245 Signalling pathways during capacitation

Corresponding author E-mail: pvisconti@vasci.umass.edu

Signalling pathways involved in sperm capacitation
Ana M. Salicioni
1
, Mark D. Platt
2
, Eva V. Wertheimer
1
, Enid Arcelay
1
, Alicia
Allaire
1
, Julian Sosnik
1
and Pablo E. Visconti
1
1
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003,
USA;
2
Rensselaer Polytechnic Institute, Biotech Building, Troy, NY, USA
After ejaculation, mammalian sperm have not yet acquired full fertilising
capacity. They will require a finite period of residence in the female
reproductive tract before they become fertilisation competent. The
molecular, biochemical, and physiological changes that occur to sperm
while in the female tract are collectively referred to as capacitation. During
capacitation, changes in membrane properties, enzyme activities, and
motility render spermatozoa responsive to stimuli that induce the
acrosome reaction and prepare spermatozoa for penetration of the egg
investments prior to fertilisation. These changes are facilitated by the
activation of cell signalling cascades in the female reproductive tract in
vivo or in defined media in vitro. The purposes of this review are to
consider some recent contributions towards our understanding of
capacitation, to summarise open questions in this field, and to discuss
future avenues of research.
Introduction
Mammalian sperm are not able to fertilise eggs immediately after ejaculation. Chang (1951)
and Austin (1951) independently demonstrated that sperm acquire fertilisation capacity after
residing in the female tract for a finite period of time. Following these original observations,
many studies confirmed that the environment of the female tract induces a series of physiologi-
cal changes in the sperm; these changes are collectively called capacitation. Taking these
initial investigations into account capacitation became defined using fertilisation as end-point.
However, various pieces of evidence suggest that the functional changes occurring in sperma-
tozoa during capacitation are not one event, but a combination of sequential processes. Thus,
capacitation can be divided in: (a) fast events such as the initiation of sperm motility, occurring
as soon as the sperm are released from the epididymis, and (b) slow events such as changes in
the motility pattern (e.g. hyperactivation) and the acquisition of the sperm capacity to undergo
agonist-stimulated acrosome reaction, which are activated only after a certain period of time in
conditions that support the sperm ability to fertilise the egg. In addition to these sequential
events, capacitation is also the result of concomitant processes involving changes at the mo-
lecular level occurring both in the head (i.e., preparation for the acrosome reaction) and in the
tail (i.e., motility changes). Although more than 50 years have passed since sperm capacitation
was first reported, it is noteworthy that the molecular basis of this process is still today not well
understood.
Spermatology. SRF Vol. 65. ERS Roldan and M Gomendio (eds) Nottingham University Press, Nottingham
17-Visconti.p65 3/20/2007, 11:19 AM 245
246 A.M. Salicioni et al.
Molecular basis of capacitation
Fast processes
The molecular basis of fast occurring processes in sperm has been recently the focal point of several
studies (Fig. 1).
CatSper
NBC
sAC
cAMP
nHCO
3
-
Na
+
PKA
Ca
2+
pH
Transient
hyperpolarization
Scramblase
Testable endpoints
- Increase cholesterol availability for acceptors
- Activation of flagellum
PE PS
PDE
Phosphorylation
of other substrates
Figure 1. Fast capacitation-associated processes. Although it is controversial whether events
that occur immediately after ejaculation are part of capacitation, it is important to notice
that, physiologically, these processes take place in the female tract and that are likely to be
necessary for fertilisation. This figure is based in work from several laboratories. (1) After
ejaculation, the HCO
3
-
concentration in the sperm surrounding milieu increased signifi-
cantly (Setchell, Maddocks and Brooks, 1994). (2) HCO
3
-
enters the sperm through a Na
+
/
HCO
3
-
cotransporter (Demarco et al., 2003). (3) Increased HCO
3
-
concentration activates
sAC and consequently PKA (Chen, Cann, Litvin, Iourgenko, Sinclair, Levin and Buck,
2000). (4) Activation of PKA modulates the response of CatSper to changes in membrane
potential (Wennemuth et al., 2003), activates a phospholipids scramblase (Harrison et al.,
1996; Gadella and Harrison, 2002) and increases the availability of cholesterol for exter-
nal acceptors (Flesch, Brouwers, Nievelstein, Verkleij, van Golde, Colenbrander and
Gadella, 2001b).
Among those, it is important to highlight work from Babcock and collaborators, and from Harrison,
Gadella and collaborators. While Babcocks group has concentrated on mechanisms regulating
flagellar movement, Harrison and Gadella have worked in the regulation of sperm membrane
dynamics. From these studies it is possible to conclude that: (1) Exposure of sperm to HCO
3
-
immediately triggers activation of their flagella and increases the depolarization-evoked rate of
rise of intracellular Ca
2+
concentration ([Ca
2+
]
i
)(Wennemuth, Carlson, Harper and Babcock, 2003).
