Human Reproduction Update, Vol.12, No.3 pp. 253–267, 2006
Advance Access publication December 7, 2005
doi:10.1093/humupd/dmi050
Calcium signalling in human spermatozoa: a specialized
‘toolkit’ of channels, transporters and stores
C.Jimenez-Gonzalez1, F.Michelangeli1, C.V.Harper1,5, C.L.R.Barratt2,3 and S.J.Publicover1,4
1
School of Biosciences, 2Reproductive Biology and Genetics Research Group, The Medical School, University of Birmingham and
Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham, UK
3
4
To whom correspondence should be addressed at: School of Biosciences, University of Birmingham, B15 2TT.
E-mail: [email protected]
5
Present address: School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK
Ca2+ is a ubiquitous intracellular messenger which encodes information by temporal and spatial patterns of concentration. In spermatozoa, several key functions, including acrosome reaction and motility, are regulated by cytoplasmic Ca2+ concentration. Despite the very small size and apparent structural simplicity of spermatozoa, evidence is
accumulating that they possess sophisticated mechanisms for regulation of cytoplasmic Ca2+ concentration and generation of complex Ca2+ signals. In this review, we consider the various components of the Ca2+-signalling ‘toolkit’
that have been characterized in somatic cells and summarize the evidence for their presence and activity in spermatozoa.
In particular, data accumulated over the last few years show that spermatozoa possess one (and probably two) Ca2+
stores as well as a range of plasma membrane pumps and channels. Selective regulation of the various components of
the ‘toolkit’ by agonists probably allows spermatozoa to generate localized Ca2+ signals despite their very small cytoplasmic volume, permitting the discrete and selective activation of cell functions.
Key words: calcium/calcium channels/calcium pumps/calcium stores/sperm
Functional importance of sperm [Ca2+]i signalling
A human spermatozoon is a terminally differentiated and extremely
specialized cell that has only one ‘aim’, to achieve fertilization and
pass on the genetic information contained in its nucleus. The cell
faces an enormous task; upon ejaculation into the female tract it
must undertake a long and arduous journey through mucus, locate
the oocyte, penetrate the cumulus and zona pellucida, undergo
acrosome reaction and ultimately fuse with the plasmalemma of the
egg. During its residence in the tract the cell must undergo capacitation and may also spend a period in a quiescent state before reactivating. Regulation of motility (probably including activation
and cessation of flagellar beat, switching of flagellar beat mode and
directional responses to chemotactic cues) and acrosome reaction
must be rapidly responsive to external signals (timing of these
responses is crucial) but must not be activated inappropriately or
prematurely. Not surprisingly, the number of sperm that actually
reach the oocyte with the potential to fertilize is believed to be very
small (Williams et al., 1993; De Jonge, 2005).
Since spermatozoa are apparently transcriptionally and translationally inactive, all activities of the cell are carried out by the
suite of proteins inherited during differentiation. Control of these
activities in mature sperm must be exerted purely by regulation of
these proteins through second messengers. There is little doubt
that spermatozoa use a range of such messengers. Production of
cAMP and the role of downstream serine–threonine and tyrosine
phosphorylation has been the focus of an enormous amount of
study. However, data accumulated over the last few years have
shown that [Ca2+]i also plays a major role in all the important
sperm functions that occur after ejaculation. The use of Ca2+ signalling in somatic cells has been the subject of intense research
over a number of years (Berridge, 2005). [Ca2+]i can change by
orders of magnitude, causing activation of Ca2+-binding proteins
and consequent cellular responses. Such responses are often
‘immediate’, including activation of secretory and contractile
machinery (either directly or through kinase cascades), as well as
more long-term effects on gene transcription. Because there are
few aspects of cell physiology that are not subject to some form of
regulation by Ca2+, [Ca2+]i is subject to strict spatio-temporal control, allowing specific Ca2+-sensitive responses that can be activated discretely (Berridge et al., 2000). In ejaculated spermatozoa,
[Ca2+]i regulates motility and hyperactivation (Carlson et al.,
2003; Suarez and Ho, 2003), chemotaxis (Eisenbach, 1999; Spehr
et al., 2003), acrosome reaction (Kirkman-Brown et al., 2002) and
is a key player in the process of capacitation. These processes
must occur at the appropriate time so sperm must, like somatic
cells, be able separately to control many Ca2+-activated activities.
This may, at least in part, be achieved by regulation of the Ca2+
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C.Jimenez-Gonzalez et al.
sensitivity of specific targets, but it is highly likely that spatiotemporal control of the [Ca2+]i is at least as important. In spermatozoa the scale on which this must be achieved is much smaller
and precision in the control of [Ca2+]i may, therefore, be far more
crucial.
In somatic cells, the control of [Ca2+]i is often complex and
sophisticated. A somatic cell’s Ca2+-signalling ‘toolkit’ (Berridge
et al., 2000) includes a range of pumps and channels at the plasmalemma that can be regulated in response to intracellular or
extracellular events and signals and a complex, structured array of
Ca2+ stores (Berridge, 2005; Michelangeli et al., 2005). The Ca2+storage organelles possess many Ca2+ uptake ATPases, at least
two families of Ca2+-release channels that are controlled by second
messengers (and by the activity of second–messenger-regulated
enzymes) and luminal Ca2+-binding proteins. Structured spatial
distribution of Ca2+ stores in combination with tight buffering of
cytoplasmic [Ca2+] permits both spatial and temporal restriction
and shaping of [Ca2+]i signals. Heterogenous expression of Ca2+
release channels, in combination with positive feedback and channel–
channel cascades of activation can generate ‘[Ca2+]i spikes’ and
waves. The development of this understanding of [Ca2+]i
signalling over the past 35 years has recently been reviewed
(Berridge, 2005). In contrast, an understanding of Ca2+ signalling
in the highly specialized sperm cell (despite its great importance)
is only now developing. In comparison to most somatic cells, spermatozoa are not only very small but appear to be comparatively
simple. Their cytoplasmic volume is minute and they possess a
nucleus, an acrosome and many mitochondria arranged helically
around the outer dense fibres in the mid-piece. Thus, although
[Ca2+]i is of great importance in spermatozoa, and the timing and
precision of the activation of Ca2+-regulated events is crucial,
sperm appear to be ‘under-equipped’ for the job! Figure 1 shows
three simple models of Ca2+ signalling. The simple pump-leak
model is attractive for spermatozoa. Due to the small size of the
cell, diffusion is unlikely to be a limiting factor, and it is reasonable to assume that Ca2+ influx from the extracellular compartment can effect a rapid rise in [Ca2+]i in any part of the cytoplasm.
Although evidence for expression of mechanisms for Ca2+ store
mobilization in mammalian spermatozoa has been available for
some time (Walensky and Snyder, 1995; Kuroda et al., 1999), direct
demonstration of store mobilization proved difficult, possibly,
because the store was labile or only partially filled (Publicover and
Barratt, 1999). However, investigations on the induction of the
acrosome reaction by zona strongly implicated a mechanism
involving mobilization of a Ca2+ store (probably the acrosome—
see Actions of Ca2+-mobilizing agonists) and activation of capacitative Ca2+ influx (Blackmore, 1993; O’Toole et al., 2000; Evans and
Florman, 2002) consistent with the model summarized in Figure 1b.
More recently, it has become apparent that a second, separately
regulated store may exist, which functions primarily to regulate
flagellar beat (Ho and Suarez, 2001, 2003; Harper et al., 2004).
Thus sperm may well possess a relatively complex Ca2+-signalling
apparatus including pump-leak and multiple stores (Figure 1c).
Sperm present a particularly difficult system for the study of
physiology. The small size of spermatozoa presents inherent difficulties in detecting and understanding the roles (and even the presence) of molecules of low abundance. Also the difficulty of
studying oocyte-induced responses is considerable, particularly
with human cells where material is very difficult to obtain and
often available only because it has ‘failed’ at IVF and is therefore
potentially flawed. Consequently much work on sperm Ca2+ signalling has used ‘substitute’ agonists and activators (such as
Figure 1. ‘Toolkits’ for [Ca2+]i signalling. Plasma membrane Ca2+ ATPases (PMCA) and Na+–Ca2+ exchangers (NCX) are shown by green boxes; Ca2+ channels
in the plasma membrane are shown by red boxes; sarcoplasmic–endoplasmic reticulum ATPases (SERCAs) on intracellular stores are shown in blue and channels
for mobilization of stored Ca2+ are shown in orange. (a) Shows a simple pump-leak system based entirely upon plasma membrane transport. [Ca2+]i is maintained
by PMCA/NCX and signalling is by gating of channels in the plasma membrane regulated by ligand-binding, second messengers and/or membrane potential. Different types of channel may be localized, allowing the cell to generate different [Ca2+]i signals according to the nature of the stimulus. (b) Shows a system including an intracellular Ca2+ store, potentially giving more complex [Ca2+]i signals and including store-operated channels to provide prolonged Ca2+ influx at the
plasma membrane. Current models for sperm [Ca2+]i signalling incorporate all these components (see text). (c) Shows a system including two (or more) Ca2+
stores. In this system, there is the potential for localized mobilization of stored Ca2+ and for interaction between stores.
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Calcium signalling in human spermatozoa
A23187 and progesterone), and our models for the action of agonists such as zona strongly very dependent on evidence from animal
studies. Our understanding of sperm Ca2+ signalling, its underlying mechanisms and the roles that it plays has progressed considerably over the last few years and is currently in a state of change.
In this review we aim to provide an update of available evidence
for the expression of Ca2+-‘toolkit’ components in spermatozoa
and their probable functions.
Ca2+ flux at the plasmalemma (ion channels)
Voltage-operated Ca2+ channels
Voltage-operated Ca2+ channels (VOCCs) are a family of transmembrane, channel-forming proteins which show strong structural
similarity to each other and to the voltage-operated sodium channels (see Catterall, 2000, and Ertel et al., 2000, for reviews). The
pore-forming unit of all VOCCs is the α1 subunit, which comprises four homologous domains (I–IV) connected by cytoplasmic
linker regions. Ten α1 genes are currently known, α1A–α1I and
α1S. Each domain of the α1 subunit is made up of six transmembrane helices (S1–S6). Between each S5 and S6 segments there is
a non-helical region (the P-loop) which forms part of the lining of
the channel pore and determines ion conductance and selectivity
(Catterall et al., 2003). In addition to the α1 subunits, VOCCs
normally include three or four auxiliary subunits which contribute
to channel characteristics, regulation and localization. The resulting family of channels shows great diversity in biophysical characteristics such as voltage dependence, kinetics of activation and
inactivation and also in pharmacological sensitivity. VOCCs were
initially classified on this basis into L, N, P/Q, R and T types (see
Catterall et al., 2003, for explanation of nomenclatures). Current
nomenclature recognizes three subfamilies (Cav1, Cav2 and Cav3).
