered
medium, designated MJB, described by Bailey and Storey (1994),
with composition: 109 mM NaCl, 5 mM KCl, 25 mM NaHCO
3
,
25 mM HEPES, 25 mM sodium lactate, 5.6 mM glucose, 1.2 mM
MgCl
2
, 1.8 mM CaCl
2
, 1.0 mM sodium pyruvate, and 3 mg/ml
bovine serum albumin (BSA). The medium was first prepared in the
absence of calcium, bovine serum albumin, NaHCO
3
, and pyruvate,
then sterilized by filtration through a 0.22 mm tissue culture filter
unit (Nalgene, Rochester, NY) and frozen at "208C in aliquots for
single use. The working ‘‘complete’’ medium was prepared by
adding CaCl
2
, pyruvate, NaHCO
3
, and BSA, followed by adjusting
the pH to 7.4 and gassing with 5% CO
2
.
Preparation of sperm from male mice expressing EGFP in
the sperm acrosomes
Male mice (strain B6SJF1) expressing EGFP in the sperm acrosomes
were re-derived by Dr. Ralph DeBeradinis and Dr. Haig Kazazian
(Department of Genetics, University of Pennsylvania Medical
Center) using the Acr3-EGFP9 plasmid kindly provided by
Dr. Tadashi Baba (University of Tsukuba, Tsukuba City, Ibaraki,
Japan). EGFP expression, driven by the proacrosin promoter, was
targeted specifically to the mouse sperm acrosome using the
proacrosin signal sequence and part of the N-terminus of
proacrosin (Nakanishi et al., 1999). The mice were bred in-house
to provide the animals used for these studies; they are designated
‘EGFP mice’ in this report. Animals were used in accordance with
the International Guiding Principles for Biomedical Research
Involving Animals as promulgated by the Society for the Study of
Reproduction. Research protocols involving mice for this study
were approved by the University of Pennsylvania Institutional
Animal Care and Use Committee. Cauda epididymal sperm were
collected from mature (8-week-old) mice by placing minced caudae
epididymides in 1 ml of medium MJB without Ca
2þ
, BSA, and
NaHCO
3
. After 5 min incubation to allow sperm to swim out, the
sperm were washed in 1 ml of the same medium by centrifugation at
800g for 10 min at room temperature. The sperm were
resuspended to a final concentration of 2 # 10
7
sperm/ml in the
presence of different amounts of solubilized ZP. For analysis of
percentage of sperm with EGFP in their acrosomes, 10 ml aliquots
of the sperm suspensions were placed on polylysine-coated glass
slides and covered by coverslips. The presence of EGFP in sperm
acrosomes was quantitated by fluorescence microscopy, using a
Nikon TE2000 inverted microscope with fluorescence optics
(excitation 480 nm, emission 515 nm). Because of variations in the
percent acrosomal exocytosis due to the spontaneous reaction,
the data have been reported as percent acrosomal exocytosis over
baseline (Delta AE %), this being the total percent acrosome
reactions in a given sample minus the percent acrosome reactions
in the absence of solubilized ZP in the same sperm sample
incubated under identical conditions.
JOURNAL OF CELLULAR PHYSIOLOGY
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Preparation of purified solubilized zona pellucida protein
Zonae pellucidae were obtained from ovaries of 3-week-old female
mice by differential centrifugation of homogenates, based on the
original method of Bleil and Wassarman (1986) with sequential
modifications by Tanphaichitr et al. (1993) and Thaler and Cardullo
(1995), and subsequently used in kinetic studies by Rockwell
and Storey (2000). The homogenization buffer (HB) had the
composition 150 mM NaCl, 25 mM triethanolamine (TEA), 1 mM
MgCl
2
, 1 mM CaCl
2
, pH 8.5, containing, in 50 ml, 10 mg aprotinin,
10 mg DNase, and 1 tablet Complete Protease Inhibitor.
