Department of Cell Biology, Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA
* Author for correspondence (e-mail: barry{at}cellbio.emory.edu)
Accepted 29 September 2003
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SUMMARY |
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Key words: Capacitation, Fertilization, Sperm, Acrosome reaction, cAMP
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Introduction |
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One of the sperm proteins thought to function during fertilization is
ß1,4-galactosyltransferase I (GalT I; B4galt1 Mouse Genome
Informatics). GalT I normally serves as a biosynthetic enzyme in the Golgi
apparatus; however, in certain cell types, it also functions as a cell-surface
receptor for extracellular glycoside ligands (reviewed by
Rodeheffer and Shur, 2002).
Mouse sperm are unusual in that all of their GalT I is present on the plasma
membrane overlying the acrosome, where it functions as a receptor for the
major sperm-binding ligand in the zona pellucida, ZP3. ZP3-dependent
aggregation of GalT I initiates pertussis toxin-sensitive signaling pathways
that contribute to induction of the acrosome reaction
(Gong et al., 1995
;
Lopez et al., 1985
;
Miller et al., 1992
;
Scully et al., 1987
;
Shi et al., 2001
;
Shur and Hall, 1982a
;
Shur and Neely, 1988
).
Consistent with this, sperm that overexpress GalT I bind more ZP3, have higher
rates of G-protein activation, and undergo accelerated acrosomal exocytosis,
relative to normal sperm (Youakim et al.,
1994
). Similarly, sperm from males devoid of all GalT I, or devoid
of just the surface isoform of GalT I, fail to bind ZP3 or undergo a
ZP3-induced acrosome reaction. However, despite their inability to bind ZP3,
GalT I-null sperm are still able to bind to the egg coat and fertilize eggs in
vitro, albeit at very low efficiency (Lu
and Shur, 1997
). The fertility of GalT I-null mice suggests that
sperm binding to the zona pellucida requires a GalT-ZP3-independent
interaction (Rodeheffer and Shur,
2004
). Furthermore, there is reason to believe that the process of
capacitation may be accelerated in GalT I-null sperm, relative to wild-type
sperm. The capacitation phenotype of GalT I-null sperm is the subject of this
report.
Capacitation is defined as the complement of physiological changes that
sperm undergo in the female reproductive tract before gaining the competence
to fertilize the egg (Austin,
1952; Chang, 1951
).
Capacitation can also occur in vitro in medium mimicking the fluid of the
female reproductive tract (Oliphant and
Brackett, 1973
). In vitro capacitation results in increased sperm
metabolism (Fraser and Herod,
1990
; Hoppe,
1976
), alterations in plasma membrane fluidity
(Wolf et al., 1986
) and lectin
reactivity (Johnson and Hunter,
1972
; Talbot and Franklin,
1978
), hyperactivated motility
(Ho and Suarez, 2001
),
elevated intracellular pH (Zeng et al.,
1996
), membrane hyperpolarization
(Demarco et al., 2002
) and
protein tyrosine phosphorylation (Visconti
et al., 1995a
). In many species, these events are negatively and
positively regulated by factors in seminal plasma and in the fluid of the
female reproductive tract, respectively. Membrane fluidity is dependent on the
proportion of sterols (cholesterol and desmesterol) in the lipid bilayer,
which is regulated by lipid vesicles in seminal plasma and cholesterol sinks,
such as albumin, in the reproductive tract
(Davis, 1981
;
Nimmo and Cross, 2003
;
Visconti et al., 1999
).
Ca2+ is essential for capacitation
(Yanagimachi, 1982
) and
controls the maintenance of sperm motility, the display of hyperactivation
(Ho et al., 2002
;
Ren et al., 2001
) and the
membrane fusion that occurs during the acrosome reaction
(Yanagimachi and Usui, 1974
).
HCO3 is also required for capacitation
(Lee and Storey, 1986
) and
functions with Ca2+ to regulate cAMP production
(Garbers et al., 1982
;
Litvin et al., 2003
).
Additionally, HCO3 appears to mediate a wide
range of processes via its direct stimulation of adenylyl cyclase
(Chen et al., 2000
;
Okamura et al., 1985
).
HCO3, and possibly the cAMP produced by this
enzyme, leads to increased protein tyrosine phosphorylation
(Visconti et al., 1995b
),
phospholipid disorder on the plasma membrane
(Gadella and Harrison, 2002
)
and changes in motility (Wennemuth et al.,
2003
).