Cav1 and Cav2 types form high-voltage activated channels but
Cav3 (α1G [Cav3.1], α1H [Cav3.2] and α1I [Cav3.3]) all encode
T-type channels which activate in response to relatively modest
depolarization (Catterall et al., 2003). VOCCs similar to those of
somatic cells have also been detected in mature and immature
sperm cells (Arnoult et al., 1996a,b). An important function of
VOCCs in sperm is likely to be mediation of the bicarbonatecAMP signal. In mouse sperm, depolarization-evoked Ca2+ entry
is enhanced by bicarbonate through a mechanism that is dependent
on protein kinase A (as it is blocked by H-89, a PKA inhibitor) and
potentiated by the phosphodiesterase inhibitor isobutyl methylxanthine and the membrane-permeable cAMP analogue cAMP–AM
(Wennemuth et al., 2000, 2003).
Functional studies
Assessment of the functional importance of VOCC expression in
sperm is limited by the technical difficulties of working on these
cells. Application of electrophysiological techniques to spermatozoa is still extremely difficult, but patch clamping of immature
male germ cells (from both rodent and human) has shown consistently that these cells display typical low-voltage activated (LVA),
fast-inactivating (T-type) currents (Hagiwara and Kawa, 1984;
Arnoult et al., 1996a; Lievano et al., 1996; Jagannathan et al., 2002).
No high–voltage-activated currents have been observed using this
method. Electrophysiological recordings in α1G (CaV3.1 under
newer nomenclature) knockout mice showed that the loss of this
T-type channel had little effect on the currents, suggesting that
α1H (CaV3.2) is the main functional VOCC in wild-type male germ
cells (Stamboulian et al., 2004). Most pharmacological analyses of
these channels shows a characteristic T-channel profile, though
there is an unusually high sensitivity to dihydropyridines (Arnoult
et al., 1998), which may have been misinterpreted as evidence for
participation of L-type VOCCs in some studies. Wennemuth et al.
(2000) reported that Ca2+ currents in mouse spermatogenic cells
were highly sensitive to the application of ω-conotoxin GVIA and
blockers of N-type Ca2+ channels, their data suggesting that the
current may have two distinct components. Functional expression
of T channels in mature spermatozoa is yet to be confirmed, though
photometric measurements of the responses of murine sperm to
solubilize zona pellucida shows a [Ca2+]i transient consistent with
T-channel activation (Arnoult et al., 1999; see Actions of Ca2+mobilizing agonists).
Molecular studies and localization
Using RT–PCR and RNase protection assays, Park et al. (2003)
detected and quantified different mRNA Ca2+ α1 subunits in preparations of motile human sperm. Though mRNAs for α1C [Cav1.2]
and α1I [Cav3.3] were present, they proposed that T-type (mainly
α1H [Cav3.2] and α1G [Cav3.1]) and non-L-type (α1E [Cav2.3] and
α1B [Cav2.2]) Ca2+ channels would be the main Ca2+ entry pathways involved in the acrosome reaction. Immunostaining experiments using specific antibodies detected the presence and
distribution of T-type (all three forms), L-, R- and P/Q-type
(Lievano et al., 1996; Serrano et al., 1999; Trevino et al., 2004;
Westenbroek and Babcock, 1999) and N-type channels (Wennemuth
et al., 2000) in rodent sperm cells. The channels appear to show
distinct distributions. In human cells, the three different T-type
channels are differentially distributed, α1H [Cav3.2] being localized to the principal piece of the tail and also in the back of the
sperm head and α1I [Cav3.3] being restricted to the mid-piece
(Serrano et al., 2004). High–voltage-activated channels show similar localization in mouse and human sperm (Trevino et al., 1998,
2004; Westenbroek and Babcock, 1999; Wennemuth et al., 2000).
L-type channels have been detected in all studies, using RT–PCR
and/or immunostaining techniques (Goodwin et al., 1997, 1998,
2000). However, the sensitivity of sperm T-type channels to drugs
previously considered L-channel-specific (see above) means that
pharmacological data used to identify these channels must be
treated cautiously. Table I summarizes the range of VOCC α1 subunits that have been detected in mammalian spermatozoa.
Store-operated channels
In both non-excitable (Parekh and Penner, 1997) and excitable cells
(Zhu et al., 1996; Garcia and Schilling, 1997; Philipp et al., 1998;
Fomina and Nowicky, 1999; Li et al., 1999; Liman et al., 1999),
Ca2+ efflux from intracellular stores is believed to activate Ca2+permeable ion channels [store-operated channels (SOCs)] in the
plasmalemma, a process called capacitative Ca2+ entry (Putney,
1990). Though the identity of SOCs is still unknown, members of
the canonical transient receptor potential (TRPC) family are the
most likely candidates (Padinjat and Andrews, 2004). The presence
of several mRNAs for TRPCs (TRPC1-7) in mouse spermatogenic
cells and TRPC1, three and six proteins in mature mouse sperm
were reported by Trevino et al. (2001). Jungnickel et al. (2001)
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C.Jimenez-Gonzalez et al.
Table I. Summary of studies which have illustrated expression of voltage-operated Ca2+ channels in male germ cells of rodent and human male germ cells
Tissue
Method
CaV subunit present
Reference
Mouse
Testis
RT–PCR, in situ hybridization
CaV2.1, CaV2.2, CaV1.2, CaV1.3,
CaV2.3 (836–240 bp), CaV3.2
CaV3.1 (520 bp), CaV3.2 (351 bp),
CaV2.3 (240 bp), CaV2.1 (753 bp),
CaV1.2
CaV3.3 (404 bp)
CaV1.2 detected (data not shown)
CaV2.1, CaV1.2 detected; CaV2.2,
CaV1.3 not detected
CaV3.1, CaV3.2, CaV3.3
CaV2.1, CaV1.2 detected; CaV2.2,
CaV1.3 not detected
CaV2.1, CaV1.2, CaV2.3-detection/
regional localization, CaV2.2detection/regional localization
CaV3.1, CaV3.2, CaV3.3-detection/
regional localization
Lievano et al. (1996); Son
et al. (2002)
Lievano et al. (1996);
Espinosa et al. (1999)
Germ cells
RT–PCR
Germ cells
Germ cells
Germ cells
RT–PCR
Northern blot
Immunostaining
Germ cells
Spermatozoa
Immunostaining
Immunostaining
Spermatozoa
Immunostaining
immunoblot
Spermatozoa
Immunostaining
Rat
Testis
Spermatozoa
Spermatozoa
Testis/spermatozoa
Testis sections
Testis sections
Testis
Multitissue northern blot panel
Testis sections
Human
Spermatozoa
Spermatozoa
RT–PCR
Immunostaining
In situ RT–PCR
Western blot
Immunostaining
In situ RT–PCR
RT–PCR
Northern blot
Immunostaining, in situ
hybridization
Sperm RNA
Testis/male germ cells
Testis
In situ RT–PCR
RT–PCR, Rnase protection
essays
Immunogold transmission
EM, immunostaining (confocal
microscope)
RT–PCR
PCR
In situ hybridization
Multitissue northern blot panel
Northern blot
Testis
RT–PCR
Spermatozoa
CaV1.2 (full sequence)
CaV1.1 (antibody against α1S)
CaV1.2
CaV1.1 (antibody against α1S)
CaV1.1 (antibody against α1S)
CaV1.2
CaV3.1 (domain IV and C-terminus,
domains I-III not detected)
CaV3.1 not detected
CaV2.1, CaV2.2 detected; CaV2.3 not
detected (data not shown)
Trevino et al. (2004)
Espinosa et al. (1999)
Serrano et al. (1999)
Trevino et al. (2004)
Serrano et al. (1999)
Westenbroek and Babcock
(1999); Wennemuth et al.
(2000)
Trevino et al. (2004)
Goodwin et al. (1997)
Goodwin et al. (1997)
Goodwin et al. (1997)
Goodwin et al. (1998)
Goodwin et al. (2000)
Goodwin et al. (2000)
Jacob and Benoff (2000)
Perez-Reyes et al. (1998)
Fragale et al. (2000)
CaV1.2 detected
CaV3.1, CaV3.2, CaV3.3, CaV1.2,
CaV2.3, CaV2.2
CaV3.1, CaV3.2, CaV3.3, CaV1.2,
CaV2.3-detection/regional localization
Goodwin et al. (2000)
Park et al. (2003)
CaV3.1 not detected
CaV3.1 and CaV3.2 full sequence
CaV3.1 and CaV3.2 in germ cells and
somatic cells
CaV3.1 detected (small amounts);
CaV3.3 not detected
CaV3.2 detected (489 bp); CaV3.1 and
CaV3.3 not detected
Jacob and Benoff (2000)
Jagannathan et al. (2002)
Jagannathan et al. (2002)
Trevino et al. (2004)
Monteil et al. (2000a,b)
Son et al. (2000)
Column one shows the tissue/cells studied, columns two and three summarize the methods used and the main findings, and column four gives the relevant references.
showed by immunohistochemistry with anti-Trp2 antibodies that
Trp2 was distributed mainly in the anterior head of the sperm and
to a lesser extent in the region of the posterior head. However,
Castellano et al. (2003) found that in human sperm the distribution
of TRP(s) was much wider. They were not only expressed in the
sperm head but also in the flagellum, which suggests that these
channels are involved in human sperm motility.
Using pharmacological mobilization of stored Ca2+ (thapsigargin and cyclopiazonic acid) capacitative Ca2+ influx has been
shown to occur in non-capacitated human sperm (Blackmore,
1993), spermatogenic and sperm cells of mouse, bull and ram
256
(Santi et al., 1998; Dragileva et al., 1999; O’Toole et al., 2000;
Rossato et al., 2001). Ca2+ efflux from intracellular stores
produces a signal (gating signal) that opens a SOC in the plasma
membrane. Through these channels Ca2+ ions flow into the cytosol
producing a sustained rise in the intracellular Ca2+ which is
believed to lead to the acrosome reaction in mammalian and nonmammalian spermatozoa (O’Toole et al., 2000; GonzalezMartinez et al., 2001; Hirohashi and Vacquier, 2003) and regulates chemotactic behaviour in ascidian sperm (Yoshida et al.,
2003). Jungnickel et al. (2001) proposed TRP2 as a necessary
component of the channel in mouse sperm responsible for the
Calcium signalling in human spermatozoa
sustained rise in intracellular Ca2+ (triggered by ZP3) that leads to
acrosome reaction. The TRP channel could be regulated either by
the depletion of Ca2+ from internal stores (O’Toole et al., 2000) or
through receptor activation (Harteneck et al., 2000).
Cyclic nucleotide-gated channels and CatSpers
Cyclic nucleotide signalling is pivotal in the functioning of all
spermatozoa. In invertebrate cells (cGMP) is of importance in regulation of motility and the acrosome reaction (Kaupp et al., 2003).
In sperm of mammals (including humans), levels of cGMP are
very low and cAMP appears to be of greater importance (Ain
et al., 1999; Lefievre et al., 2000). Manipulation of cGMP or
cAMP levels in mouse spermatozoa induced a transient elevation
of [Ca2+]i lasting 20–60 s (Kobori et al., 2000). This effect was
greatly reduced in low-Ca2+ saline or in the presence of Ca2+channel blockers, indicating that Ca2+ is mobilized through cyclic
nucleotide-gated channels, cGMP being significantly more effective
in elevating [Ca2+]i. Weyand et al. (1994) cloned a cyclic nucleotide gated (CNG), Ca2+-permeable channel, of the type found in
vertebrate photoreceptors and olfactory neurons, from bovine testis. The channel is expressed in spermatozoa, occurring in the principal piece of the flagellum, and is more sensitive to cGMP than
cAMP (Weyand et al., 1994; Wiesner et al., 1998). Intriguingly,
olfactory receptors, which activate a cyclic nucleotide-mediated
Ca2+ influx and control chemotactic activity, have recently been
described in human sperm (Spehr et al., 2003; see Actions of Ca2+mobilizing agonists).