(2) Physiological levels of HCO
3
-
induce a rapid collapse of the sperm phospholipid asymmetry
mediated by scramblases (Harrison, Ashworth and Miller, 1996; Gadella and Harrison, 2002). (3) In
17-Visconti.p65 3/20/2007, 11:19 AM 246
247 Signalling pathways during capacitation
both cases, HCO
3
-
actions appear to be mediated by a cAMP pathway through activation of the
soluble adenylyl cyclase (sAC, see below). (4) As a consequence of the increase in cAMP levels,
protein kinase A (PKA) is activated resulting in the fast phosphorylation of a subset of proteins
(Harrison, 2004). (5) A new family of Ca
2+
channels, named CatSper, appears to mediate the
depolarization-induced increase in [Ca
2+
]
i
(Carlson, Westenbroek, Quill, Ren, Clapham, Hille,
Garbers and Babcock, 2003). (6) Interestingly, contrary to other slower capacitation-associated
events, these fast processes do not require the presence of cholesterol-acceptors such as bovine
serum albumin (BSA). Although this review will be focused mainly on processes that occur after a
significant period of capacitation, it is important to consider these rapidly occurring events; first,
initial fast signalling in sperm capacitation is likely to be essential for the slower processes to take
place; second, both fast and slow processes appear to be regulated by similar molecules (e.g.
HCO
3
-
, sAC, cAMP).
Slow processes
Despite the importance of fast occurring events in capacitation, until recently, most researchers
have considered only the slow processes (Fig. 2) as part of sperm capacitation.
pHe

Ca
2+
channel
NBC
sAC
cAMP
nHCO 3
-
Na
+
PKA
Ca
2+
PDE
Cholesterol efflux
BSA
Na
+
BSA -chol.
ENaC
K
+
channel

K
+
?
Protein Tyrosine Phosphorylation
Hyperpolarization
Amiloride

Phosphorylation
of other
substrates
Low Voltage
Ca
2+
channel
Inactive
Ability to undergo
agonist-induced AR
Close
Figure 2. Slow capacitation-associated processes. Capacitation is a complex series of
molecular events that occurs in sperm after epididymal maturation and confers on sperm
the ability to fertilise an egg (Yanagimachi, 1994). In most cases, capacitation media con-
tain energy substrates, such as pyruvate, lactate and glucose, a cholesterol acceptor (usu-
ally serum albumin), NaHCO
3
, Ca
2+
, low K
+
, and physiological Na
+
concentrations. The
mechanism of action by which these compounds promote capacitation is poorly under-
stood at the molecular level; however, some molecular events significant to the initiation of
capacitation have been identified and are represented in this model.
What follows is a list of endpoints that have been associated to capacitation over the years: (1)
Ability of the sperm to fertilise the egg: this is the initial definition of capacitation and still, the
17-Visconti.p65 3/20/2007, 11:19 AM 247
248 A.M. Salicioni et al.
most important evidence that sperm are capacitated. (2) Sperm hyperactivation: this change in
the motility pattern is considered necessary for fertilisation; therefore, in this review we will
consider hyperactivation as part of capacitation. (3) Preparation of the sperm to undergo an
agonist-induced acrosome reaction: despite some controversy about the exact nature of the
physiological inducer of the sperm acrosome reaction, two of the more likely agonists are the
zona pellucida (ZP) of the egg and progesterone. In both cases, only capacitated sperm will
react in the presence of these reagents. (4) Sperm chemotaxis: although chemotaxis in mamma-
lian sperm is still controversial, it has been proposed that only capacitated sperm undergo
chemotactic behaviour (Eisenbach, 1999). At the molecular level, during the last ten years, a
series of studies have established that some signalling pathways are activated during sperm
capacitation. These signalling pathways are summarised in Figure 2. This figure is based on
work from many laboratories ( Langlais and Roberts, 1985; Espinosa and Darszon, 1995; Visconti,
Bailey, Moore, Pan, Olds-Clarke and Kopf, 1995a; Visconti, Moore, Bailey, Leclerc, Connors,
Pan, Olds-Clarke and Kopf, 1995b; Zeng, Clark and Florman, 1995; Hernandez-Gonzalez,
Sosnik, Edwards, Acevedo, Mendoza-Lujambio, Lopez-Gonzalez, Demarco, Wertheimer,
Darszon and Visconti, 2006). Different aspects of this model have been the focus of our
investigations and are summarised below.