We suspected that GalT I-null sperm may undergo precocious capacitation
based on three observations: (1) on wild-type sperm, epididymal
glycoconjugates normally occupy or `mask' the GalT I active site and maintain
sperm in an `uncapacitated' state (Shur
and Hall, 1982b); (2) washing wild-type sperm releases these
glycoconjugates and results in precocious binding to the zona pellucida
without a requirement for in vitro capacitation; and (3) GalT I-null sperm
appear as single, motile cells upon release from the epididymis, whereas
wild-type sperm are initially clumped and relatively immotile
(Lu and Shur, 1997
). As GalT
I-null sperm should be unable to bind these epididymal `decapacitating'
glycoconjugates, their rate of capacitation may be accelerated relative to
wild-type sperm in which capacitation is dependent upon the removal of these
epididymal factors.
The goal of this study is to test the hypothesis that GalT I-null sperm are precociously capacitated relative to wild-type sperm. By two distinct criteria, GalT I-null sperm appear to exhibit precocious capacitation: (1) GalT I-null sperm bind maximally to the zona pellucida independent of capacitation in vitro and (2) they are more sensitive to Ca2+ ionophore-induced acrosome reactions prior to and throughout capacitation, than are wild-type sperm. The accelerated rate of capacitation in GalT I-null sperm is correlated with both their inability to bind `decapacitating' factors and to constitutively activated Ca2+- and HCO3 -dependent signaling cascades. The precocious capacitation phenotype of GalT I-null sperm is specific to events occurring in the sperm head, whereas tail-specific events, such as motility, are unaltered.
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Materials and methods |
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Sperm collection
One cauda epididymis of strain-matched wild-type and long isoform GalT
I-null mice (Lu and Shur,
1997) was dissected into 2 ml complete medium, and the other into
2 ml incomplete medium and shredded. The epididymides were incubated for 15
minutes at 37°C and the sperm were collected after filtration (3-35/27
Nitex, Sefar America, Kansas City, MO). The sperm suspension was centrifuged
at 66 g for 5 minutes at 24°C and resuspended in fresh
media (complete or incomplete) at a final concentration of
2x106 sperm/ml. Sperm were capacitated at 37°C for
varying time periods in complete or incomplete medium before assaying in
complete medium (Fig. 1). To
determine whether decapacitating factors in sperm fluid alter capacitation,
sperm were released from the epididymis by trituration (not swim out),
filtered, centrifuged and resuspended in fresh media. The suspension was
divided into two equal volumes and both pelleted a second time at 66
g for five minutes. One sample was resuspended in fresh media
(`washed') while the second sample was resuspended in the original supernatant
(`unwashed').
|
A23187-induced acrosome reaction
Sperm were collected, and at specific points during in vitro capacitation
(Fig. 1) a 100 µl aliquot of
sperm was added to 0.5 mM A23187 in DMSO to give a final concentration of 10
µM A23187. An equal volume of DMSO alone was used as a control. For sperm
capacitated in incomplete medium, the suspension was `spiked' with the
appropriate cofactor at the same time A23187 was added. The sperm were further
incubated at 37°C for 10 minutes and fixed with 4% paraformaldehyde at
24°C. The acrosomes of the sperm were stained with Coomassie Blue
following the procedure described (Larson
and Miller, 1999). Three drops of 200 sperm each were counted for
each time point and averaged. The data presented are the average of at least
three experiments (±s.e.m.).
Measurement of cAMP
Sperm were collected as described. After filtration and centrifugation, the
sperm were resuspended at a final concentration of 1x107
sperm/ml, and capacitated in vitro. The sperm were then centrifuged at 1000
g for 4 minutes and the supernatant was discarded. The cAMP
was extracted from sperm with 100 µl 65% ice-cold ethanol for 15 minutes
and then at 20°C for 1 hour. The insoluble material was washed with
100 µl 65% ethanol and the extracts were combined and centrifuged at 2000
g for 15 minutes at 4°C. The supernatants were dried under
vacuum and the cAMP was detected using the cAMP Biotrak Enzyme immunoassay kit
(Amersham Biosciences, Piscataway, NJ). Each time point was analyzed in
duplicate samples and the data shown reflects the mean±s.e.m. for four
experiments.
Anti-phosphotyrosine immunoblot
Sperm were collected as described, with the exception that the `swim-out'
period lasted for only 5 minutes instead of 15 minutes. Sperm lysates were
prepared and analyzed as described
(Visconti et al., 1995a).