Recently CatSpers, a novel family of ion channels, expressed
exclusively in sperm, has been described. CatSper family members contain six transmembrane segments and have a putative
voltage-sensor S4 domain [similar to KV, CNG, hyperpolarization
activated cyclic nucleotide-modulated channels (HCN) and TRP
channels]. The pore region is Ca2+ selective, and its transmembrane sequence resembles that of voltage-gated calcium and
sodium channels. Four different subunits have been identified:
CatSper1 (Ren et al., 2001), CatSper2 (Quill et al., 2001), 3 and 4
(Lobley et al., 2003). CatSper expression in the testis is first
observed when round spermatids appear during spermatogenesis
(Ren et al., 2001; Nikpoor et al., 2004). In mature cells, CatSper2
protein is localized to the sperm flagellum (Quill et al., 2001) and
CatSper1 to the principal piece of the tail (Ren et al., 2001), suggesting that CatSper channels may be involved in regulating sperm
motility. Consistent with this idea, initial studies suggest that Catsper expression is reduced in human sperm which lack of motility
(Nikpoor et al., 2004).
Expression of CatSper1 and 2 in different heterologous systems
failed to reveal whether the channels (presumably a tetramer as in
other members of the superfamily) are homo- or heterotetrameric,
although it is known that expression of only one subunit does not
result in functional channels (Quill et al., 2001; Ren et al., 2001).
These authors suggested that CatSper proteins probably need additional subunits and/or additional factors to form a fully functional
tetrameric channel.
Studies with CatSper-knockout mice showed that in mutant
sperm lacking Catsper1 motility was severely decreased and, as a
consequence, the sperm could not fertilize (Ren et al., 2001). Furthermore, in Catsper1 –/– cells, the rapid increase in tail [Ca2+]i
that occurs upon application of cell-permeant cAMP/cGMP (see
above; Kobori et al., 2000) did not occur. This observation suggested that the channel might be cAMP-gated (though CatSper
does not contain any identifiable cyclic nucleotide-binding region;
Ren et al., 2001).
Subsequently Carlson et al. (2003) and Quill et al. (2003) confirmed the importance of CatSper1 and Catsper2 in sperm motility
(more specifically in the hyperactivated movement required for
zona penetration). It was also shown that Catsper1 is required for
depolarization-evoked Ca2+ entry, suggesting that CatSper1 functions as voltage-gated Ca2+ channel (facilitated by cyclic nucleotides) which regulates hyperactivated motility. In addition,
Carlson et al. (2003) propose that this Ca2+ entry could be due to
channel facilitation instead of direct gating.
Ca2+ clearance mechanisms in sperm
In all eukaryotic cells, including sperm, Ca2+ clearance to maintain
low (resting) levels of intracellular [Ca2+]i or to decrease [Ca2+]i
back to resting levels post-stimulation is fundamental for stringent
control of cell signalling events (Berridge et al., 2000). In most
cells this process of Ca2+ clearance is undertaken to a great extent
by ATP requiring Ca2+ pumps (Ca2+-ATPases) or Na+–Ca2+
exchangers (NCXs), which extrude Ca2+ either out of the cell, or
into intracellular Ca2+ stores (Michelangeli et al., 2005). Analysis
of Ca2+ clearance in mouse sperm suggests that both Ca2+ pumps
and Ca2+ exchangers are important contributors to Ca2+ clearance
in mammalian sperm. Wennemuth et al. (2003) investigated in
detail the contributions and kinetics of the different Ca2+ clearance
mechanisms in mouse sperm following a membrane depolarizing
stimulus. Quantitative analysis showed that plasma membrane
Ca2+ ATPase (PMCA) pumps are the fastest Ca2+ extrusion mechanism in sperm, whereas NCX and mitochondrial Ca2+ uniporter
(MCU) are about a third as fast. Contribution of sarcoplasmic–
endoplasmic reticulum ATPases (Ca2+ store Ca2+ pumps) could
not be detected (see SERCA in sperm). They further concluded, in
agreement with earlier studies (Santi et al., 1998; Wennemuth et al.,
2000; Ren et al., 2001), that these cells are not adapted for rapid
signalling, decay times of [Ca2+]i transients typically being 40–60s,
more than an order of magnitude slower than in many somatic
cells. Two studies on mouse (Arnoult et al., 1999) and hamster sperm
(Suarez et al., 1993) have shown much more rapid Ca2+-clearance
rates (time constants of ∼50 min), but this may reflect other mechanisms such as local intracellular diffusion and equilibration with
cytoplasmic buffers.
Ca2+ pumps
To date, three types of ATP-utilizing Ca2+ pumps have been identified: the PMCA; the sarcoplasmic–endoplasmic Ca2+ ATPase
(SERCA) and the secretory pathway Ca2+ ATPase (SPCA)
(Michelangeli et al., 2005). These three types of Ca2+ ATPases
show around a 30% sequence similarity to each other (GunteskiHamblin et al., 1992) and are believed to have similar structures
and similar mechanisms of actions. They all belong to the P-type
family of ATPases, which become transiently phosphorylated to
transport ions across the membrane through an E1 to E2 conformational change, as proposed by de Meis and Vianna (1979).
More recently the elucidation of the crystal structure of the
SERCA1a isoform of the Ca2+ ATPase, has both identified the
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C.Jimenez-Gonzalez et al.
major domains and has shown how they move relative to each
other for translocation of Ca2+ across the membrane to take place
(Toyoshima and Inesi, 2004). SERCA1a and (it is believed) all
other types of Ca2+ ATPase consist of three large cytoplasmic
domains: ATP binding; phosphorylation and an actuator domain
which contributes to the re-arrangement of the transmembrane
helices, allowing the Ca2+ to move from one side of the membrane
to the other (Toyoshima and Inesi, 2004).
PMCA in sperm
PMCAs are the largest of the three types of Ca2+ ATPases, being
between 130 and 140 kDa MW. The larger size has been attributed
to an additional calmodulin-binding region located at the C-terminus of the protein which is involved in the regulation of ATPase
activity (Carafoli and Brini, 2000). There are four isoforms of the
PMCA (designated PMCA1–4) and approximately a dozen splice
variants (Carafoli and Brini, 2000). PMCA1 and PMCA4 are found
in most mammalian tissues, suggesting a general housekeeping role
as well as possibly more specialized functions in some cell types.
Western blotting and immunofluorescent labelling revealed the
presence of PMCA protein in rat spermatids and mouse spermatozoa (Berrios et al., 1998; Wennemuth et al., 2003). Western and
northern analysis showed that PMCA4 is the main isoform present
in testis and sperm. More than 90% of the PMCA protein in sperm
is PMCA4 (Okunade et al., 2004). Initially immunolocalization of
PMCA in mouse sperm showed staining in the postacrosomal segment and some labelling in the flagellum (Adeoya-Osiguwa and
Fraser, 1996). More recently, Wennemuth et al. (2003) mapped
PMCA to the principal piece of the sperm flagellum, with little or
no staining in the head and mid-piece, and the principal piece was
subsequently confirmed as the primary location of PMCA4 (Okunade
et al., 2004; Schuh et al., 2004).
With the introduction of genome manipulation to specifically
‘knockout’ targeted genes, it has been possible to assess the effects
of disrupting these genes on phenotype characteristics. Such a procedure was undertaken with the gene coding for several PMCA
isoforms in mice (Prasad et al., 2004). Unlike null mutant for
PMCA1 (which proved to be lethal during embryonic development), the null knockout animals for PMCA4 were relatively
healthy (Okunade et al., 2004; Prasad et al., 2004; Schuh et al.,
2004). The major phenotype observed in these mice was male
infertility (Okunade et al., 2004; Prasad et al., 2004; Schuh et al.,
2004), yet they showed normal spermatogenesis and mating
behaviour. Upon further investigation it was shown that the sperm,
though appearing normal before capacitation, failed to respond to
conditions that induce hyperactivated motility. After 90 min, most
cells were non-motile, with a few showing only very weak hyperactivated motility compared to wild type (Okunade et al., 2004;
Prasad et al., 2004). Measurement of [Ca2+]i showed that, after
60 min of incubation in capacitating medium, resting [Ca2+]i was
increased from 157 to 370 nM in PMCA4-deficient sperm (Schuh
et al., 2004). Schuh et al. (2004) mimicked the effect of PMCA
knockout using the PMCA inhibitor, 5-(and-6)-carboxyeosin diacetate succinimidyl ester, on wild-type mice. A similar failure of
hyperactivated motility was observed.
SERCA in sperm
In contrast to the persuasive evidence for the importance of
PMCA in sperm function, the grounds for attributing a major role
258
to activity of SERCA in mature sperm appear more controversial.
As pointed out earlier, the study of Wennemuth et al. (2003) suggested that Ca2+ clearance in mature sperm is unlikely to involve
activity of SERCA. Evidence for a role of SERCA in human
mature sperm was presented by Rossato et al. (2001), where they
showed that both Ca2+ mobilization and the acrosome reaction in
sperm could be induced by the potent SERCA-specific inhibitor
thapsigargin (Wictome et al., 1992) in the 10–100 nM concentration range. Localization studies using BODIPY-FL-thapsigargin
(a fluorescent analogue) appeared predominantly to stain the acrosome and mid-piece of sperm (Rossato et al., 2001).
In a more recent study by our laboratory, using an anti-SERCA
antibody (which recognized all known mammalian SERCA isoforms), no cross-reactivity was detected in western blots using
human sperm (Harper et al., 2005). Furthermore this study also
showed that thapsigargin only induced Ca2+ mobilization and disruption of Ca2+ signalling in sperm in the 1–10 μM range, which is
far higher than concentrations used to specifically inhibit SERCA
(Wictome et al., 1992; Brown et al., 1994). Earlier studies on the
effects of thapsigargin on the acrosome reaction and on [Ca2+]i in
sperm also showed that concentrations of 0.5 μM to more than 20 μM
thapsigargin were required to induce this response (Blackmore,
1993; Meizel and Turner, 1993; Llanos, 1998; Dragileva et al., 1999
O’Toole et al., 2000; Williams and Ford, 2003; Herrick et al.,
2005). Thus, treatment of sperm with thapsigargin at concentrations
sufficient to inhibit SERCAs is largely without effect, significant
actions occurring only at high, ‘non-specific’ doses.
Studies using sperm crudely isolated from whole goat testes
have been shown to both cross-react with SERCA-specific antibodies and exhibit Ca2+-dependent ATPase activity (Bhattacharyya
and Sen, 1998). However, these sperm cells are likely to be highly
contaminated by other testicular cell types (Leydig, Sertoli, white
blood cells etc.) in addition to immature sperm germ cells.