Ovaries from 40 mice were homogenized in 2.0 ml HB in
a Wheaton-Tenbroek borosilicate glass tissue grinder,
0.10–0.15 mm clearance (Fisher Scientific, Pittsburgh, PA), at room
temperature (228C) to give a suspension visually free of coarse
particles. To the homogenate was added 0.2 ml Nonidet NP-40
(10%, w/v) and 0.2 ml sodium deoxycholate (10%, w/v), followed by
a short further homogenization to obtain uniform mixing of the
detergents. The homogenate was layered onto a three-step
discontinuous gradient of Percoll in HB, constituted as: 2 ml 2%,
2 ml 10%, and 3 ml 22% Percoll, and centrifuged in a swinging bucket
rotor at 200g for 2 h at 48C. The 10% Percoll fraction, containing
the zonae pellucidae, was collected, diluted with 45 ml HB, and
centrifuged in 24 2-ml tubes at 16,000g for 10 min at 48C to pellet
the ZP. The pellets were resuspended in HB and combined to give a
single pellet after centrifugation at 16,000g as before. This pellet
was resuspended in 2.0 ml MJB and again centrifuged at 16,000g to
obtain a ZP pellet in MJB. The supernatant was removed, and the ZP
resuspended in MJB so as to give 0.1–0.2 ml ZP suspension. Three
1-ml aliquots of the suspension were taken to count the ZP, which
were essentially all intact and so readily counted. Concentrations
of ZP were recorded as ZP/ml; stock suspensions varied in
concentration from 50 to 200 ZP/ml. Solubilized ZP protein was
obtained by reducing the pH of the ZP suspension to 2.5 with
1 N HCl, followed by incubation at 378C for 15 min to complete
solubilization. The solution of ZP was then centrifuged at 16,000g
to remove any particulate matter; in most cases this was so little as
to be barely or not visible. Volumes of 1 N HCl for acidification and
1 N NaOH for subsequent re-neutralization were determined on
separate volumes of MJB. These volumes did not exceed 2 ml, so
that ZP concentration as ZP/ml was not affected. Neutralized ZP
solutions were frozen in 12-ml aliquots in liquid N
2
and stored at
"808C. To avoid loss of ZP due to adsorption to plastic or glass
surfaces during processing, all glassware and plasticware used after
the original homogenization were siliconized, as suggested by
Thaler and Cardullo (1996).
Live imaging of acrosomal exocytosis by EGFP fluorescence loss
Sperm from EGFP mice were collected as described above, and the
motile sperm in the preparation were isolated by swim-up into MJB
medium containing 3 mg/ml BSA and incubated for 60 min to effect
capacitation. Coverslips were washed overnight with 85% ethanol,
rinsed with water, and coated with laminin by adding a drop of
20 mg/ml laminin (Sigma Chemical Co.) and allowing to air-dry. The
laminin-covered coverslips were placed into a Leiden chamber
(Medical Systems, Greenvale, NY). The mouse sperm were affixed
to coverslips for fluorescence imaging of acrosomal EGFP. The
sperm were then washed twice with 400 ml of modified MJB
medium to remove non-attached sperm. The chamber was
placed onto the temperature-controlled stage of an inverted
epifluorescence Nikon TE2000 microscope at 378C equipped with
a 100# 0.5–1.3 NA S Fluor oil objective and a Princeton
Instruments MicroMAX CCD camera (Roper Scientific, Trenton,
NJ). Basal sperm fluorescence levels were recorded, and without
interruption of imaging, either purified mouse zona pellucida at a
concentration of 1.5 ZP/ml (10–20 ml) or 10 mM ionomycin (Sigma)
was added to the coverslip and incubated for 30 sec at 378C. For
analysis, we selected sperm that were oriented such that the side of
the head could be visualized and the apical, dorsal, and posterior
regions of the sperm head could be readily identified. These regions
are depicted in Figure 1A. Fluorescence emission was processed
with MetaFluor software (Universal Imaging, West Chester, PA).
The fluorescence signal was measured every 0.5 sec for 10 min.
Kinetics of EGP fluorescence loss from the sperm acrosome
induced by solubilized ZP or ionomycin, as recorded with the
imaging system described above, were expressed as half-times
from start of induction to complete loss of fluorescence. This
measure is independent of the kinetic order of the reaction and
provides a convenient quantitation of the reaction rate. Means of
the half-times and the standard errors of the means were calculated
with the GraphPad InStat program.
Results
ZP concentration dependence of acrosomal exocytosis
The protocol to prepare solubilized zona pellucida protein to
follow acrosomal exocytosis by the loss of EGFP fluorescence
was the same as that used by Rockwell and Storey (2000) to
study Ca
2þ
entry and acrosomal lumen pH equilibration at
the onset of the ‘‘acrosome reaction.’’ The efficacy of the ZP
Fig. 1. Acrosomal exocytosis in response to solubilized zona
pellucida. A: Schematic representation of the sperm head and
subdomains of the acrosome (green). B: Capacitated mouse sperm
undergo acrosomal exocytosis in a dose-dependent manner in
response to solubilized ZP. Capacitated mouse sperm (2 T 10
6
cells/
ml) in MJB/P medium were incubated 30 min at 37-C in the presence
of varying amounts of solubilized zona pellucida equivalents. In these
experiments, sperm that had undergone acrosomal exocytosis
were detected by observing the loss of EGFP from the acrosome.