After the lysates were fractionated by SDS-PAGE, the proteins were transferred
to nitrocellulose and blocked in 3% milk, 0.01% Tween-20, 0.9% NaCl, Tris pH
7.4. The membranes were probed with 1 µg/ml of anti-phosphotyrosine mouse
monoclonal antibody, washed and subsequently incubated in a 1:10,000 dilution
of goat anti-mouse IgG-HRP (both from Upstate Biotech, Lake Placid, NY). The
membranes were washed again and developed with ECL Plus (Amersham Biosciences,
Piscataway, NJ). After each experiment, the membranes were stripped and
reprobed with 1 µg/ml of anti-
-tubulin mouse monoclonal antibody
(Sigma) to assess consistency of protein loading. The blot shown is
representative of six experiments.
Measurement of protein kinase A (PKA) activity
Sperm were collected as described and PKA activity was measured following
the procedure described (Visconti et al.,
1997) with a few exceptions. Owing to high contaminating PKA
activity in epididymal fluid, sperm were capacitated for varying periods of
time and subsequently washed three times with ice-cold medium by
centrifugation at 200 g at 4°C followed by resuspension in
fresh buffer. After the final wash, the sperm were resuspended at a
concentration of 1x107 cells/ml for the kinase assay. Owing
to potential loss of sperm during washing, each result (pmol
32P-labeled kemptide) was normalized to the quantity of protein in
each sample. Each time point was analyzed in triplicate, and the data shown
reflects the mean±s.e.m. for three separate experiments.
Computer-assisted semen (epididymal sperm) analysis
Sperm were collected and capacitated in complete medium for 10 or 60
minutes at 37°C. Each sperm suspension (10 µl) was loaded onto a 20
µm MicroCell slide (Conception Technologies, San Diego, CA) and analyzed in
duplicate using an IVOS sperm analyzer (Hamilton Thorne, Beverly, MA). The
parameters of the CASA analysis were: frames acquired, 30; frame rate, 60 Hz,
minimum contrast, 80; minimum cell size, three pixels; minimum static
contrast, 80; straightness (STR) threshold, 8.0%; low VAP cutoff, 5.0 µm/s;
low VSL cutoff, 11 µm/s; head size, non-motile, six pixels; head intensity,
non-motile, 160; static head size, 1.0-2.9; static head intensity, 0.6-1.4;
static elongation, 0-80; slow cells motile, no; magnification, 10x. The
data presented are the average of data (±s.e.m.) collected from three
males.
Statistical analysis
The probability that the data are statistically significant was calculated
using a paired t-test through the
www.graphpad.com
website. Data in which P<0.01 are indicated with an asterisk.
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Results |
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|
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As shown in Fig. 4,
wild-type and GalT I-null sperm continue to exhibit a time-dependent increase
in ionophore-induced acrosome reactions after washing. The sperm are
significantly less sensitive to ionophore after this procedure (compare
Fig. 4 with
Fig. 3) presumably because
trituration is more disruptive than allowing the sperm to swim out of the
epididymis. Nevertheless, washing wild-type sperm results in increased
sensitivity to A23187 during in vitro capacitation. By contrast, washing GalT
I-null sperm has no apparent effect, because as expected; they are already
genetically `washed' owing to the loss of GalT I, the binding site for the
decapacitating factors (Shur and Hall,
1982b). Therefore, decapacitating factors bound to wild-type sperm
appear to inhibit the initiation of capacitation; removal of these factors
during normal capacitation, by centrifugal washing or by elimination of GalT I
initiates at least some aspects of capacitation. These results suggest that
GalT I maintains sperm in an uncapacitated state until they enter the female
reproductive tract or are incubated in capacitating medium.
|
As before, capacitation was monitored by two criteria; sensitivity to A23187-induced acrosome reactions (Fig. 5) and the ability to bind the zona pellucida (Fig. 6). Sperm were capacitated in incomplete medium (i.e. absence of albumin, Ca2+ or HCO3) for varying periods of time, after which sensitivity to A23187 was determined in complete medium (i.e. containing all co-factors). This ensures that the only co-factor-dependent effects being assayed were during the capacitation period, and not during induction of the acrosome reaction. For wild-type sperm, eliminating each of these co-factors from the capacitation medium significantly reduces the rate of capacitation, such that sperm do not respond to A23187 until late in capacitation (i.e. 45 minutes). For GalT I-null sperm, the rate of capacitation is also somewhat reduced in the absence of these co-factors. However, the rate at which GalT I-null sperm undergo capacitation in the absence of each co-factor is similar to the rate of wild-type sperm capacitation in complete medium. In other words, GalT I-null sperm phenocopy wild-type sperm when capacitated in the absence of albumin, Ca2+ or HCO3, suggesting they are primed to undergo capacitation independent of these co-factors.