Evidence from our own work employing RT–PCR and using
SERCA2 and SERCA3 specific primers showed that germ cells
(spermatids) express the mRNA for both SERCA isoforms
(Hughes et al., 2000). It was also shown that rat round spermatids
responded to thapsigargin at concentrations less than 100 nM,
indicative of a specific SERCA interaction (Berrios et al., 1998).
These observations lead us to speculate that within sperm SERCA
may only be required during spermatogenesis, but then becomes
redundant in the mature sperm, either not being further expressed
or being rapidly degraded. The reason for this apparent ‘switch’ in
the expression of intracellular Ca2+-pumps as the germ cells
mature is not clear. Future work on the physiology and/or regulation of SPCAs may reveal characteristics that suit them particularly for the role that they play in spermatozoa.
SPCA in sperm
The SPCA was originally identified in mammalian cells due to its
high similarity to the vacuolar Ca2+ ATPase (PMR1) expressed in
yeast (Gunteski-Hamblin et al., 1992). In somatic cells the SPCA
are found located on the Golgi apparatus or secretory vesicles
(Wuytack et al., 2003; Wootton et al., 2004) and are believed to
control the levels of both Ca2+ and Mn2+ within the Golgi to regulate its function (Missiaen et al., 2004; Michelangeli et al., 2005).
Two isoforms of SPCA (SPCA1 and SPCA2) have been identified
which show about a 60% sequence similarity to each other
(Gunteski-Hamblin et al., 1992). Although there is no specific
Calcium signalling in human spermatozoa
inhibitor for SPCA1, a drug called bis-phenol has been shown to
be equally potent at inhibiting both SPCA1 and SERCA (Brown
et al., 1994; Harper et al., 2005).
We have shown, using RT–PCR, that rat germ cells (spermatids) express the mRNA for SPCA1 (Wootton et al., 2004) and
that mature human sperm cross-react with a SPCA1-specific antibody on western blots (Harper et al., 2005). Furthermore, immunolocalization studies on mature human sperm using this antibody
showed that labelling was localized to the anterior mid-piece,
extending into the rear of the head (Harper et al., 2005; Figure 2),
perhaps reflecting expression in the putative Ca2+ store of the
redundant nuclear envelope (RNE) (Ho and Suarez, 2003). In this
same study, parallel incubations employing a SERCA-specific
antibody showed no specific labelling.
Using bis-phenol, a SERCA and SPCA inhibitor (Brown et al.,
1994; Harper et al., 2005), we were able to demonstrate that this
drug, at doses consistent with its actions on thapsigargin-resistant,
Ca2+ store ATPase, could induce Ca2+ mobilization in sperm and
also stop progesterone-induced Ca2+ oscillations. In contrast, thapsigargin did not cause either of these responses unless applied at
very high (1–30 μM), non-specific doses (Harper et al., 2005).
The observation that thapsigargin inhibits SPCA activity with an
IC50 of between 10 and 20 μM (Reinhardt et al., 2004), may go
some way to explain some of the contradictory data regarding the
activity of SERCA in sperm when employing high ‘non-specific’
concentrations of this inhibitor.
In summary, there is strong evidence to indicate that sperm
express both PMCA and SPCA and that these Ca2+ pumps play a
major role in controlling sperm Ca2+ homeostasis. The role for
SERCA in mature sperm is more tenuous, but there appears to be
some evidence that it may play a role during spermatogenesis.
The Na+–Ca2+ exchanger
The forward action mode of the NCX exports an intracellular Ca2+
ion and imports three Na+ ions, using energy derived from the Na+
gradient at the cell membrane, and thus indirectly from activity of
the Na+, K+-ATPase (Blaustein and Lederer, 1999; Philipson and
Nicoll, 2000). This exchanger can also operate in a reverse mode,
facilitating Ca2+ influx. There are two groups (families) of Na/Ca
exchanger (NCX): NCX and K+-dependent Na/Ca exchangers
(NCKX). By using RT–PCR, two splice variants from the NCX1
gene, NCX1.3 and NCX1.7 were found in rat testis (Quednau
et al., 1997) and message for the NCKX3 isoform was detected in
mouse testis (Kraev et al., 2001).
Bradley and Forrester (1980) reported the actions of NCX in
plasma membrane vesicles from rat sperm flagella, showing
inhibition of NCX by the Ca2+ channel blocker verapamil. In
bovine sperm, Rufo et al. (1984) reported a difference in NCX
activity in epidymal sperm and in ejaculated sperm. In epididymal sperm, NCX transports Ca2+ into the cell acting in a
reverse mode. When cells are ejaculated, NCX is inhibited by a
seminal protein, thus decreasing its activity. It has been shown
recently (Su and Vacquier, 2002) that NCX expressed in the
tail of sea urchin sperm plays an important role in [Ca2+]i
homeostasis.
Figure 2. Expression of secretory pathway Ca2+ ATPases (SPCA) in human spermatozoa. Upper panels show fluorescent localization of anti-SPCA 1 (left) and
corresponding phase contrast image. Staining is concentrated in the sperm neck and mid-piece, with a few cells also showing some acrosomal staining. Lower panels show that control incubations using only the fluorescent secondary antibody, without anti-SPCA 1, do not result in staining of the cells.
259
C.Jimenez-Gonzalez et al.
Mitochondrial Ca2+ uptake
Mobilization of stored Ca2+
Inositol 1,4,5-trisphosphate receptors in sperm
The inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (commonly called the IP3 receptor or IP3R) has been studied extensively in a variety of cell types including sperm (Vermassen et al.,
2004). This channel binds the second messenger IP3, which leads
to elevation of intracellular Ca2+ concentrations (Michelangeli
et al., 1995). Three isoforms of this channels have so far been
identified from mammalian sources (identified as IP3R1, 2 and 3),
which in humans show greater than 74% sequence similarity to
each other (Taylor et al., 1999). The genes for the IP3Rs encode a
single polypeptide consisting of around 2500 amino acid residues,
which can be subdivided into three major domains. The region
closest to the amino-terminus has been identified as the IP3 binding domain, whereas the region closest to the carboxy-terminus is
260
14 0
progesterone
2,4-DNP (500 uM)
120
Δ fluorescence (%)
Accumulation of Ca2+ by mitochondria is well established as a
process that occurs under normal physiological conditions. Ca2+
uptake acts as a regulator of mitochondrial function and, in many
cells, as a contributor to the generation and shaping of [Ca2+]i signals, as well as during pathological conditions leading to Ca2+
overload (for a recent review see Bianchi et al., 2004). In human
spermatozoa, the mitochondria are restricted to the mid-piece and
potentially form an important Ca2+ buffer in this region. Many
studies on permeabilized cells have demonstrated that sperm mitochondria are able to accumulate Ca2+ in situ (Storey and Keyhani,
1973, 1974; Babcock et al., 1975), but data obtained from sperm
of various mammalian species and at various stages of maturation
suggest that the nature of mitochondrial Ca2+ accumulation varies
and may be regulated. Ca2+ uptake of rabbit epididymal sperm
mitochondria is inefficient compared to that in mitochondria of
somatic cells and shows little sensitivity to inhibition by ruthenium red (a widely used blocker of the mitochondrial Ca2+-uptake
pathway; Storey and Keyhani, 1974). In contrast, mitochondria of
ejaculated bovine spermatozoa accumulate Ca2+ and show an
increase in Ca2+ uptake in response to treatment with the dye
Cibacron Blue FG3A. This uptake is highly sensitive to ruthenium
red (Schoff, 1995). The mitochondria of bovine sperm isolated
from the caput epididymis are reported to show significantly
(2–3 times) higher rates of Ca2+ uptake than mitochondria of cells
in the cauda (Vijayaraghavan and Hoskins, 1990). Analysis of
Ca2+ clearance in caudal mouse spermatozoa showed that mitochondria contribute only slightly under normal conditions but their
activity was clearly demonstrated when membrane clearance
mechanisms were simultaneously inhibited (Wennemuth et al.,
2003). Similarly, mitochondrial uncoupling had very little effect
on [Ca2+]i signalling in human spermatozoa stimulated with progesterone. In response to progesterone stimulation, up to 50% of
cells generate [Ca2+]i oscillations due to mobilization of Ca2+ from
a store situated in the neck region of the cell (see Ryanodine
receptors in sperm). Application of 0.1–0.5 mM 2,4-dinitrophenol
to cells responding to progesterone had very little effect, slightly
enlarging/accelerating oscillations in a small proportion of cells
(Harper et al., 2004; Figure 3). Thus it appears that sperm mitochondria are capable of contributing to [Ca2+]i homeostatic mechanisms, but their role is limited.
10 0
80
60
40
20
0
-20
0
5
10
15
20
25
time (min)
2+
Figure 3. [Ca ]i oscillations in human sperm are insensitive mitochondrial
uncoupling. Traces show two single-cell records from cells loaded with Oregon
green BAPTA-1 acetoxy methylester to report changes in [Ca2+]i (expressed as
percentage change in fluorescent intensity). Upon application of progesterone
(3 μM), the cells generate a [Ca2+]i transient followed by a series of oscillations. These oscillations are not generated by Ca2+ influx and therefore reflect
mobilization of stored Ca2+ (Harper et al., 2004, 2005) but are not inhibited by
high concentrations of the mitochondrial uncoupler 2,4-dinitrophenol (2,4-DNP).
the location of the membrane-spanning channel domain. In
between, there is a large domain which contains a variety of phosphorylation sites, ATP binding sites and other regulatory protein
binding sites, which has been termed the modulatory or coupling
domain (Bultynck et al., 2003). In its native state, the IP3R exists
as a tetramer and a recent study employing electron microscopy
and single-particle analysis has shown it to have a flower-like
appearance with four-fold symmetry, in which the four IP3 binding domains, form the ‘stigma’ in the centre of the structure and
the four modulatory domains are arranged as ‘petals’ surrounding
it (da Fonseca et al., 2003).
Round spermatids from rat have a releasable intracellular Ca2+
store (Berrios et al., 1998). Using RT–PCR, we were able to detect
the presence of mRNA for all three isoforms of the IP3R in these
cells, although the type 1 isoform was much less prominent than
the other isoforms (Tovey et al., 1997). It appears that, as the germ
cells mature, IP3Rs are maintained, though the balance of expression shifts. Several studies have shown that sperm from a variety
of mammals, including humans, contain proteins which crossreact with different IP3R-specific antibodies (Walensky and
Snyder, 1995; Kuroda et al., 1999; Minelli et al., 2000; Ho and
Suarez, 2003). The studies by Walensky and Snyder (1995) and
Kuroda et al. (1999) also showed that mammalian sperm contain
the G-protein (Gq) and phospholipase α (PLCα) and thus have the
required proteins for agonist-stimulated production of IP3. Immunolocalization experiments in these studies showed that the IP3Rspecific antibodies extensively labelled the anterior of the acrosome region within the sperm head indicating that this could be a
mobilizable Ca2+ store. Use of isoform-specific IP3R antibodies
also showed that it was the type 1 isoform that was predominantly
located in this region (Kuroda et al., 1999; Ho and Suarez, 2003).