HCO
3
-
and the cAMP pathway in mammalian sperm
Capacitation is a HCO
3
-
-dependent process (Lee and Storey, 1986; Boatman and Robbins, 1991;
Shi and Roldan, 1995; Gadella and Harrison, 2000; Visconti, Westbrook, Chertihin, Demarco,
Sleight and Diekman, 2002). Recent evidence strongly suggests that HCO
3
-
transport in these
cells is mediated at least in part by a member of the Na
+
/HCO
3
-
cotransporter family (Romero
and Boron, 1999). This conclusion is based on findings that HCO
3
-
transport in sperm has the
following properties (Demarco, Espinosa, Edwards, Sosnik, De La Vega-Beltran, Hockensmith,
Kopf, Darszon and Visconti, 2003): (1) it is electrogenic, (2) it is Na
+
-dependent, (3) it in-
creases pH
i
, and (4) it is blocked by stilbenes, such as DIDS. The transmembrane movement of
HCO
3
-
has been associated with the increase in intracellular pH (pH
i
) observed during capacita-
tion (Parrish, Susko-Parrish and First, 1989; Zeng, Oberdorf and Florman, 1996). However,
another more likely target for HCO
3
-
action in sperm is the regulation of cAMP metabolism
(Garbers, Tubb and Hyne, 1982) through stimulation of a unique type of adenylyl cyclase
(Okamura, Tajima, Soejima, Masuda and Sugita, 1985; Garty, Galiani, Aharonheim, Ho, Phillips,
Dekel and Salomon, 1988; Visconti, Muschietti, Flawia and Tezon, 1990). Two types of adenylyl
cyclases are responsible for cAMP synthesis in eukaryotes: transmembrane adenylyl cyclases
(tmAC), and the recently isolated sAC (Buck, Sinclair, Schapal, Cann and Levin, 1999). sAC
and tmACs are regulated by different pathways; sAC is insensitive to G-protein or forskolin
regulation and is more active in the presence of Mn
2+
than Mg
2+
. Although it is still controver-
sial how many tmACs are present in sperm (Baxendale and Fraser, 2003), multiple evidence
suggests that sAC is activated during capacitation. This conclusion is supported by recent stud-
ies (Esposito, Jaiswal, Xie, Krajnc-Franken, Robben, Strik, Kuil, Philipsen, van Duin, Conti and
Gossen, 2004; Hess, Jones, Marquez, Chen, Ord, Kamenetsky, Miyamoto, Zippin, Kopf, Suarez,
Levin, Williams, Buck and Moss, 2005) indicating that sperm from sAC null mutant mice are
not able to capacitate and consequently are infertile. In addition, a specific sAC inhibitor is able
to block capacitation (Hess et al., 2005). One of the targets for cAMP action is protein kinase A
(PKA). Once activated, PKA phosphorylates various target proteins which are presumed to
initiate several signalling pathways (Harrison and Miller, 2000; Harrison, 2004). In pig sperm
exposed to HCO
3
-
, cAMP rises to a maximum within 60 sec (Harrison and Miller, 2000), and
17-Visconti.p65 3/20/2007, 11:19 AM 248
249 Signalling pathways during capacitation
the increase in PKA-dependent phosphorylation begins within 90 sec (Harrison, 2004). It is
interesting that cAMP levels fall after their initial rise and then, after 7 min, begin to rise again;
PKA-catalysed protein phosphorylation follows a similar time course. This second rise in cAMP
levels appears to be a sustained response to HCO
3
-
.