|
|
The precocious capacitation of GalT I-null sperm correlates with elevated cAMP levels
The downstream targets of Ca2+ and
HCO3 during capacitation have not been
extensively characterized. However, both Ca2+ and
HCO3 directly modulate the intracellular levels
of cAMP (Garbers et al., 1982;
Litvin et al., 2003
). cAMP is
necessary and sufficient for multiple events during capacitation, including
tyrosine phosphorylation (Visconti et al.,
1995b
). Sperm adenylyl cyclases are regulated by
Ca2+-binding proteins, such as calmodulin
(Gross et al., 1987
), as well
as directly by Ca2+ and HCO3
(Garbers et al., 1982
;
Garty and Salomon, 1987
;
Litvin et al., 2003
).
Owing to the Ca2+ and HCO3 -independent nature of capacitation in GalT I-null sperm, we predicted that they may have elevated levels of cAMP, and therefore compared cAMP levels during in vitro capacitation of wild-type and GalT I-null sperm (Fig. 7). Four points deserve emphasis. First, GalT I-null sperm have constitutively higher cAMP levels than do wild-type sperm. Second, cAMP levels are highest in GalT I-null sperm at 0-15 minutes of capacitation when GalT I-null sperm display the greatest difference from wild-type sperm with respect to zona binding and sensitivity to acrosomal exocytosis. Third, eliminating HCO3 from the capacitation medium reduces cAMP levels in both wild-type and GalT I-null sperm, but GalT I-null sperm retain approximately twice the levels of cAMP. Fourth, the quantity of cAMP in GalT I-null sperm capacitated in the absence of HCO3 is almost identical to cAMP levels in wild-type sperm capacitated in complete medium. Thus, the relative cAMP levels closely mirror the capacitative state of GalT I-null sperm.
|
|
Because the profiles of protein tyrosine phosphorylation are similar in wild-type and GalT I-null sperm, we suspected that PKA activity would also be similar between wild-type and GalT I-null sperm. As shown in Fig. 8C, PKA activity increases during capacitation in a HCO3 -dependent manner to the same extent in wild-type and GalT I-null sperm lysates.
Sperm motility is unaltered in GalT I-null sperm
The appearance of a specific pattern of motility, called hyperactivation,
has been associated with the capacitated state
(Ho and Suarez, 2001). To
determine whether wild-type and GalT I-null sperm exhibit different motility
patterns during the course of in vitro capacitation, the progressive velocity
of sperm was measured by computer-assisted sperm analysis (CASA)
(Fig. 9). Motility values were
similar for both wild-type and GalT I-null sperm during early (10 minutes) and
late (60 minutes) stages of in vitro capacitation. When individual sperm were
observed following the procedure of Tessler et al.
(Tessler et al., 1981
), no
differences in motility were observed (data not shown). Individual sperm
within each population exhibited hyperactivated motility (figure-of-eight
swimming pattern), but there was no difference in the number of hyperactivated
wild-type or GalT I-null sperm. This result, along with those in
Fig. 8, suggests that not all
aspects of capacitation are globally accelerated in GalT I-null sperm. Rather,
processes dependent on GalT I function (zona pellucida-binding and the
acrosome reaction) are accelerated, whereas GalT I-independent events
(tyrosine phosphorylation and motility) are unaltered.
|
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Discussion |
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Capacitation is often assayed by successful fertilization in vitro. We did
not use this assay here as GalT I-null sperm fertilize eggs at only 7% of the
efficiency of wild-type sperm because of to their inability to undergo
zona-induced acrosomal exocytosis (Lu and
Shur, 1997). Zona pellucida binding is an obligatory step during
mammalian fertilization and this process is capacitation dependent
(Si and Olds-Clarke, 1999
).
Using this criteria, we determined that wild-type sperm show a time-dependent
increase in zona-binding during capacitation. By contrast, GalT I-null sperm
bind to eggs without any requirement for capacitation
(Fig. 2). Thus, by this
measure, GalT I-null sperm are precociously capacitated.