Interestingly, the IP3R labelling was lost or reduced once the
sperm had undergone the acrosome reaction further confirming the
IP3R location as being the outer acrosomal membrane (Walensky
and Snyder, 1995 and Kuroda et al., 1999).
Calcium signalling in human spermatozoa
Kuroda et al. (1999) showed that the posterior region of the
head, the mid-piece and part of the tail was specifically labelled
with an IP3R3-specific antibody in human sperm, while the presence of IP3R2 was not detected at all. However, this is in direct
contrast with the study of Ho and Suarez (2003) that showed that
in bull sperm the region at the back of the head which they identified as the RNE was labelled with the IP3R1-specific antibody.
Anti-IP3R staining also localizes to this region in approximately
half of human spermatozoa, though the most intense staining was
observed in the acrosomal region (>90% of cells; Naaby-Hansen
et al., 2001).
To further characterize the IP3R isoforms present in sperm,
[3H]IP3 binding studies were undertaken on membranes
extracted from mature sperm. Two studies have shown that the
binding curves obtained form these experiments were consistent
with two classes of binding sites; one having high affinity (Kd of
20–30 nM) and the other having much lower affinity (Kd of 1–2
μM) (Walensky and Snyder, 1995; Kuroda et al., 1999). These
different classes of binding sites may well be due to the presence
of two IP3R isoforms, since IP3R1 and IP3R3 are known to have
very different IP3 binding affinities (Wojcikiewicz and Luo,
1998) and different IP3 sensitivities for Ca2+ release (Dyer and
Michelangeli, 2001). To assess whether Ca2+ mobilization from
IP3R containing Ca2+ stores contribute to sperm function, thimerosal, an IP3R activator, (Bootman et al., 1992; Sayers et al.,
1993) was used. Herrick et al. (2005) showed that thimerosal
was able to induce the acrosome reaction, confirming a role for
IP3R in sperm physiology.
From the studies outlined here it is clear that two IP3R containing Ca2+ stores are present within sperm, one in the acrosome and
the other a much smaller Ca2+ store located within the RNE at the
back of the head. To further support the notion that both these
compartments are bona fide Ca2+ stores, studies by Naaby-Hansen
et al. (2001) and Ho and Suarez (2003) showed that both regions
also contained calreticulin (a low affinity, high capacity Ca2+buffering protein) that is always associated with IP3R containing
Ca2+ stores in somatic cells. Interestingly, Ca2+ stored in the RNE
of human spermatozoa is apparently mobilized upon progesterone
stimulation by Ca2+-induced Ca2+ release, in an IP3-independent
manner (Harper et al., 2004; see Actions of Ca2+-mobilizing
agonists).
Ryanodine receptors in sperm
The ryanodine receptor (RyR) was first identified in skeletal
muscle sarcoplasmic reticulum membranes where it was characterized as a Ca2+-induced Ca2+ release channel which plays a
pivotal role in excitation–contraction coupling of striated muscle
(Fill and Copello, 2002). The genes for three mammalian isoforms of the RyR have been identified which code for a large
protein containing approximately 5000 amino acids and which
show a high degree of sequence homology with each other
(about 70% overall) (Brini, 2004). The three isoforms are
denoted as RyR 1, which was initially identified in skeletal muscle; RyR 2, initially identified from cardiac muscle and RyR 3,
identified in brain but having a much wider tissue distribution
generally (Brini, 2004).
Sequence analysis suggests that the RyR is composed of two
major domains: the amino-terminal region consisting most of the
protein forms a large cytoplasmic structure which is believed to
contain ligand and modulatory protein binding sites and the
carboxy-terminal region (consisting of about 1000 amino acids)
forming the transmembrane channel domain and probably containing four transmembrane helices (Brini, 2004). From cryo electronmicroscopy analysis of this channel it forms a homotetrameric
complex with a ‘four-leaf clover’ type appearance (Radermacher
et al., 1992).
The RyR Ca2+ channel is believed to be activated by changes in
[Ca2+], the putative second messenger cyclic adenosine diphosphateribose (CADPR), and through conformational coupling with other
associated proteins (Zucchi and Ronca-Testoni, 1997). Pharmacological modulators of this channel include caffeine and low concentrations of ryanodine which activate the channel and local
anaesthetics, ruthenium red and high concentrations of ryanodine,
which close the channel (Zucchi and Ronca-Testoni, 1997).
Initial evidence for the presence of RyR in sperm came from
a study by Giannini et al. (1995), who employed both in situ
hybridization methods using ribo-probes specific for the different RyR isoforms and immunohistochemical methods using
RyR isoform-specific antibodies. From the analysis of mouse
testis sections they concluded that cells believed to be germ
cells, such as spermatocytes and spermatids expressed both RyR
1 and RyR 3 but not RyR 2 (Giannini et al., 1995). This was followed up by many later studies using RT–PCR and isoformspecific RyR antibodies again in mouse sperm, where Trevino
et al. (1998) and Chiarella et al. (2004) showed that developing
spermocytes and spermatids expressed both RyR 1 and RyR 3.
Furthermore, Trevino et al. (1998) showed that only RyR 3
could be detected in mature fully developed sperm. This study
also showed similar staining patterns in both intact and acrosome-reacted sperm, indicating that the localization of the RyR
3 was unlikely to be on the acrosome (Trevino et al., 1998). We
have also shown that human sperm can be specifically labelled
with a fluorescent analogue of ryanodine (BODIPY-FL-X-ryanodine).
This labelling, which appears to be mainly focussed to the rear
of the sperm head, i.e. around the head and mid-piece junction,
with only low levels of labelling around the acrosome (Harper
et al., 2004), co-localizes with SPCA1 and with the oscillations
of [Ca2+]i that occur in response to progesterone stimulation
(Figure 4).
Several pharmacological-based studies have also indicated the
presence and functionality of RyR in mature sperm. Minelli et al.
(2000) using digitonin permeabilized bovine sperm showed that
both caffeine and ryanodine decreased 45Ca2+ accumulation
within the sperm in a similar manner to IP3, indicating the activation of Ca2+ efflux channel with RyR-like properties. In intact
human sperm, we have also demonstrated that the progesteroneinduced intracellular [Ca2+] oscillations are IP3-independent but
could be modified by ryanodine, with low doses increasing the
frequency and higher doses reducing the frequency (Harper et al.,
2004). In addition, the RyR inhibitor tetracaine could abolish
these Ca2+ oscillations altogether (Harper et al., 2004). The findings presented in Chiarella et al. (2004) that showed that high
doses of ryanodine could reduce spermatogonial proliferation and
increase cell meiosis, emphasize that sperm contain functional
RyR that not only plays an important physiological role in
regulating agonist-induced Ca2+ changes but also in sperm
development.
261
C.Jimenez-Gonzalez et al.
Actions of Ca2+-mobilizing agonists
response to ZP3-induced depolarization (Florman, 1994; Arnoult
et al., 1996b), causing a large [Ca2+]1 transient (9 μM), with kinetics
(rise time ∼40 ms; duration 100–200 ms) reminiscent of wholecell currents induced by step depolarization of spermatogenic cells
(Arnoult et al., 1999).
Following the initial large [Ca2+]i transient, a much slower but
sustained rise in [Ca2+]i develops, from the resting value of 160 to
∼400 nM. The sustained [Ca2+]i elevation is dependent on the initial [Ca2+]i transient and is required for induction of acrosome
reaction (O’Toole et al., 2000). This second phase is also dependent on Ca2+ influx and, in mouse, is known to involve TRPC2
(Jungnickel et al., 2001), a member of the canonical TRP family
(Padinjat and Andrews, 2004) which forms store-operated Ca2+
channels when transfected into cultured cells (Vannier et al.,
1999). However, the mechanism by which this channel is activated in vivo is not clear. Initially it appeared likely that sustained
Ca2+ influx was through a store-activated mechanism (O’Toole
et al., 2000). Thapsigargin, at high doses, causes Ca2+ influx and
acrosome reaction, which is consistent with this model (Dragileva
et al., 1999; O’Toole et al., 2000; Jungnickel et al., 2001). Mobilization of a store in response to ZP stimulation might occur by activation of PLC or by sensitization of IP3 receptors (or both)
(O’Toole et al., 2000; others). Furthermore, the acrosome itself
acts as an IP3-mobilized Ca2+ store, and its mobilization is apparently a key event in induction of acrosome reaction. Nevertheless,
it is now known that TRPC channels can be activated by various
mechanisms (Padinjat and Andrews, 2004) and the mechanism by
which zona activates sustained Ca2+ influx awaits clarification
(Evans and Florman, 2002). An interesting point with regard to the
mechanism by which ZP induces acrosome reaction in human
spermatozoa is that human TRPC2 is apparently a psueudogene
(Wes et al., 1995; Vannier et al., 1999). Relatively, little is known of
the ZP-induced [Ca2+]i signal in human cells (Patrat et al., 2000),
but it is probable that in human spermatozoa another member of
the TRPC family plays that role taken by TRPC2 in mouse sperm.
Zona pellucida-induced acrosome reaction
Ca2+ mobilization induced by progesterone
The action of zona pellucida to mobilize Ca2+, leading to acrosome reaction, is the best-characterized agonist-induced response
in mammalian sperm. Binding of ZP3 or solubilized zona induces
a series of Ca2+-dependent events which lead to acrosome reaction.
Though this process has been investigated in many mammalian
species (including humans), our knowledge of events in the mouse
is by far the most detailed. Initially, activation of a pertussis-sensitive
Gq causes cytoplasmic alkalinization (Arnoult et al, 1996b;
Florman et al., 1998), and a separate pertussis toxin-insensitive
mechanism activates a poorly selective cation channel, leading to
depolarization (Florman, 1994; Arnoult et al., 1996a). These
events cause activation of a voltage-operated channel (Florman
et al., 1992; Florman, 1994). Though this channel has some characteristics of L-type VOCCs, application of patch clamp to spermatogenic cells has shown that it is a T-type channel sensitive to
dihydropyridines (normally considered to be L-channel specific),
this channel is clearly a T-type (LVA) VOCC (Arnoult et al.,
1996a,b; Lievano et al., 1996; Santi et al., 1996) which has unusual
pharmacological properties (Arnoult et al., 1998). It is released
from inactivation by the hyperpolarization that occurs during
capacitation (Zeng et al., 1995; Arnoult et al., 1999) and opens in
In human spermatozoa, the [Ca2+]i response to progesterone has
been studied in great detail, partly due to the great difficulty of
working with human zona. In fact, human spermatozoa appear to
be unusually sensitive to progesterone (Kirkman-Brown et al.,
2002). When stimulated with 3 μM progesterone, believed to be
representative of concentrations present in the vicinity of the
oocyte-cumulus, human sperm generate a biphasic [Ca2+]i
response consisting of a transient (lasting 1–2 min) followed by a
sustained elevation. Both parts of the response involve influx of
extracellular Ca2+ and presumably reflect gating of membrane
Ca2+-permeable channels. However, the nature of the channels
involved is largely unknown. We have suggested previously that
the response to progesterone may be similar to that induced by
ZP, activating a VOCC (though probably not T-type; Blackmore,
1999; Blackmore and Eisoldt, 1999) and possibly converging
with the ZP-activated pathway on activation of store-operated influx
(Barratt and Publicover, 2001). Extracellular La8+ (Ca2+ channel
antagonist) can completely inhibit the response to progesterone
(Blackmore et al., 1990), confirming the importance of membrane
Ca2+ channels, and a late component of the initial [Ca2+]i transient
(that is particularly sensitive to occlusion by prior progesterone
Figure 4. Co-localization of [Ca2+]i oscillations, SPCA1 and binding of
BODIPY ryanodine. Diagram on left shows stylized human spermatozoan—
area of co-localization is shown by red box. Upper picture shows phase image
of live cells with overlay of Oregon green BAPTA-1 fluorescence recorded
during generation of [Ca2+]i oscillations. Central picture shows phase image of
fixed cells with overlay of fluorescently localized SPCA1. Lower picture
shows phase image of live cells with overlay of staining pattern observed with
fluorescently tagged BODIPY ryanodine. Staining was inhibited by competitive incubation with unlabelled ryanodine.