The percent acrosomal exocytosis over baseline (Delta AE %) is
plotted as a function of the concentration of solubilized zona pellucida
equivalents (ZP/ml). Error bars represent SEM for n U 4
determinations per point. The solid curve fitted to the points is a
hyperbola, with the form of the equation: y U 58.1x/
[1.7 R x], r
2
U 0.792.
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613
Page 3
preparation was evaluated in that study by titration of the
ZP-stimulated ‘‘acrosome reaction’’ as monitored with the
Coomassie Blue protein staining protocol of Thaler and
Cardullo (1995) which likely assesses the concentrated protein
of the acrosomal matrix. The titration yielded a hyperbolic
curve with an apparent ‘‘K
M
’’ for ZP equivalents of 0.78 ZP/ml
and a maximal induced ‘‘acrosome reaction’’ value above
baseline of 51.2%. The comparable titration curve for ZP
dependence of acrosomal exocytosis as monitored by loss of
EGFP fluorescence, which represents the secretory release
of a soluble component from the acrosome, is shown in
Figure 1B. The apparent ‘‘K
M
’’ for ZP equivalents was 1.7 ZP/ml
and the maximal exocytosis value above baseline was 58.1%.
The values for the two titrations were in good agreement given
the difference in assay and strain of mice, so that the two
endpoints may be taken to be stages of the same ZP-stimulated
process. From the titration curve of Figure 1B, we chose the ZP
equivalent concentration of 2 ZP/ml as giving a near maximal
effect for use in all experiments.
ZP- and ionomycin-stimulation of EGFP
fluorescence loss
The loss of EGFP fluorescence stimulated by the ionophore
ionomycin was observed to progress randomly from the
posterior, anterior, or middle regions of the acrosomal arc
(Fig. 2, Fig. 4A, and Supplementary videos 1 and 2). The
progression of fluorescence loss did not show a sharp
boundary, consistent with ionophore-mediated pore formation
with no definite point of initiation. Loss of fluorescence
following ionomycin addition was rapid (the mean half-time
was 4.8 sec) but with substantial variation (SEM ¼ 3.1 sec). The
time course of EGFP loss in response to ionomycin was in
accordance with the previous reports of Nakanishi et al. (1999)
and Yamashita et al. (2007).
In contrast to the results with ionomycin, the stimulation of
acrosomal exocytosis by ZP protein routinely produced a loss
of EGFP fluorescence from the mouse sperm acrosome in
an orderly spatial progression almost exclusively from the
posterior point of the acrosomal arc (Fig. 3 and Supplementary
video 3). The boundary between fluorescence emission and
darkness due to fluorescence loss was sharp in all cells
examined, implying that pore opening leading to EGFP loss from
the acrosomal lumen proceeded in a graded manner from the
point of initiation. This pattern was observed in 90% of the
evaluated cells (Fig. 4B). The other 10% of the sperm displayed a
starting point of fluorescence loss in the anterior part of the
acrosome whereas the fluorescence loss was never observed
to originate in the middle region of the soluble ZP-treated cells.
Loss of the fluorescence was less rapid than sperm treated with
ionomycin. The mean half-time was 9.6 sec, with a wide range of
values, yielding an SEM of 7.6 sec.
We quantitated the total fluorescence present in the heads
of individual sperm as shown in Figure 5. The variations in the
time courses observed with both ionomycin and ZP are evident
from the curves. Of interest is the observation that some of the
curves show a transient increase in fluorescence just prior to
the onset of rapid fluorescence loss (Fig. 5). This enhancement
in fluorescence reported by the pH-sensitive EGFP may result
from a brief increase in intra-acrosomal pH just prior to or
coincident with the initial exposure of the acrosomal contents
to the more alkaline extracellular milieu.
We also analyzed the different patterns of EGFP
fluorescence loss in individual sperm using the ImageJ software.
To get an objective view on the loss of EGFP, sperm images
were placed in the same orientation with the posterior end of
the head to the left and the anterior region of the sperm to the
right and a rectangular region of interest was drawn around
the head of each sperm. The computer program was used to
create a histogram of fluorescence intensity along the sperm
head at each time point and the resulting graphs were
overlayed. In this manner we could quantitatively assess the loss
of EGFP fluorescence following stimulation by ionomycin or
soluble ZP. Examples of the random progression from the
posterior, middle, or anterior regions of the acrosomal arc
(arrows) of sperm treated with ionomycin are shown in
Figure 6A–C, respectively. In contrast, sperm stimulated with
Fig. 2. Acrosomal exocytosis of mouse sperm stimulated by the
calcium ionophore ionomycin progresses from random points.