A second method to monitor capacitation status is to assay for the ability to undergo a ligand-induced acrosome reaction. Neither of the two physiological inducers of the acrosome reaction (ZP3 or progesterone) can induce acrosome reactions in GalT I-null sperm. Therefore, we determined whether the competence to undergo an acrosome reaction in response to Ca2+ influx was also a capacitation-dependent event. For wild-type sperm, short exposures to the Ca2+ ionophore A23187 induced the acrosome reaction only in sperm incubated for at least 30 minutes in capacitating medium. Sperm incubated with A23187 prior to capacitation, or capacitated in medium lacking albumin, Ca2+ or HCO3, failed to respond to A23187 (Figs 3 and 5). These results suggest that A23187 induces the acrosome reaction in a capacitation-dependent manner. In this assay, GalT I-null sperm showed both a higher basal level of sensitivity to A23187 (at 0 minutes) as well as an increased rate of A23187-induced acrosome reactions within the first 30 minutes of capacitation (Fig. 3). Thus, by a second, independent assay, GalT I-null sperm display a precociously capacitated phenotype.
Capacitation of GalT I-null sperm is independent of cofactors normally required for capacitation
We explored the mechanisms that contribute to the precocious capacitation
phenotype of GalT I-null sperm. First, we hypothesized that GalT I-null sperm
undergo precocious capacitation because they are uninhibited by decapacitating
epididymal glycoconjugates normally bound to GalT I on the surface of
wild-type sperm (Oliphant et al.,
1985; Shur and Hall,
1982b
). Consistent with this, washing sperm to completely remove
these factors increases the capacitation rate of wild-type sperm, but has no
appreciable effect on capacitation in GalT I-null sperm
(Fig. 4). This suggests that
washing wild-type sperm causes them to approach the behavior of GalT I-null
sperm, which are genetically `washed' and maximally capacitated. At least some
decapacitating factors are known to function as substrates for GalT I;
however, they may also be vesicular in nature, and washing sperm may promote
capacitation by altering the sterol:phospholipid ratio in the sperm plasma
membrane (Davis, 1981
).
Second, we hypothesized that GalT I inhibits the activation of signaling pathways normally required for capacitation. This predicts that in the absence of GalT I, capacitation would occur at an accelerated rate and may be independent of regulatory cofactors in the female reproductive tract (or in vitro). We tested this prediction by measuring the rate of capacitation in wild-type and GalT I-null sperm incubated in the absence of the obligatory cofactors albumin, Ca2+ and HCO3. As expected, wild-type sperm require each of these co-factors to undergo capacitation (Figs 5 and 6). Interestingly, in the absence of Ca2+ or HCO3, GalT I-null sperm still display maximal binding to the zona pellucida and sensitivity to ionophore. In addition, GalT I-null sperm require albumin for zona pellucida-binding but not for the A23187-induced acrosome reaction. GalT I-null sperm may display a differential requirement for albumin because the state of a plasma membrane competent to undergo vesicle fusion may not be the same as the state of a membrane competent to undergo ligand binding.
Precocious capacitation of GalT I-null sperm correlates with elevated cAMP levels
As GalT I-null sperm undergo capacitation in the absence of Ca2+
and HCO3 at the same rate as wild-type sperm
undergoing capacitation in complete medium, it is possible that processes
regulated by these two co-factors are constitutively active in GalT I-null
sperm. We initially focused on a target that is influenced by both
Ca2+ and HCO3: cAMP produced by the
soluble isoform of adenylyl cyclase
(Garbers et al., 1982;
Litvin et al., 2003
). We
determined whether this enzyme (or other sources of cAMP) might be
constitutively active in GalT I-null sperm by measuring the cAMP content of
sperm during capacitation.
cAMP levels correlate closely with the capacitation phenotype of wild-type and GalT I-null sperm (Fig. 7). Basal levels of cAMP are constitutively elevated in GalT I-null sperm consistent with their constitutively higher rates of zona pellucida binding and acrosomal exocytosis. Furthermore, capacitating GalT I-null sperm in the absence of HCO3 leads to cAMP levels similar to that in wild-type sperm capacitated in complete medium; conditions in which GalT I-null sperm behave similar to wild-type sperm during capacitation (Figs 5 and 6). This result suggests that the precocious capacitation of GalT I-null sperm could be because of increased levels of cAMP and/or other intracellular targets of Ca2+ and HCO3.