Changes that occur at the level of the plasma membrane
Capacitation is correlated with changes in the sperm plasma membrane architecture. These
changes can be rapid (Harrison, 1996; Harrison and Miller, 2000; Gadella and Harrison, 2002)
or slow (Visconti, Ning, Fornes, Alvarez, Stein, Connors and Kopf, 1999; Flesch, Wijnand, van
de Lest, Colenbrander, van Golde and Gadella, 2001a). Interestingly, rapid changes in the
sperm plasma membrane appear to be mediated by a fast activation of the HCO
3
-
/sAC/PKA
pathway. On the other hand, the late effects are related to the presence of cholesterol acceptors
in the in vitro incubation medium. Bovine serum albumin (BSA) is a critical component of in
vitro capacitation media; it is believed to function as a sink for cholesterol by removing it from
the sperm plasma membrane (Davis, Byrne and Hungund, 1979; Langlais and Roberts, 1985;
Cross, 1996; Visconti et al., 1999). How cholesterol efflux couples to the regulation of signal
transduction pathways intrinsic to capacitation is not clear at present. One possibility is that
before capacitation, cholesterol concentrates in specialised plasma membrane microdomains or
lipid rafts. Current concepts attribute important signalling properties to the existence of these
rafts, acting to bring protein assemblies together. Taking this into consideration, depletion or
supplementation of cholesterol within the plasma membrane will have profound effects on the
behaviour of the raft complexes (Zajchowski and Robbins, 2002). In somatic cells, cholesterol
removal is thought to disrupt lipid rafts, thus activating signalling events involving tyrosine
kinases, G proteins, and/or other molecules (Kabouridis, Magee and Ley, 1997; Brown and
London, 1998; Roy, Luetterforst, Harding, Apolloni, Etheridge, Stang, Rolls, Hancock and Par-
ton, 1999). Because the activation of similar signalling events during capacitation correlates
with the removal of cholesterol from the plasma membrane, it can be hypothesised that in the
sperm, cholesterol may likewise be concentrated in lipid rafts and its efflux is related to changes
in sperm lipid rafts. Supporting this hypothesis, caveolin has been detected in the plasma
membrane overlying the acrosomal region and the flagellum of mouse and guinea pig sperm
(Travis, Foster, Rosenbaum, Visconti, Gerton, Kopf and Moss, 1998; Trevio, Serrano, Beltran,
Felix and Darszon, 2001) suggesting the presence of a special type of lipid raft, called caveolae,
in these cells. More recent studies have shown that a 2-h incubation in HCO
3
-
and BSA-contain-
ing capacitation medium induces lateral redistribution of raft marker proteins (Cross, 2004;
Shadan, James, Howes and Jones, 2004) as well as disruption of lipid rafts (Sleight, Miranda,
Plaskett, Maier, Lysiak, Scrable, Herr and Visconti, 2005). Among others, a possible result of
cholesterol loss could be related to alterations in the steady-state intracellular ion concentra-
tions with the resultant modification of the sperm resting membrane potential.
Sperm membrane potential
Mouse sperm capacitation is accompanied by the hyperpolarization of its plasma membrane
potential (Zeng, Clark and Florman, 1995). This change results from a combination of electro-
genic ion permeability changes that shift the membrane potential towards the K
+
equilibrium
potential. Although the functional role of the capacitation-associated hyperpolarization is not
clear, Flormans group (Florman, Arnoult, Kazam, Li and OToole, 1998) has proposed that
since capacitation prepares the sperm for the acrosome reaction, hyperpolarization may regu-
17-Visconti.p65 3/20/2007, 11:19 AM 249
250 A.M. Salicioni et al.
late the ability of sperm to generate transient intracellular Ca
2+
elevations during the acrosome
reaction induced by physiological agonists (e.g. ZP). The major function of the voltage sensi-
tive Ca
2+
(Ca
v
) channels is to convert changes in membrane potential into a Ca
2+
signal. The
Ca
V
channel permeation pathway is formed by its
1
subunit, encoded by a family of at least 10
genes. Ca
v
channels fall into two major functional classes: high voltage-activated (HVA) and
low voltage activated (LVA). LVA channels open following weak depolarizations and are
encoded by the Ca
v
3 subfamily of genes (Ca
v
3.1 to Ca
v
3.3) (Perez-Reyes, 2003). A property of
Ca
v
3 channels is that they are inactive at a membrane potential equivalent to those of non-
capacitated sperm (~-30 mV) (Lievano, Santi, Serrano, Trevino, Bellve, Hernandez-Cruz and
Darszon, 1996). Thus, if Ca
v
3 channels are involved in the regulation of the acrosome reaction,
the sperm membrane potential should hyperpolarise before becoming able to undergo this
exocytotic event (Florman et al., 1998). Interestingly, single sperm studies (Arnoult, Kazam,
Visconti, Kopf, Villaz and Florman, 1999) indicated that after capacitation, sperm can be di-
vided in two fractions. Approximately 50% of the sperm remain at a membrane potential close
to the uncapacitated population while the rest hyperpolarise to -80 mV, a potential that can
remove inactivation from Ca
v
3 channels. Only this last population was able to undergo the
acrosome reaction when exposed to solubilised ZP.