Capacitated epididymal sperm were attached to laminin-coated
slides. After washing to remove the non-attached sperm, the cells
were exposed to 1 mM ionomycin and the disappearance of the EGFP
fluorescence from the acrosome was monitored. The numbers
indicate the elapsed time in second s since the addition of the reagent.
The arrows indicate the posterior end of the acrosomal cap regions.
The magnification bar represents 5 mm. Videos illustrating the effect
of ionomycin on sperm acrosome EGFP are available in the
supplemental material of this publication.
JOURNAL OF CELLULAR PHYSIOLOGY
614 B U F F O N E E T A L .
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ZP consistently showed a progression from the posterior end
of the acrosomal arc (Fig. 6D, arrow). We also found that the
level of fluorescence at the extreme anterior tip of the sperm
treated with ionomycin did not vary in the three examples
shown here. In contrast, there was a gradual loss of
fluorescence at the extreme anterior end of the acrosome of
ZP-treated sperm (oval) even though the majority of EGFP
fluorescence was lost in an anterograde direction from the
posterior end. This indicates that there is a slight loss of EGFP
from the anterior tip of the sperm that precedes or coincides
with the more prominent anterograde loss of fluorescence.
Discussion
Binary versus analog models for acrosomal dynamics
These experiments demonstrate that the zona pellucida-
stimulated process of acrosomal exocytosis is not random.
The prevalent Acrosome Reaction Model is a binary paradigm
consisting of two states: acrosome-intact and acrosome-
reacted. These two states of acrosomal dynamics are frequently
depicted to result from the random fusion of the PM and OAM
with the consequent, rapid release of acrosomal contents
(Yanagimachi, 1994). However, our results indicate that, as a
consequence of the exposure to ZP proteins, fusion of these
membranes and the subsequent loss of acrosomal contents
occurs in a predictable and orderly fashion. Previously, we
proposed that the exposure of acrosomal matrix components
is initiated at the apical tip of the acrosome and is followed
by the uncovering of matrix material in the more posterior
domains of the acrosome (Kim and Gerton, 2003). Those
experiments were carried out with different aliquots of live
sperm that were removed from the incubation mixture and
examined at various time points; in other words, we did not
follow individual sperm over the course of the incubation.
Those experiments identified four classes of sperm acrosomes
(1: matrix not exposed, EGFP present; 2: matrix exposed,
EGFP present; 3: matrix exposed, EGFP absent; and 4: matrix
absent, EGFP absent). In the studies presented in this report,
we were able to track the same sperm over the course of
the experiment, which allowed us to visualize the loss of the
EGFP from each acrosome, in essence more finely resolving
intermediate stages of acrosomal exocytosis between Classes 2
Fig. 4. Points of progression of EGFP fluorescence loss from the
mouse sperm acrosome. A total of 79 and 85 cells were analyzed for
ionomycin and solubilized ZP, respectively. Of these, acrosomal
exocytosis of individual cells was stimulated by ionomycin (A; n U 8) or
solubilized mouse zona pellucida (B; n U 7).
Fig. 3. Acrosomal exocytosis of mouse sperm stimulated by zona
pellucida consistently progresses from the posterior end of the
acrosome. Capacitated epididymal sperm were attached to
laminin-coated slides. After washing to remove the non-attached
sperm, the cells were exposed to 2 ZP/ml (solubilized zona pellucida
equivalents). We monitored the disappearance of the EGFP
fluorescence from the acrosome. The numbers indicate the elapsed
time in seconds since the addition of the ZP solution. The
magnification bar represents 5 mm. Videos illustrating the effect
of ionomycin on sperm acrosome EGFP are avai lable in the
supplemental material of this publication
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and 3. These findings further support the concept that
acrosomal exocytosis is an analog (continuous variable) process
with defined intermediate stages in sequence, rather than a two
point binary event.