262
Calcium signalling in human spermatozoa
stimulation; Harper et al., 2003) is sensitive to nifedipine (KirkmanBrown et al., 2003). However, the balance of evidence from studies which have specifically attempted to demonstrate a role for
VOCCs in the response to progesterone does not support this
model (Blackmore and Eisoldt, 1999; Garcia and Meizel, 1999;
Bonaccorsi et al., 2001; Fraire-Zamora and Gonzalez-Martinez,
2004). Stimulation of sperm from PLCδ4-knockout mice with
50–100 μM progesterone generates a response of reduced amplitude
and greatly reduced duration compared to that of wild-type cells
(Fukami et al., 2003), consistent with a requirement for emptying
of an IP3-sensitive store, though the high doses required to evoke
large responses in murine sperm may be acting by a different pathway to that normally studied in human spermatozoa, which saturates at approximately 300 nM progesterone (Baldi et al., 1991;
Harper et al., 2003) Attempts to demonstrate pharmacologically
that the sustained elevation of [Ca2+]i is due to activation of SOCs
have produced equivocal data (Blackmore, 1999; Harper et al.,
2004, 2005). Recently it has been shown that progesterone also
activates repeated [Ca2+]i oscillations in human spermatozoa
which are the result of store mobilization (Harper et al., 2004;
Kirkman-Brown et al., 2004). If progesterone is applied as a gradient (to represent more closely the stimulus encountered as a spermatozoon approaches the oocyte) then the initial [Ca2+]i transient,
a characteristic of all previous studies, does not occur, but [Ca2+]i
oscillations occur in many cells (Harper et al., 2004). Ca2+ influx
induced by progesterone apparently activates a ryanodine (like)
receptor located in the sperm neck/mid-piece (probably on the
RNE; Figure 4) leading to repetitive bursts of Ca2+-induced Ca2+
release. Though IP3Rs have been localized to this area of the
sperm, the [Ca2+]i oscillations are resistant to pharmacological
treatments designed to inhibit PLC or IP3Rs, suggesting that IP3
generation is not required for their generation (Harper et al.,
2004). Re-uptake of Ca2+ during oscillations is thapsigargininsensitive and apparently is dependent (at least in part) on activity
of SPCA1 (Harper et al., 2005).
spermatozoa, apparently generated by mobilization from a store in
the sperm neck region (see above), similarly affect the flagellar
activity but fail to induce acrosome reaction.
Ca2+-signalling ‘toolkit’ in sperm
From the data summarized above a complex model for sperm Ca2+
homeostasis involving several types of Ca2+-permeable channel in
the plasma membrane and at least two stores is appropriate (Figure 5).
Furthermore, it is clear that these toolkit components are distributed to allow localization of [Ca2+]i signals. As well as a range of
VOCCs, which are clearly localized to sperm regions, the
CatSpers, which are essential for activation of hyperactivated
mobility, are expressed specifically in the principal piece of the
sperm tail, as is PMCA4. It appears that the acrosome functions as
an IP3-releasable store activated by agonists linked to PLC. Recent
studies suggest mobilization of acrosomal Ca2+ is intimately
Ca2+ signals induced by activation of olfactory receptors
Spermatozoa in externally fertilizing animals must locate the
oocyte and the occurrence of sperm chemotaxis in such organisms
is well documented (Neill and Vacquier, 2004; Yoshida, 2004). In
human sperm, there is also good evidence for the occurrence of
chemotaxis to components of follicular fluid (and therefore to the
oocyte), but the nature of the chemo-attractant and its effects on
sperm motility are disputed (Eisenbach, 1999). Recently it has
been shown that agonists of olfactory receptors expressed in
human spermatozoa act as chemo-attractants by regulation of
Ca2+-influx. The receptor hOR17-4, which is strongly activated by
burgeonal and antagonized by undecanal, activates adenyl cyclase
(Spehr et al., 2003), probably the membrane-associated mAC III,
activated through Gαolf (Spehr et al., 2004a). The same receptor
apparently performs a ‘normal’ olfactory function in the human
nasal mucosa (Spehr et al., 2004b). The resulting Ca2+ signal originates in the mid-piece, a larger signal then occurring in the head
with an average latency of >2 s (Spehr et al., 2004a). Interestingly,
whilst affecting flagellar beat and causing clear chemotactic
effects (Spehr et al., 2003, 2004a), the burgeonal-activated [Ca2+]i
signal does not appear to cause acrosome reaction. The [Ca2+]i
oscillations that occur in progesterone–gradient-stimulated human
Figure 5. Two-store model for Ca2+ signalling in human spermatozoa. Plasma
membrane Ca2+ ATPases (PMCA) and Na+–Ca2+ exchangers (NCX) are shown
in green; Ca2+ channels in the plasma membrane are shown in red; sarcoplasmic–
endoplasmic reticulum ATPases (SERCAs) on intracellular stores are shown in
blue and channels for mobilization of stored Ca2+ are shown in orange. Identified
or putative components of the Ca2+-signalling toolkit are labelled (using the
same colour coding) adjacent to their localization.
263
C.Jimenez-Gonzalez et al.
involved in activation of acrosome reaction (De Blas et al., 2002;
Herrick et al., 2005). A separate store, probably the RNE, exists in
the neck region of the sperm and plays a key role in regulation of
flagellar beat mode (see above). Future work must address several
questions to do with interrelatedness of these mechanisms. For
instance, how do the roles of the CatSper channels, the Golf /
cAMP-regulated channels and the mobilization of stored Ca2+
interact in the control of flagellar beat?
Acknowledgements
The support of The Wellcome Trust for CJG is gratefully acknowledged.
References
Adeoya-Osiguwa SA and Fraser LR (1996) Evidence for Ca2+-dependent
ATPase activity, stimulated by decapacitation factor and calmodulin, in
mouse sperm. Mol Reprod Dev 44,111–120.
Ain R, Uma Devi K, Shivaji S and Seshagiri PB (1999) Pentoxifylline-stimulated
capacitation and acrosome reaction in hamster spermatozoa: involvement
of intracellular signalling molecules. Mol Hum Reprod 5,618–626.
Arnoult C, Cardullo RA, Lemos JR and Florman HM (1996a) Activation of
mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida.
Proc Natl Acad Sci USA 93,13004–13009.
Arnoult C, Zeng Y and Florman HM (1996b) ZP3-dependent activation of
sperm cation channels regulates acrosomal secretion during mammalian
fertilization. J Cell Biol 134,637–645.
Arnoult C, Kazam IG, Visconti PE, Kopf GS, Villaz M and Florman HM
(1999) Control of the low voltage-activated calcium channel of mouse
sperm by egg ZP3 and by membrane hyperpolarization during capacitation. Proc Natl Acad Sci USA 96,6757–6762.
Arnoult C, Villaz M and Florman HM (1998) Pharmacological properties of
the T-type Ca2+ current of mouse spermatogenic cells. Mol Pharmacol
53,1104–1111.
Babcock DF, First NL and Lardy HA (1975) Transport mechanism for succinate and phosphate localized in the plasma membrane of bovine spermatozoa. J Biol Chem 250,6488–6495.
Baldi E, Casano R, Falsetti C, Krausz C, Maggi M and Forti G (1991) Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 12,323–330.
Barratt CL and Publicover SJ (2001) Interaction between sperm and zona pellucida in male fertility. Lancet 358,1660–1662.
Berridge MJ (2005) Unlocking the secrets of cell signalling. Annu Rev Physiol
67,1–21.
Berridge MJ, Lipp P and Bootman MD (2000) The versatility and universality
of calcium signalling. Nat Rev Mol Cell Biol 1,11–21.
Berrios J, Osses N, Opazo C, Arenas G, Mercado L, Benos DJ and Reyes JG
(1998) Intracellular Ca2+ homeostasis in rat round spermatids. Biol Cell
90,391–398.
Bhattacharyya D and Sen PC (1998) Purification and functional characterization of a low-molecular-mass Ca2+, Mg2+- and Ca2+-ATPase modulator
protein from rat brain cytosol. Biochem J 330,95–101.
Bianchi K, Rimessi A, Prandini A, Szabadkai G and Rizzuto R (2004) Calcium
and mitochondria: mechanisms and functions of a troubled relationship.
Biochim Biophys Acta 1742,119–131.
Blackmore PF (1993) Thapsigargin elevates and potentiates the ability of progesterone to increase intracellular free calcium in human sperm: possible
role of perinuclear calcium. Cell Calcium 14,53–60.
Blackmore PF (1999) Extragenomic actions of progesterone in human
sperm and progesterone metabolites in human platelets. Steroids 64,
149–156.
Blackmore PF, Beebe SJ, Danforth DR and Alexander N (1990) Progesterone
and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in
human sperm. J Biol Chem 265,1376–1380.
Blackmore PF and Eisoldt S (1999) The neoglycoprotein mannose-bovine
serum albumin, but not progesterone, activates T-type calcium channels in
human spermatozoa. Mol Hum Reprod 5,498–506.
Blaustein MP and Lederer WJ (1999) Sodium/calcium exchange: its physiological implications. Physiol Rev 79,763–854.
Bonaccorsi L, Forti G and Baldi E (2001) Low-voltage-activated calcium
channels are not involved in capacitation and biological response to progesterone in human sperm. Int J Androl 24,341–351.
264
Bootman MD, Taylor CW and Berridge MJ (1992) The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5trisphosphate receptor. J Biol Chem 267,25113–25119.
Bradley MP and Forrester IT (1980) A sodium-calcium exchange mechanism
in plasma membrane vesicles isolated from ram sperm flagella. FEBS Lett
121,15–18.
Brini M (2004) Ryanodine receptor defects in muscle genetic diseases. Biochem
Biophys Res Commun 322,1245–1255.
Brown GR, Benyon SL, Kirk CJ, Wictome M, East JM, Lee AG and Michelangeli F
(1994) Characterisation of a novel Ca2+ pump inhibitor (bis-phenol) and
its effects on intracellular Ca2+ mobilization. Biochim Biophys Acta
1195,252–258.