Sperm maintain an internal ion concentration markedly different from that in the extracellu-
lar medium. This difference is determined by the relative permeability of the plasma mem-
brane to the ions found in the media, to their gradients and to the metabolic state of the cell. It
is likely that the capacitation-associated hyperpolarization results from changes in the activity
of ion-selective channels and transporters that control the extent of ion flow. Consistent with
this hypothesis, different components of the capacitation media play important roles in the
regulation of the sperm membrane potential. In mouse sperm, it has been shown that in the
absence of BSA or HCO
3
-
the changes in membrane potential do not occur (Demarco et al.,
2003). These data suggest that HCO
3
-
as well as cholesterol efflux have a role in controlling
events leading to hyperpolarization. Additionally, Muoz-Garay, De la Vega-Beltran, Delgado,
Labarca, Felix and Darszon (2001) demonstrated with patch clamp techniques that inward rec-
tifying K
+
(Kir) channels are expressed in mouse spermatogenic cells and proposed that these
channels may be responsible for the capacitation-associated hyperpolarization. Supporting this
hypothesis, pharmacological experiments suggested that the K
ATP
channels contribute to the
capacitation-associated hyperpolarization (Acevedo, Mendoza-Lujambio, de la Vega-Beltran,
Trevino, Felix and Darszon, 2006). In addition to these findings, our studies have revealed that
amiloride sensitive Na
+
channels are present in mouse sperm (Hernandez-Gonzalez et al.,
2006). These channels constitute a new class of proteins known as epithelial Na
+
channels
(ENaCs) (Kellenberger and Schild, 2002) that are expressed in many tissues of invertebrate and
vertebrate organisms. Interestingly, ENaC family members may be regulated by pH, Ca
2+
,
Na
+
, Cl
-
, and their state of phosphorylation or that of proteins that regulate them (Kellenberger
and Schild, 2002). Some of these parameters change during capacitation. ENaCs have been
implicated in reproductive and early development processes in Drosophila (Darboux, Lingueglia,
Champigny, Coscoy, Barbry and Lazdunski, 1998). How HCO
3
-
, BSA and other ions integrate
to regulate the changes in the sperm membrane potential is not known.
Phosphorylation events in mammalian sperm
Protein phosphorylation plays a role in the regulation of many intracellular events such as
transduction of extracellular signals, intracellular transport, and cell cycle progression. Condi-
tions favouring capacitation of mouse sperm promote tyrosine phosphorylation of a subset of
17-Visconti.p65 3/20/2007, 11:19 AM 250
251 Signalling pathways during capacitation
proteins of Mr 40 120 Kda. In the absence of BSA, Ca
2+
or NaHCO
3
neither capacitation nor
the increase in tyrosine phosphorylation are observed (Visconti et al., 1995a). Interestingly, the
increase in tyrosine phosphorylation is regulated by a cAMP-dependent pathway in mouse
sperm (Visconti et al., 1995b) and other species (Leclerc, de Lamirande and Gagnon, 1996;
Galantino-Homer, Visconti and Kopf, 1997; Kalab, Peknicova, Geussova and Moos, 1998).
Because PKA is a serine/threonine kinase and not a tyrosine kinase, these experiments strongly
suggest that capacitation is regulated by a protein kinase cascade (see Fig. 2). Despite this
knowledge, except for PKA, the identity and functions of other kinases and their targets in
sperm are not well defined. An initial approach to investigate the role of phosphorylation in
capacitation is to identify proteins phosphorylated during this process and to characterise the
kinases involved in their phosphorylation. In this respect, the use of two dimensional (2-D)
polyacrylamide gel electrophoresis (PAGE) followed by tandem mass spectrometry (MS/MS)
provides a comprehensive approach to the analysis of proteins involved in cell signalling
(Blomberg, 1997; Alms, Sanz, Carlson and Haystead, 1999; Soskic, Gorlach, Poznanovic,
Boehmer and Godovac-Zimmermann, 1999; Lewis, Hunt, Aveline, Jonscher, Louie, Yeh,
Nahreini, Resing and Ahn, 2000). Specifically, changes in tyrosine phosphorylation can be
monitored using 2-D gel electrophoresis (OFarrell, 1975; Gorg, Postel and Gunther, 1988)
followed by Western blot analysis with anti phosphotyrosine ( PY) antibodies. Proteins that
undergo changes in tyrosine phosphorylation during cellular processes can then be isolated
from a duplicated gel stained with silver and sequenced by MS/MS. We have used this strategy
to identify several sperm proteins that undergo tyrosine phosphorylation during capacitation
(Ficarro, Chertihin,Westbrook, White, Jayes, Kalab, Marto, Shabanowitz, Herr, Hunt and Visconti,
2003).
Identification of phosphorylation substrates using 2-D PAGE is a powerful approach. How-
ever, this methodology has some limitations. (1) It is not completely appropriate to identify
proteins that undergo phosphorylation in serine or threonine residues since antibodies against
those phosphorylated residues have not achieved sufficient quality and sensitivity. (2) MS/MS
on proteins isolated by PAGE has detection limits several orders of magnitude lower than MS/
MS performed on proteins not embedded in gels. (3) Although in some cases it is possible to
get the exact site of phosphorylation of a candidate protein, more often the phosphorylation
site remains elusive due to the presence of more abundant peptides that do not present the
phosphorylation site.