Kinetics of acrosomal lumen pH increase and EGFP loss
induced by solubilized ZP and ionomycin
The acrosome is an acidic organelle that becomes alkalinized
during the time leading up to exocytosis (Allison and Hartree,
1970; Meizel and Deamer, 1978). It is useful to note that EGFP
can be used as an indicator of pH and has an overall pK
a
of 5.8
(Haupts et al., 1998). Taking advantage of the fact that EGFP
fluorescence increases at more alkaline pH values, Nakanishi
et al. (2001) demonstrated that the pH within the acrosome
rises from pH 5.3 to pH 6.2 over the course of sperm
capacitation. In several of the sperm we tracked, we detected a
transient increase in fluorescence just prior to the onset of
rapid fluorescence loss (Fig. 5). This enhancement in
fluorescence may result from a brief increase in intra-acrosomal
pH just prior to or coincident with the initial exposure of
the EGFP to the more alkaline extracellular milieu.
Alternatively, the fluorescence of intact acrosomes may be
slightly quenched if the EGFP is tightly packed with other
proteins in the acrosome; as the EFGP begins to diffuse away
following PM and OAM fusion, the quenching may be relieved
just before it is diluted into the medium surrounding the sperm
acrosome.
In accordance with the reports of Nakanishi et al. (1999) and
Yamashita et al. (2007), the time course of EGFP loss during the
course of acrosomal exocytosis is on the order of seconds
(Fig. 5C). The mean rate, as expressed by the half-time to
complete fluorescence loss, is twice as rapid for the loss
induced by ionophore as for that stimulated by solubilized ZP.
This is hardly unexpected, given that ionomycin would be
predicted to cause a massive influx of calcium all over the sperm
cell whereas ZP protein may act in a restricted area to effect
a localized calcium increase. The wide range of half-times for
the loss of EGFP from each sperm exposed to solubilized ZP
is remarkable and implies that sperm become primed for
acrosomal exocytosis at varying rates during incubation under
capacitating conditions.
As described by Storey and colleagues (Lee and Storey, 1989;
Rockwell and Storey, 2000), the kinetics of the ‘‘onset of the
acrosome reaction’’ triggered by soluble ZP or ionomycin in
capacitated mouse sperm have previously been determined
with weakly basic fluorescent probes [N-(N-dodecyl)
aminoacridine (NDAA) and dapoxyl-2-aminoethyl sulfonamide
(DAES)]. They found rates an order of magnitude slower
(%2 min) than those we observed for EGFP loss (% 10 sec). The
same half-time of 2 min was obtained in the earlier study (Lee
and Storey, 1989), in which structurally intact ZP were used and
the later study, in which acid-solubilized ZP, prepared as in this
study, were used. Greve and Wassarman (1985) had shown
that acid-solubilized ZP had lost the cross-linking of the intact
ZP, but had left the individual ZP2-ZP3 filaments intact (also see
Appendix in Rockwell and Storey (2000)). This preparation is
thus a reliable substitute for the structurally intact ZP, and far
closer to physiological than is isolated ZP3, which must be
prepared by sodium dodecyl sulfate denaturation followed by
electrophoresis. Since NDAA and DAES show bright
fluorescence only when they are bound to protein, a reasonable
explanation for the observed half-times is that the weakly basic
probes distribute to the acidic acrosomal compartment in
acrosome-intact sperm and there bind to acrosomal matrix
proteins. Upon equilibration of the compartment pH with the
suspending medium, the probes dissociate from matrix protein
at a characteristic rate that is much slower, but far more
consistent, than the rate observed for EGFP loss in the initial
onset of exocytosis. The lower intensities of NDAA or DAES
fluorescence observed after stimulation of the sperm with
soluble ZP or ionomycin reflects probe bound to matrix
protein remaining on the IAM after the completion of OAM and
PM fusion.
Fig. 5. Kinetics of EGFP disappearance during acrosomal
exocytosis. A total of 79 and 85 sperm were analyzed after treatment
with ionomycin and solubilized ZP, respectively. Of these, acrosomal
exocytosis was stimulated in sperm by ionomycin (A; n U 8) or
solubilized zona pellucida (B; n U 7). Using imaging software, a region
of interest (ROI) was selected around the head of a sperm undergoing
acrosomal exocytosis and the fluorescence intensity within the box
was determined. An equivalent sized ROI of a background region was
selected to determine the baseline fluorescence. For normalization of
the data, an ROI was also selected that encompassed a sperm head
that did not undergo acrosomal exocytosis in response to the
stimulant. For each sperm, the background fluorescence was
subtracted and the value of the exocytotic sperm was divided by the
non-responsive sperm to give the normalized fluorescence intensity
in relative units (ordinate). In C, half-times (t
1/2
) of the decrease in
EGFP fluorescence observed on treatment of capacitated mouse
sperm with ionomycin or solubilized zona pellucida are presented.
Values given are means W SEM. The decrease in fluorescence was
allowed to proceed for 10 min at which time a new steady state
had been reached.