Bultynck G, Sienaert I, Parys JB, Callewaert G, De Smedt H, Boens N, Dehaen
W and Missiaen L (2003) Pharmacology of inositol trisphosphate receptors. Pflugers Arch 445,629–642.
Carafoli E and Brini MR (2000) Calcium pumps: structural basis for and
mechanism of calcium transmembrane transport. Curr Opin Chem Biol
4,152–161.
Carlson AE, Westenbroek RE, Quill T, Ren D, Clapham DE, Hille B, Garbers
DL and Babcock DF (2003) CatSper1 required for evoked Ca2+ entry and
control of flagellar function in sperm. Proc Natl Acad Sci USA
100,14864–14868.
Castellano LE, Trevino CL, Rodriguez D, Serrano CJ, Pacheco J, Tsutsumi V,
Felix R and Darszon A (2003) Transient receptor potential (TRPC) channels in human sperm: expression, cellular localization and involvement in
the regulation of flagellar motility. FEBS Lett 541,69–74.
Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels.
Annu Rev Cell Dev Biol 16,521–555.
Catterall WA, Streissnig J, Snutch T and Perez-Reyes E (2003) International
Union of Pharmacology. XL. Compendium of voltage-gated ion channels:
calcium channels. Pharmacol Rev 55,579–581.
Chiarella P, Puglisi R, Sorrentino V, Boitani C and Stefanini M (2004) Ryanodine receptors are expressed and functionally active in mouse spermatogenic cells and their inhibition interferes with spermatogonial
differentiation. J Cell Sci 117,4127–4134.
da Fonseca PC, Morris SA, Nerou EP, Taylor CW and Morris EP (2003)
Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as
revealed by single-particle analysis. Proc Natl Acad Sci USA 100,3936–
3941.
de Meis L and Vianna AL (1979) Energy interconversion by the Ca2+dependent ATPase of the sarcoplasmic reticulum. Annu Rev Biochem
48,275–292.
De Blas G, Michaut M, Trevino CL, Tomes CN, Yunes R, Darszon A and
Mayorga LS (2002) The intraacrosomal calcium pool plays a direct role in
acrosomal exocytosis. J Biol Chem 277,49326–49331.
De Jonge C (2005) Biological basis for human capacitation. Hum Reprod
Update 11,205–214.
Dragileva E, Rubinstein S and Breitbart H (1999) Intracellular Ca2+-Mg2+ATPase regulates calcium influx and acrosomal exocytosis in bull and
ram spermatozoa. Biol Reprod 61,1226–1234.
Dyer JL and Michelangeli F (2001) Inositol 1,4,5-trisphosphate receptor isoforms show similar Ca2+ release kinetics. Cell Calcium 30,245–250.
Eisenbach M (1999) Sperm chemotaxis. Rev Reprod 4,56–66.
Ertel EA, Campbell KP, Harpold MM, Hofmann F, Mori Y, Perez-Reyes E,
Schwartz A, Snutch TP, Tanabe T, Birnbaumer L et al. (2000) Nomenclature of voltage-gated calcium channels. Neuron 25,533–535.
Espinosa F, Lopez-Gonzalez I, Serrano CJ, Gasque G, de la Vega-Beltran JL,
Trevino CL and Darszon A (1999) Anion channel blockers differentially
affect T-type Ca2+ currents of mouse spermatogenic cells, α1E currents
expressed in Xenopus oocytes and the sperm acrosome reaction. Dev
Genet 25,103–114.
Evans JP and Florman HM (2002) The state of the union: the cell biology of
fertilization. Nat Cell Biol 4,s57–s63.
Fill M and Copello JA (2002) Ryanodine receptor calcium release channels.
Physiol Rev 82,893–922.
Florman HM (1994) Sequential focal and global elevations of sperm intracellular Ca2+ are initiated by the zona pellucida during acrosomal exocytosis.
Dev Biol 165,152–164.
Florman HM, Tombes RM, First NL and Babcock DF (1989) An adhesionassociated agonist from the zona pellucida activates G protein-promoted
elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis. Dev Biol 135,133–146.
Florman HM, Corron ME, Kim TD and Babcock DF (1992) Activation of
voltage-dependent calcium channels of mammalian sperm is required
for zona pellucida-induced acrosomal exocytosis. Dev Biol 152,304–314.
Calcium signalling in human spermatozoa
Florman HM, Arnoult C, Kazam IG, Li C and O’Toole CM (1998) A perspective on the control of mammalian fertilization by egg-activated ion channels in sperm: a tale of two channels. Biol Reprod 59,12–16.
Fomina AF and Nowicky MC (1999) A current activated on depletion of intracellular Ca2+ stores can regulate exocytosis in adrenal chromaffin cells.
J Neurosci 19,3711–3722.
Fragale A, Aguanno S, Kemp M, Reeves M, Price K, Beattie R, Craig P,
Volsen S, Sher E and D’Agostino A (2000) Identification and cellular
localisation of voltage-operated calcium channels in immature rat testis.
Mol Cell Endocrinol 162,25–33.
Fraire-Zamora JJ and Gonzalez-Martinez MT (2004) Effect of intracellular pH
on depolarization-evoked calcium influx in human sperm. Am J Physiol
Cell Physiol 287,C1688–C1696.
Fukami K, Yoshida M, Inoue T, Kurokawa M, Fissore RA, Yoshida N,
Mikoshiba K and Takenawa T (2003) Phospholipase Cdelta4 is required
for Ca2+ mobilization essential for acrosome reaction in sperm. J Cell
Biol 14,79–88.
Garcia MA and Meizel S (1999) Progesterone-mediated calcium influx and
acrosome reaction of human spermatozoa: pharmacological investigation
of T-type calcium channels. Biol Reprod 60,102–109.
Garcia RL and Schilling WP (1997) Differential expression of mammalian
TRP homologues across tissue and cell lines. Biochem Biophys Res Commun 239,279–283.
Giannini G, Conti A, Mammarella S, Scrobogna M and Sorrentino V (1995) The
ryanodine receptor/calcium channel genes are widely and differentially
expressed in murine brain and peripheral tissues. J Cell Biol 128,893–904.
Gonzalez-Martinez MT, Galindo BE, de De La Torre L, Zapata O, Rodriguez E,
Florman HM and Darszon A (2001) A sustained increase in intracellular
Ca(2+) is required for the acrosome reaction in sea urchin sperm. Dev
Biol 236,220–229.
Goodwin LO, Leeds NB, Hurley I, Mandel FS, Pergolizzi RG and Benoff S
(1997) Isolation and characterization of the primary structure of testisspecific L-type calcium channel: implications for contraception. Mol Hum
Reprod 3,255–268.
Goodwin LO, Leeds NB, Hurley I, Cooper GW, Pergolizzi RG and Benoff S
(1998) Alternative splicing of exons in the alpha1 subunit of the rat testis
L-type voltage-dependent calcium channel generates germ line-specific
dihydropyridine binding sites. Mol Hum Reprod 4,215–226.
Goodwin LO, Karabinus DS, Pergolizzi RG and Benoff S (2000) L-type voltagedependent calcium channel α1C subunit mRNA is present in ejaculated
human spermatozoa. Mol Hum Reprod 6,127–136.
Gunteski-Hamblin AM, Clarke DM and Shull GE (1992) Molecular cloning
and tissue distribution of alternatively spliced mRNAs encoding possible
mammalian homologues of the yeast secretory pathway calcium pump.
Biochemistry 31,7600–7608.
Hagiwara S and Kawa K (1984) Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules. J Physiol (Lond)
356,135–149.
Harper CV, Kirkman-Brown JC, Barratt CL and Publicover SJ (2003) Encoding of progesterone stimulus intensity by intracellular [Ca2+] ([Ca2+]i) in
human spermatoza. Biochem J 372,407–417.
Harper CV, Barratt CL and Publicover SJ (2004) Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte.
Induction of [Ca2+]i oscillations and cyclical transitions in flagellar beating. J Biol Chem 279,46315–46325.
Harper C, Wootton L, Michelangeli F, Lefievre L, Barratt C and Publicover S
(2005) Secretory pathway Ca2+-ATPase (SPCA1) Ca2+ pumps, not SERCAs,
regulate complex [Ca2+]i signals in human spermatozoa. J Cell Sci
118,1673–1185.
Harteneck C, Plant TD and Schultz G (2000) From worm to man: three subfamilies of TRP. Channels Trends Neurosci 23,159–166.
Herrick SB, Schweissinger DL, Soo-Woo K, Bayan KR, Mann S and Cardullo
RA (2005) The acrosomal vesicle of mouse sperm is a calcium store.
J Cell Physiol 202,663–671.
Hirohashi N and Vacquier D (2003) Store-operated calcium channels trigger
exocytosis of the sea urchin sperm acrosomal vesicle. Biochem Biophys
Res Commun 304,285–292.
Ho HC and Suarez SS (2001) An inositol 1,4,5-trisphosphate receptor-gated
intracellular Ca2+ store is involved in regulating sperm hyperactivated
motility. Biol Reprod 65,1606–1615.
Ho HC and Suarez SS (2003) Characterization of the intracellular calcium
store at the base of the sperm flagellum that regulates hyperactivated
motility. Biol Reprod 68,1590–1596.
Hughes PJ, McLellan H, Lowes DA, Kahn SZ, Bilmen JG, Tovey SC, Godfrey
RE, Michell RH, Kirk CJ and Michelangeli F (2000) Estrogenic alkylphe-
nols induce cell death by inhibiting testis endoplasmic reticulum Ca2+
pumps. Biochem Biophys Res Commun 277,568–574.
Jacob A and Benoff S (2000) Full length low voltage-activated (‘T-type’) calcium Ca2+ channel α1G mRNA is not detected in mammalian testis and
sperm. J Androl 56,48.
Jagannathan S, Punt EL, Gu Y, Arnoult C, Sakkas D, Barratt CL and Publicover SJ (2002) Identification and localization of T-type voltage-operated
calcium channel subunits in human male germ cells. Expression of multiple isoforms. J Biol Chem 277,8449–8456.
Jungnickel MK, Marrero H, Birnbaumer L, Lemos JR and Florman HM (2001)
Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.
Nature Cell Biol 3,499–502.
Kaupp UB, Solzin J, Hildebrand E, Brown JE, Helbig A, Hagen V, Beyermann M,
Pampaloni F and Weyand I (2003) The signal flow and motor response
controlling chemotaxis of sea urchin sperm. Nat Cell Biol 5,109–117.
Kirkman-Brown JC, Punt EL, Barratt CL and Publicover SJ (2002) Zona pellucida and progesterone-induced Ca2+ signalling and acrosome reaction in
human spermatozoa. J Androl 23,306–315.
Kirkman-Brown JC, Barratt CL and Publicover SJ (2003) Nifedipine reveals
the existence of two discrete components of the progesterone-induced
[Ca2+]i transient in human spermatozoa. Dev Biol 259,71–82.
Kirkman-Brown JC, Barratt CL and Publicover SJ (2004) Slow calcium oscillations in human spermatozoa. Biochem J 378,827–832.
Kobori H, Miyazaki S and Kuwabara Y (2000) Characterization of intracellular Ca2+ increase in response to progesterone and cyclic nucleotides in
mouse spermatozoa. Biol Reprod 63,113–120.