Recently, several methods for the selective detection and enrichment of phosphopeptides
have been developed (Porath, 1992; Cao and Stults, 1999; Posewitz and Tempst, 1999; Cao
and Stults, 2000; Annan, Huddleston, Verma, Deshaies and Carr, 2001). However, most of
them have been applied only on a protein-by-protein basis. Another method for enrichment of
phosphoproteins is the use of Fe
3+
-immobilised metal affinity chromatography (IMAC) prior to
MS/MS to enrich digests for peptides containing phosphorylated amino acids. This technique
was used by a number of investigators (Cao and Stults, 1999; Cao and Stults, 2000; Zarling,
Ficarro, White, Shabanowitz, Hunt and Engelhard, 2000) but proved to generate false positives
as acidic residues (i.e. glutamic and aspartic acid) will readily bind to IMAC (Muszynska,
Dobrowolska, Medin, Ekman and Porath, 1992). To increase the selectivity of the IMAC col-
umn for phosphopeptides, we have used a modification of this technique (Ficarro, McCleland,
Stukenberg, Burke, Ross, Shabanowitz, Hunt and White, 2002) in which acidic residues are
converted to methyl esters to block their binding to iron before IMAC is employed. A flow
diagram of the method is summarised in Fig. 3. Using this methodology, we have characterised
5 sites of tyrosine, 56 sites of serine and 2 sites of threonine phosphorylation in capacitated
human sperm (Ficarro et al., 2003).
17-Visconti.p65 3/20/2007, 11:19 AM 251
252 A.M. Salicioni et al.
Capacitated Sperm Protein
extract
Tryptic
Digest
Methylation of
protein digest
IMAC
Elute phosphopeptides
from IMAC to HPLC reverse
phase column
MS/MS
C18 capillary column
with ESI emitter tip
Phosphopeptides
IMAC
Figure 3. Flow chart of the IMAC/RP-HPLC/ESI/MS/MS analysis of capacitated sperm.
Carboxy-methylated phosphopeptides were enriched using IMAC and then identified
using nano-flow reverse phase (RP)-HPLC micro-electrospray ionization (nHPLC-mESI)
mass spectrometry (MS) on a hybrid linear quadrupole ion trap/Fourier transform (LTQ/FT)
ion cyclotron resonance mass spectrometer.
Although the combination of IMAC and MS/MS is ideally suited for the characterization of
phosphorylation sites on proteins in complex mixtures, it is also important to determine which
sites are phosphorylated in response to a particular signalling pathway. Common methods to-
ward this goal include comparison of proteins labelled in vitro with [
32
P]ATP, use of PY
antibodies or other phospho-specific antibodies, and in vivo labelling using inorganic [
32
P]
followed by immunoprecipitation and/or 2D gel analysis. We have developed an alternative
method to compare phosphorylation of defined sequences in two different cell populations by
differential isotopic labelling (Ficarro et al., 2003). Details on this method are given below.
The evaluation of differential phosphorylation added to the knowledge of the exact phosphory-
lated sequence goes beyond the sperm capacitation field and could be used to understand
signalling mechanisms in multiple biological systems.
Differential MS/MS analysis of phosphopeptides during the capacitation process
Similarly to other high throughput methodologies such as microarrays, the advantage of a glo-
bal MS/MS approach to identify phosphopeptides is that this methodology is able to generate
large amount of data in a relatively short time. On the minus side, these high throughput
techniques are often regarded as a descriptive analysis of a particular problem. More informa-
tion can be achieved when these techniques are used to analyse functional changes occurring
in a biological process. One interesting approach used differential isotopic labelling to com-
pare phosphorylated sequences from capacitated and non-capacitated sperm populations (Ficarro
et al., 2003) (Fig. 4). This analysis is based in mass spectrometry; therefore, differential label-
ling is achieved by the use of isotopes of different mass for each sample. In our case, to
compare non-capacitated with capacitated sperm populations by differential labelling, the afore-
mentioned carboxymethylation reaction of acidic residues was used. Briefly, tryptic peptides
from each cell population were converted to peptide methyl esters with deuterated (CD
3
OH)
(d3) and nondeuterated (CH
3
OH) (d0) methanol, respectively. Both samples are then mixed in
equal proportions and the mixture purified by IMAC to ensure that only phosphopeptides are
retained. Signals for phosphopeptides present in both samples appear as doublets separated by
a mass calculated as: n(3Dalton)/z (where n is the number of carboxylic acid groups in the
17-Visconti.p65 3/20/2007, 11:20 AM 252
253 Signalling pathways during capacitation
peptide and z is the charge on the peptide). The ratio of the two signals in the doublet changes
as a function of the phosphorylation or dephosphorylation that occurs during capacitation. Pep-
tides of interest are then targeted for sequence analysis subsequently performed on the ion trap
instrument. Moreover, comparison of doublets will give a quantitative estimate of the level of
phosphorylation of each sequence. The differential isotopic labelling of phosphopeptides gives
for the first time the possibility to analyse any phosphorylation site in a single experiment. In
addition, this technique completes the information given by Western blot analysis since it
identifies the exact sequence in a particular protein that becomes phosphorylated after a spe-
cific stimulus.