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Fig. 6. Schematic representation of the different patterns of EGFP fluorescence loss. For this experiment, a rectangular ROI was drawn around
the head of each sperm with the posterior end of the head to the left and the anterior region of the sperm to the right; using imaging software,
a histogram of fluorescence intensity at each time point was produced and the graphs overlayed in each part. The loss of EGFP fluorescence
stimulated by ionomycin was observed to progress randomly from the posterior (A), middle (B), or anterior (C) regions of the acrosomal arc
(arrows). In contrast, the stimulation with ZP consistently showed a progression from the posterior end of the acrosomal arc (D, arrow). Also note
that the anterior fluorescence of the extreme anterior tip of the sperm treated with ionomycin did not vary in the three examples shown here. In
contrast, there was a gradual loss of fluorescence at the extremeanterior end ofthe acrosome ofZP treated sperm(oval) even thoughthe majority
of EGFP fluorescence was lost in an anterograde direction from the posterior end.
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Role of calcium in acrosomal exocytosis
While extracellular Ca
2þ
is obligate for acrosomal exocytosis,
there is still controversy about where calcium is stored in
sperm. One scheme postulates that the acrosome is a calcium
store and that the depletion of Ca
2þ
from the acrosome
activates store-operated channels (SOCs) that allow sustained
entry of Ca
2þ
from the medium (Herrick et al., 2005). The influx
of Ca
2þ
through SOCs could drive the fusion events between
the PM and OAM. The ordered progression of EGFP loss
implies that the SOCs are localized near the posterior region of
the acrosome and that the Ca
2þ
wave proceeding from SOCs at
this point drives the orderly formation of site(s) of PM and
OAM fusion through which EGFP is lost over a period of several
seconds. In contrast to the concept of the acrosome as a silo for
calcium ions, Ho and Suarez (2001) identified the redundant
nuclear envelope at the posterior end of the sperm head as a
calcium store. Bedu-Addo et al. (2008) have elaborated on this
theme, providing evidence that an inositol 1,4,5-trisphosphate
(IP3) receptor-gated Ca
2þ
store exists near the base of the
flagellum and provides Ca
2þ
to initiate hyperactivation. Along
these lines, Xia and Ren (2009) have recently captured
intriguing video images that indicate that CATSPER calcium
channels localized in the principal piece of the sperm flagellum
are required for [Ca
2þ
]
i
changes that occur in response to
soluble ZP. Curiously, the ZP-induced [Ca
2þ
]
i
increase starts in
the sperm tail (near the annulus, the demarcation point
between the principal piece and midpiece of the tail) and
propagates toward the head. In the head, a prominent wave of
calcium then occurs in an anterograde direction from the base
of the head, reminiscent of the loss of EGFP we have
demonstrated in the study reported here. Although these
laboratories have focused on the importance of their results on
(hyperactive) motility, the physical proximity of such a calcium
store to the posterior region of the sperm acrosome and the
anterograde wave of calcium in response to soluble ZP suggests
to us that a calcium store near the head-tail junction may be
associated with ZP-stimulated acrosomal exocytosis.
Directionality of EGFP loss stimulated by ZP: evidence
for a ZP ligand ‘‘trigger’’?
Loss of EGFP fluorescence from the mouse sperm acrosome
upon stimulation of acrosomal exocytosis by soluble ZP
occurred as an orderly anterograde progression initiating from
the posterior point of the acrosomal arc. Of the sperm cells
examined, 90% showed initiation of EGFP loss at the posterior
point; no initiation at the midpoint was observed. When
the Ca
2þ
ionophore, ionomycin, acted as the trigger, loss of
EGFP fluorescence occurred from the posterior, anterior, and
midpoints of the acrosomal arc at approximately the same
proportion (Fig. 4). The random ionophore result was
expected: ionomycin mediates Ca
2þ
entry through the plasma
membrane into the sperm interior by creation of a lipophilic
cation carrier complex, without regard to cellular localization.
The former result with solubilized ZP was unanticipated: the
defined spatial anterograde progression of EGFP loss from the
posterior acrosome implies the presence of a localized
receptor site for a ZP ligand. One may thus conceive of a zona
ligand ‘‘trigger’’ with a localized target near the posterior
acrosomal PM site (Kligman et al., 1991; Kerr et al., 2002).