The elevated cAMP levels in GalT I-null sperm may also reflect signal
transduction pathways in addition to those regulated by Ca2+ and
HCO3. Adenosine receptors, through the action of
fertilization promoting peptide, stimulate cAMP production in a heterotrimeric
G protein-dependent manner (Baxendale and
Fraser, 2003; Fraser and
Adeoya-Osiguwa, 1999
). GalT I is functionally linked with a
pertussis toxin-sensitive G protein during the acrosome reaction and it may
interact with adenosine receptors during capacitation. In addition, cAMP
production in mammalian sperm may be stimulated by reactive oxygen species and
regulated by intracellular pH (Aitken et
al., 1998
). pH is regulated by HCO3
(Zeng et al., 1996
) and could
be altered in GalT I-null sperm. In any event, the accelerated capacitation
rate of GalT I-null sperm is positively correlated with elevated cAMP levels,
and further experiments are required to determine whether this increase is
causal or incidental to the accelerated capacitation of GalT I-null sperm.
The precocious capacitation phenotype in GalT I-null sperm is confined to functional aspects of the sperm head
We suspected that the elevated levels of cAMP would lead to increased PKA
activity and a consequent increase in PKA-dependent tyrosine phosphorylation.
However, neither PKA activity nor tyrosine phosphorylation were significantly
different between wild-type and GalT I-null sperm
(Fig. 8). The only detectable
difference in the profile of tyrosine-phosphorylated proteins was an
additional 200 kDa protein of unknown significance in GalT I-null sperm
lysates. This protein may directly or indirectly promote the accelerated
capacitation of GalT I-null sperm, but this waits further testing. The
kinetics of tyrosine phosphorylation in sperm from these mice occurs much more
rapidly than previously reported (Visconti
et al., 1995a
), which is probably due to differences in mouse
strains used or in the composition of the capacitation medium.
It is unclear why the PKA activity in GalT I-null sperm did not correlate
with the elevated levels of cAMP. Others have experimentally increased cAMP
concentration in sperm using cAMP analogs and demonstrated an effect on PKA
activity (Demarco et al., 2002;
Visconti et al., 1995b
;
Visconti et al., 1999
). One
explanation is that there are multiple steps between cAMP production and
tyrosine phosphorylation, and the amount of cAMP in the cell may not directly
correlate with every downstream target
(Harrison, 2003
).
Alternatively, the elevated cAMP in GalT I-null sperm may act on a limited
number of targets and/or in a localized manner in the sperm head. cAMP may
also influence targets in the sperm head other than PKA, such as cyclic
nucleotide-gated ion channels and cAMP-activated guanine-nucleotide exchange
proteins (Harrison, 2003
).
Collectively, these results suggest that capacitation can be segregated
into GalT I-dependent events in the head (zona pellucida binding, acrosomal
exocytosis) and GalT I-independent events in the tail (protein tyrosine
phosphorylation, sperm motility). This conclusion is supported by observations
in the literature in addition to results presented here. For example,
calmodulin antagonists prevent capacitation as assayed by in vitro
fertilization and acrosomal exocytosis, but fail to block protein tyrosine
phosphorylation (Si and Olds-Clarke,
2000). Many substrates of protein tyrosine phosphorylation are
components of the principle piece and fibrous sheath of the flagellum
(Carrera et al., 1996
;
Jha and Shivaji, 2002
;
Naaby-Hansen et al., 2002
), or
associated with microtubule function, such as AKAP3, AKAP4, dynein
intermediate chain and PKA (Ficarro et al.,
2003
). Although there is evidence that some PKA subunits localize
to the sperm head (Flesch et al.,
2001
; Harrison et al.,
2000
; Visconti et al.,
1997
), it is believed to act on substrates primarily in the sperm
tail. Consistent with this, the sperm-specific catalytic subunit of PKA has
been detected in the midpiece, mitochondria and the axoneme of murine and
ovine sperm (San Agustin and Witman,
2001
), a location where it would not be subject to regulation by
GalT I.
Although capacitation was first described over 50 years ago, the identity
and mechanism of extracellular and intracellular signaling molecules
regulating this process are still unclear. The study of capacitation has been
hindered by the lack of defined mutations that interfere with its normal
progression. To our knowledge, only one other class of mutations affects the
capacitation process in mammalian sperm: the t haplotypes. Several genes
affected by the t haplotypes have been identified
(Herrmann et al., 1999;
Samant et al., 2002
); this
group of mutations appears to overwhelmingly affect flagellar function
(Olds-Clarke and Johnson,
1993
). The present study reports the first mutation in a sperm
surface receptor that leads to a constitutively activated capacitation
phenotype, and which recapitulates wild-type capacitation in the absence of
co-factors normally required for capacitation. We expect further study of this
mutant to aid understanding of this physiologically complex process.
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ACKNOWLEDGMENTS |
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