Non -Capacitated
Population (NON)
IMAC/MS for
quantitation,
IMAC/MS/MS
for peptide
sequence
Sperm
Protein
Extract
Triptic
Digest
d0-methanolic
HCl labeled
peptides
Capacitated
Population (CAP)
Sperm
Protein
Extract
Triptic
Digest
d3-methanolic
HCl labeled
peptides
NON (d0) + CAP (d3)
Reverse Phase HPLC
with ESI emitter tip
IMAC
Figure 4. Schematic representation of the procedure for phosphopeptide comparison
between two samples. Differential labeling is achieved by the use of a different isotope of
hydrogen for each sample. Tryptic peptides from two samples of cells (i.e. non-capacitated
vs capacitated sperm) are converted to peptide methyl esters with deuterated (CD
3
OH) (d3)
and nondeuterated (CH
3
OH) (d0) methanol, respectively. Both samples are then mixed in
equal proportions and the mixture purified by IMAC to ensure that only phosphopeptides
are retained. Signals for phosphopeptides present in both samples appear as doublets
separated by n(3 Da)/z (where n is the number of carboxylic acid groups in the peptide and
z is the charge on the peptide). The ratio of the two signals in the doublet changes as a
function of the phosphorylation or dephosphorylation that occurs during capacitation.
Peptides of interest are then targeted for sequence analysis subsequently performed on the
ion trap instrument.
Kinases in mammalian sperm
Protein kinases play essential roles in the regulation of cellular processes. Therefore, it is not
surprising that several protein kinases have been shown to be involved in spermatogenesis
(Sassone-Corsi, 1997). However, it is not known which of these kinases remains in the mature
sperm and has a function during capacitation. Few kinases have been described in mature
mammalian sperm using antibodies and, apart from PKA, their functional role in sperm have not
been established. Some of these kinases are protein kinase C (PKC) (Rotem, Paz, Homonnai,
Kalina and Naor, 1990), GSK 3 (Vijayaraghavan, Mohan, Gray, Khatra and Carr, 2000), casein
kinase II (Chaudhry, Nanez and Casillas, 1991a; Chaudhry, Newcomer and Casillas, 1991b),
MAP kinase (Luconi, Barni, Vannelli, Krausz, Marra, Benedetti, Evangelista, Francavilla, Properzi,
Forti and Baldi, 1998) and at least one member of the testis specific serine kinase (Tssk) family
(Hao, Jha, Kim, Vemuganti, Westbrook, Chertihin, Markgraf, Flickinger, Coppola, Herr and
17-Visconti.p65 3/20/2007, 11:20 AM 253
254 A.M. Salicioni et al.
Visconti, 2004). Less is known about the identity of tyrosine kinases in sperm. Although a
tyrosine kinase activity has been partially purified from boar sperm (Berruti, 1994), the identity
of the kinase responsible for this activity is still not known. There is some evidence for the
presence of yes (Leclerc and Goupil, 2002) and src (Baker, Hetherington and Aitken, 2006) and
also of a tyrosine kinase receptor in human sperm (ZRK) (Burks, Carballada, Moore and Saling,
1995); these kinases could mediate the effect the capacitation-associated increase in protein
tyrosine phosphorylation. In particular, it is interesting the presence of csk in mouse sperm
(Baker et al., 2006). This kinase negatively regulates src kinase activity and it is at the same
time directly inhibited by PKA, opening the possibility that this pathway is involved in the
regulation of the capacitation-associated increase in tyrosine phosphorylation (Baker et al.,
2006). Undoubtedly, the presence of this kinase cascade in sperm warrants further investiga-
tion. Because protein kinases have become major targets for the development of novel drugs,
identification of those that regulate capacitation could offer new opportunities towards an alter-
native approach for contraception.
Capacitation and the acrosome reaction
The acrosome is a membrane-limited organelle which overlies the sperm nucleus. In response
to either physiological or pharmacological stimuli, the outer acrosomal membrane and the
overlying plasma membrane undergo fusion and vesiculation leading to the exposure of the
acrosomal contents to the extracellular environment (Yanagimachi, 1994). This exocytotic pro-
cess is called acrosome reaction and its completion is an absolute prerequisite for fertilisation.