Fig. 7. Model of sperm–ZP interaction. Capacitation results in an initial rise in acrosomal pH and is accompanied by a mobilization of Ca
2R
that
serves to mediate the fusion of the PM and OAM of the apical tip of mouse sperm acrosomes, exposing some of the underlying acrosomal matrix
protein that becomes available for ZP binding. Once initial contact to the ZP is made through the apical tip, movement of the sperm brings the
dorsalsurface of the spermheadintocontact with the zonasurface.Fusionof the PM andOAMprogressesinan anterograde direction,leadingto the
release of soluble acrosomal components and initiating the gradual dispersion of acrosomal matrix proteins. The gradual release of acrosomal
matrix proteins, which include ZP-binding proteins and potential zona hydrolases, occurs at the outer margins of this structure, and the freshly
exposed proteins then participate in another round of binding and release, enabling the motile sperm to ratchet its way through the zona.
JOURNAL OF CELLULAR PHYSIOLOGY
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Our experiments bear some similarities to a recent study
with human sperm from Harper et al. (2008). These
investigators resolved acrosomal exocytosis in live human
sperm by video imaging the accumulation of fluorescently-
tagged proteins that bind to acrosomal proteins. Soybean
trypsin inhibitor (SBTI: to detect acrosomal serine-like
proteases such as acrosin) and an antibody against CD46 (an
inner acrosomal membrane protein) were found to bind to the
acrosomal region of human sperm undergoing exocytosis. The
exposure of SBTI binding sites generally initiated at the apical tip
of the human sperm treated with progesterone or calcium
ionophore A23187 (completing in less than a minute) whereas
the exposure of CD46 appeared to occur more randomly and
at a slower rate (on the order of several minutes). This time
course for SBTI binding site detection is about 5–6 times slower
than what we saw for EGFP loss from mouse sperm exposed to
ionomycin. These differences could result from several factors:
Our laboratories used different species. We measured the loss
of a soluble component of the acrosome (EGFP) rather than the
(likely slower) binding and accumulation of a fluorescently
tagged protein (SBTI or anti-CD46) to an acrosomal matrix or
inner acrosomal membrane protein. It would be interesting to
see if human sperm exhibit a similar directionality of OAM and
PM fusion in response to ZP as we have observed with mouse
sperm.
A recent report published by Baibakov et al. (2007)
challenges models that rely on zona-mediated processes for
sperm binding and penetration through the ZP. In their
experiments, they used GFP-labeled sperm from a transgenic
mouse model similar to the one used in our study and eggs
enclosed by ZP containing human ZP2 (expressed from a
transgene either together with endogenous mouse ZP2 or in a
‘‘rescue’’ experiment in ZP2-null animals). They found that
sperm that had bound to ZP containing human ZP2 retained
GFP for extended periods of time, suggesting that sperm
binding to ZP is not sufficient to induce acrosome exocytosis. In
addition, they examined the sperm that had passed through
polycarbonate filters of decreasing sizes and found that filters
with pores sizes less than 5 mm only allowed the passage of
‘‘acrosome-reacted’’ sperm. As a consequence of these studies,
they challenged the ‘‘glycan release’’ model sperm binding to a
carbohydrate ligand of the ZP of an unfertilized egg; upon
fertilization, this ligand is postulated to be removed by a
glycosidase released from egg cortical granules, thereby
eliminating the ability of sperm to bind to the early embryo
(Wassarman, 2005; Shur et al., 2006). Instead, they proposed
that a ‘‘mechanosensory’’ signal produced by the penetration of
the sperm through the zona pellucida matrix is sufficient for
inducing acrosomal exocytosis. These results are inconsistent
with the ligand-receptor signal transduction model in
acrosome exocytosis, supported by many observations that
acid-solubilized ZP or purified ZP3 can stimulate acrosomal
exocytosis (Bleil and Wassarman, 1980; Bleil and Wassarman,
1983; Kopf, 2002). From our perspective, any model for
acrosomal exocytosis must take into account the
microenvironment at the site of sperm-zona pellucida
interaction, the capacitation state of the sperm, and the kinetics
of acrosomal exocytosis compared to conditions utilizing native
ZP. Spontaneous acrosomal exocytosis naturally occurs at a
slow rate under capacitating conditions but in the presence of
ZP, PM, and OAM fusion is stimulated and leads to an
accelerated rate of GFP loss from the sperm acrosomes.