It is noteworthy that a physiologically induced acrosome reaction cannot occur in sperm that
have not undergone capacitation. Recent work has shown that N-ethyl maleimide-sensitive
fusion protein (NSF) and soluble NSF attachment protein receptors (SNAREs) (Jahn and Sudhof,
1999) are present in sea urchin (Schulz, Wessel and Vacquier, 1997; Schulz, Sasaki and Vacquier,
1998) and mammalian sperm (Michaut, Tomes, De Blas, Yunes and Mayorga, 2000; Ramalho-
Santos, Moreno, Sutovsky, Chan, Hewitson, Wessel, Simerly and Schatten, 2000; Yunes,
Michaut, Tomes and Mayorga, 2000). These observations support the idea that the sperm acrosome
reaction might be regulated in similar ways as exocytotic processes in somatic cells. However,
the acrosome reaction also presents differences with other known exocytotic events. Some of
these differences are: (1) The acrosome is a single secretory vesicle. (2) There are multiple
fusion points between the outer acrosomal membrane and the plasma membrane. (3) Both
these membranes form mixed vesicles that are shed during the acrosome reaction, resulting in
membrane loss. (4) The acrosome reaction is a one-shot fusion event; thus there is no mem-
brane recycling.
One may postulate that components of the sperm exocytotic machinery are modified during
capacitation. Some of these alterations may involve changes in the phosphorylation status of
certain proteins, changes in protein localization, and/or modification of protein-protein interac-
tions. Experiments leading to the identification and characterization of these effector mol-
ecules will further increase our understanding of capacitation. Among the proteins that undergo
tyrosine phosphorylation during capacitation, we identified valosin containing protein (VCP).
The 97-kDa VCP is a member of the type II AAA (ATPases associated with a variety of activi-
ties) ATPases, which are characterised by the presence of two conserved ATPase domains (Wang,
Song and Li, 2004). As other AAA proteins, VCP is an enzymatic machine. It catalyses ATP
hydrolysis to generate energy and uses the energy to perform mechanical work in cells. VCP,
also known as p97, is highly evolutionarily conserved and homologues of this protein can be
found in archaebacteria (VAT), in yeast (CDC48) and in Drosophila (TER94) (Woodman, 2003).
17-Visconti.p65 3/20/2007, 11:20 AM 254
255 Signalling pathways during capacitation
In the last few years, emerging biochemical and genetic evidence has associated VCP with a
range of functions. Among them, VCP is related to retranslocation of unfolded protein from the
endoplasmic reticulum and to fusion events during homotypic fusion of smooth endoplasmic
reticulum membranes and in the reformation of Golgi cisternae that occurs as the cell exits
mitosis.
Since capacitation prepares sperm to undergo a regulated exocytosis (e.g. acrosome reac-
tion), phosphorylation of proteins involved in fusion events may regulate this process and are of
particular interest. VCP undergoes tyrosine phosphorylation during capacitation. This conclu-
sion is based on two independent lines of evidence. First, using 2-D gel electrophoresis, a
protein spot that undergoes tyrosine phosphorylation during human sperm capacitation was
identified as VCP (Ficarro et al., 2003). Second, PY immunoprecipitates of capacitated hu-
man sperm contain more VCP than the equivalent immunoprecipitates from a non-capacitated
population (Ficarro et al., 2003). Tyrosine phosphorylation of VCP during sperm capacitation has
also been reported in boar sperm (Geussova, Kalab and Peknicova, 2002). In addition, in human
sperm VCP changes its immunofluorescence pattern during capacitation (Ficarro et al., 2003). It
can be hypothesised that VCP function in sperm is regulated by phosphorylation during capacita-
tion and that changes in localization of VCP during capacitation are involved in the regulation of
the acrosome reaction. More work will be necessary to evaluate this hypothesis.
Conclusions
This review is an attempt to summarise the current knowledge on sperm capacitation. Although
this process was discovered more than 50 years ago, its molecular basis is still not well defined.
Technical advances in the last years as well as knock out studies on sperm proteins warrant new
discoveries in the mechanisms of capacitation and in the regulation of sperm function in gen-
eral. In addition, it is important to notice that most discoveries in sperm capacitation come from
in vitro experiments. Despite the importance of these observations, it should be taken into
account for future research the ability of the female tract to control the speed of capacitation
and the delivery of capacitated sperm to the site of fertilisation.
Acknowledgements
This review was supported by the National Institutes of Health, Grants HD38082 and HD44044
(to PEV).
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