Model for sperm zona interactions
Acrosomal exocytosis encompasses several distinctive events:
the fusion of the OAM and PM (which leads to the exposure of
acrosomal contents), the rapid loss of soluble acrosomal
components, and the more gradual but slower dispersion of the
acrosomal matrix proteins. Exposure and delayed release of the
acrosomal matrix proteins means that these components
remain associated with the sperm head and can participate in
zona pellucida binding for a longer period of time. Their
progressive dispersal likely takes place over a time scale long
enough to allow the sperm to enter and penetrate the zona
pellucida under physiological conditions (Kim and Gerton,
2003). In addition, Kerr et al. (2002) fluorescently labeled ZP2
and ZP3 and used quantitative image analysis to characterize the
saturable binding of ZP2 and ZP3 to distinct sites on living,
capacitated, acrosome-intact mouse sperm. They found about
20% of the ZP3 binding sites were localized over the acrosomal
cap, whereas the remaining sites were located over the
postacrosomal region of the head. In contrast, ZP2 binding sites
were detected only over the postacrosomal region.
Based on these findings and those of others, we propose the
following working model: Capacitation results in an initial rise in
acrosomal pH and is accompanied by a mobilization of Ca
2þ
that serves to mediate the fusion of the PM and OAM of the
apical tip of mouse sperm acrosomes, exposing some of the
underlying acrosomal matrix protein that becomes available for
ZP binding. This initial fusion site is metastable but is sustained
when a sperm interacts with the zona pellucida. Once initial
contact to the ZP is made through the apical tip, movement of
the sperm brings the posterior region of the sperm head into
contact with the zona surface. ZP-responsive signaling
molecules on the surface of the posterior region of the
acrosome (ZP2 and/or ZP3 binding sites of the postacrosomal
region?) are activated, leading the mobilization of internal
stores of calcium, which, in turn, stimulate SOCs and the fusion
of the PM and OAM in the posterior domain of the acrosome.
Fusion of the PM and OAM progresses in an anterograde
direction, leading to the release of soluble acrosomal
components and initiating the gradual dispersion of acrosomal
matrix proteins. The gradual release of acrosomal matrix
proteins, which include ZP-binding proteins and putative zona
hydrolases, occurs at the outer margins of this structure, and
the freshly exposed proteins then participate in subsequent
rounds of binding and release, enabling the motile sperm to
ratchet its way through the zona.
Acknowledgments
We thank Tanya Merdiushev for her assistance with the
Metamorph imaging and our fellow laboratory members for
encouragement. This work was supported by National
Institutes of Health. Grant numbers: R01 HD-41552 (to
G.L.G.); Fogarty 5D43TW000671 (to M.G.B. and E.R.M.).
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  • Source
    • "This confirmed an earlier report that rabbit sperm recovered from the perivitelline space can fertilize zona-intact oocytes (Kuzan et al., 1984). When taken together with the observations that AE of sperm is minimal or nonexistent when the ZP proteins are assembled in the native three-dimensional structure (Baibakov et al., 2007; Buffone et al., 2009), these findings strongly suggest that ZP is not the primary physiological inducer of the acrosome reaction (Yanagimachi, 2011). Furthermore, the findings raise the question of where fertilizing spermatozoa initiate AE. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.
    Full-text · Article · Feb 2016 · Developmental Biology
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    • "In contrast, AR induced by a more physiological stimulus like Progesterone typically starts at the anterior region of the sperm head. A similar conclusion was reached in mouse spermatozoa regarding the starting point of AR promoted by either zona pellucida or Ca 2+ ionophore[31]. Similarly to what we observed in mouse sperm, in humans, most of sperm undergoing[Ca 2+ ]i oscillations after Progesterone addition fail to undergo AR. "
    [Show abstract] [Hide abstract] ABSTRACT: During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatio-temporal correlation between the changes in ([Ca(2+)]i and AR in single mouse spermatozoa in response to Progesterone. We found that Progesterone stimulates an ([Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory and sustained. We also observed that the ([Ca(2+)]i increase promoted by Progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in ([Ca(2+)]i and the process of exocytosis in response to Progesterone and found that only a specific transitory increase in ([Ca(2+)]i originated in the sperm head promotes the initiation of AR.
    Full-text · Article · Jan 2016 · Biology of Reproduction
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    • "Men or mice carrying mutations affecting the process of acrosomal exocytosis are infertile or display some degree of subfertility (Dam et al., 2007; Kang-Decker et al., 2001; Lin et al., 2007). To penetrate the zona pellucida (ZP), the extracellular matrix surrounding the egg, mammalian sperm must undergo acrosomal exocytosis in an orderly manner (Buffone et al., 2009; Yanagimachi, 1994 ). In addition, only acrosome-reacted sperm are able to relocalize Izumo1, a protein essential for sperm egg-fusion, to the equatorial segment (Miranda et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 are essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jul 2015 · Developmental Biology
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