Kraev A, Quednau BD, Leach S, Li XF, Dong H, Winkfein R, Perizzolo M,
Cai X, Yang R, Philipson KD et al. (2001) Molecular cloning of a third
member of the potassium-dependent sodium-calcium exchanger gene
family, NCKX3. J Biol Chem 276,23161–23172.
Kuroda Y, Kaneko S, Yoshimura Y, Nozawa S and Mikoshiba K (1999) Are
there inositol 1,4,5-triphosphate (IP3) receptors in human sperm? Life Sci
65,135–143.
Lefievre L, De Lamirande E and Gagnon C (2000) The cyclic GMP-specific
phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility
and capacitation but not acrosome reaction. J Androl 21,929–937.
Li HS, Xu XZS and Montell C (1999) Activation of a TRPC3-dependent cation current through the neurotrophin BDNF. Neuron 24,261–273.
Lievano A, Santi CM, Serrano CJ, Trevino CL, Bellve AR, Hernandez-Cruz A
and Darszon A (1996) T-type Ca2+ channels and alpha1E expression in
spermatogenic cells, and their possible relevance to the sperm acrosome
reaction. FEBS Lett 388,150–154.
Liman ER, Corey DP and Dulac C (1999) TRP2: a candidate transduction
channel for mammalian pheromone sensory signalling. Proc Natl Acad
Sci USA 96,5791–5796.
Llanos MN (1998) Thapsigargin stimulates acrosomal exocytosis in hamster
spermatozoa. Mol Reprod Dev 51,84–91.
Lobley A, Pierron V, Reynolds L, Allen L and Michalovich D (2003) Identification of human and mouse CatSper3 and CatSper4 genes: characterisation of a common interaction domain and evidence for expression in testis.
Reprod Biol Endocrinol 1,53.
Meizel S and Turner KO (1993) Initiation of the human sperm acrosome reaction by thapsigargin. J Exp Zool 267,350–355.
Michelangeli F, Mezna M, Tovey S and Sayers LG (1995) Pharmacological
modulators of the inositol 1,4,5-trisphosphate receptor. Neuropharmacology 34,1111–1122.
Michelangeli F, Ogunbayo OA and Wootton LL (2005) A plethora of interacting organellar Ca2+ stores. Curr Opin Cell Biol 17,135–140.
Minelli A, Allegrucci C, Rosati R and Mezzasoma I (2000) Molecular and
binding characteristics of IP3 receptors in bovine spermatozoa. Mol
Reprod Dev 56,527–533.
Missiaen L, Raeymaekers L, Dode L, Vanoevelen J, Van Baelen K, Parys JB,
Callewaert G, De Smedt H, Segaert S and Wuytack F (2004) SPCA1
pumps and Hailey-Hailey disease. Biochem Biophys Res Commun
322,1204–1213.
Monteil A, Chemin J, Bourinet E, Mennessier G, Lory P and Nargeot J (2000a)
Molecular and functional properties of the human alpha (1G) subunit that
forms T-type calcium channels. J Biol Chem 275,6090–6100.
Monteil A, Chemin J, Leuranguer V, Altier C, Mennessier G, Bourinet E,
Lory P and Nargeot J (2000b) Specific properties of T-type calcium
channels generated by the human alpha 1I subunit. J Biol Chem 275,
16530–16535.
Naaby-Hansen S, Wolkowicz MJ, Klotz K, Bush LA, Westbrook VA,
Shibahara H, Shetty J, Coonrod SA, Reddi PP, Shannon J et al. (2001) Colocalization of the inositol 1,4,5-trisphosphate receptor and calreticulin in
265
C.Jimenez-Gonzalez et al.
the equatorial segment and in membrane bounded vesicles in the cytoplasmic droplet of human spermatozoa. Mol Hum Reprod 10,923–933.
Neill AT and Vacquier VD (2004) Ligands and receptors mediating signal
transduction in sea urchin spermatozoa. Reproduction 127,141–149.
Nikpoor P, Mowla SJ, Movahedin M, Ziaee SA and Tiraihi T (2004) CatSper
gene expression in postnatal development of mouse testis and in subfertile
men with deficient sperm motility. Hum Reprod 19,124–128.
O’Toole CMB, Arnoult C, Darszon A, Steinhardt RA and Florman HM (2000)
Ca2+ entry through store-operated channels in mouse sperm is initiated by
egg ZP3 and drives the acrosome reaction. Mol Biol Cell 11,1571–1584.
Okunade GW, Miller ML, Pyne GJ, Sutliff RL, O’Connor KT, Neumann JC,
Andringa A, Miller DA, Prasad V, Doetschman T et al. (2004) Targeted
ablation of plasma membrane Ca2+-Atpase (PMCA) 1 and 4 indicates a
major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem
279,33742–33750.
Padinjat R and Andrews S (2004) TRP channels at a glance. J Cell Sci
117,5707–5709.
Parekh AB and Penner R (1997) Store depletion and calcium influx. Physiol
Rev 77,901–930.
Park JY, Ahn HJ, Gu JG, Lee KH, Kim JS, Kang HW and Lee JH (2003)
Molecular identification of Ca2+ channels in human sperm. Exp Mol Med
35,285–292.
Patrat C, Serres C and Jouannet P (2000) The acrosome reaction in human
spermatozoa. Biol Cell 92,255–266.
Perez-Reyes E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP,
Fox M, Rees M and Lee JH (1998) Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 391,896–900.
Philipp S, Hambrecht J, Braslavski L, Schroth G, Freichel M, Murakami M,
Cavalie A, and Flockerzi V (1998) A novel capacitative calcium entry
channel expressed in excitable cells. EMBO J 17,4274–4282.
Philipson KD and Nicoll DA (2000) Sodium-calcium exchange: a molecular
perspective. Annu Rev Physiol 62,111–133.
Prasad V, Okunade GW, Miller ML and Shull GE (2004) Phenotypes of
SERCA and PMCA knockout mice. Biochem Biophys Res Commun
322,1192–1203.
Publicover SJ and Barratt CL (1999) Voltage-operated Ca2+ channels and the
acrosome reaction: which channels are present and what do they do? Hum
Reprod 14,873–879.
Putney JW Jr (1990) Receptor-regulated calcium entry. Pharmacol Ther
48,427–434.
Quednau BD, Nicoll DA and Philipson KD (1997) Tissue specificity and
alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2,
and NCX3 in rat. Am J Physiol 272,C1250–C1261.
Quill TA, Ren D, Clapham DE and Garbers DL (2001) A voltage-gated ion
channel expressed specifically in spermatozoa. Proc Natl Acad Sci USA
98,12527–12531.
Quill TA, Sugden SA, Rossi KL, Doolittle LK, Hammer RE and Garbers DL
(2003) Hyperactivated sperm motility driven by CatSper2 is required for
fertilization. Proc Natl Acad Sci USA 100,14869–14874.
Radermacher M, Wagenknecht T, Grassucci R, Frank J, Inui M, Chadwick C
and Fleischer S (1992) Cryo-EM of the native structure of the calcium
release channel/ryanodine receptor from sarcoplasmic reticulum. Biophys
J 61,936–940.
Reinhardt TA, Horst RL and Waters WR (2004) Characterization of Cos-7
cells overexpressing the rat secretory pathway Ca2+-ATPase. Am J Physiol Cell Physiol 286,C164–C169.
Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q, Tilly JL and Clapham
DE (2001) A sperm ion channel required for sperm motility and male fertility. Nature 413,603–609.
Rossato M, Di Virgilio F, Rizzuto R, Galeazzi C and Foresta C (2001) Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential. Mol Hum Reprod
7,119–128.
Rufo GA Jr, Schoff PK and Lardy HA (1984) Regulation of calcium content in
bovine spermatozoa. J Biol Chem 259,2547–2552.
Santi CM, Darszon A and Hernandez-Cruz A (1996) A dihydropyridine-sensitive
T-type Ca2+ current is the main Ca2+ current carrier in mouse primary
spermatocytes. Am J Physiol 271,C1583–C1593.
Santi CM, Santos T, Hernandez-Cruz A and Darszon A (1998) Properties of a
novel pH-dependent Ca2+ permeation pathway present in male germ cells
with possible roles in spermatogenesis and mature sperm function. J Gen
Physiol 112,33–53.
Sayers LG, Brown GR, Michell RH and Michelangeli F (1993) The effects
of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-
266
induced calcium release in cerebellar microsomes. Biochem J 289,
883–887.
Schoff PK (1995) Mitochondrial calcium uptake stimulated by Cibacron blue
F3GA in bovine sperm. Arch Biochem Biophys 318,349–355.
Schuh K, Cartwright EJ, Jankevics E, Bundschu K, Liebermann J, Williams
JC, Armesilla AL, Emerson M, Oceandy D, Knobeloch KP and Neyses L
(2004) Plasma membrane Ca2+ ATPase 4 is required for sperm motility
and male fertility. J Biol Chem 279,28220–28226.
Serrano CJ, Trevino CL, Felix R and Darszon A (1999) Voltage-dependent
Ca(2+) channel subunit expression and immunolocalization in mouse
spermatogenic cells and sperm. FEBS Lett 462,171–176.
Son WY, Lee JH, Lee JH and Han CT (2000) Acrosome reaction of human
spermatozoa is mainly mediated by α1H T-type calcium channels. Mol
Hum Reprod 6,893–897.
Son WY, Han CT, Lee JH, Jung KY, Lee HM and Choo YK (2002) Developmental expression patterns of alpha1H T- type Ca2+ channels during spermatogenesis and organogenesis in mice. Dev Growth Differ 44,181–190.
Spehr M, Gisselmann G, Poplawski A, Riffell JA, Wetzel CH, Zimmer RK and
Hatt H (2003) Identification of a testicular odorant receptor mediating
human sperm chemotaxis. Science 299,2054–2058.
Spehr M, Schwane K, Riffell JA, Barbour J, Zimmer RK, Neuhaus EM and
Hatt H (2004a) Particulate adenylate cyclase plays a key role in human
sperm olfactory receptor-mediated chemotaxis. J Biol Chem 279,40194–
40203.
Spehr M, Schwane K, Heilmann S, Gisselmann G, Hummel T and Hatt H
(2004b) Dual capacity of a human olfactory receptor. Curr Biol 14,R832–
R833.
Stamboulian S, Kim D, Shin HS, Ronjat M, De Waard M and Arnoult C
(2004) Biophysical and pharmacological characterization of spermatogenic T-type calcium current in mice lacking the CaV3.1 (alpha1G) calcium channel: CaV3.2 (alpha1H) is the main functional calcium channel in
wild-type spermatogenic cells. J Cell Physiol 200,116–124.
Storey BT and Keyhani E (1973) Interaction of calcium ion with the mitochondria of rabbit spermatozoa. FEBS Lett 37,33–36.
Storey BT and Keyhani E (1974) Energy metabolism of spermatozoa. II. Comparison of pyruvate and fatty acid oxidation by mitochondria of rabbit
epididymal spermatozoa. Fertil Steril 10,857–864.
Su YH and Vacquier VD (2002) A flagellar K+-dependent Na+/Ca2+ exchanger
keeps Ca2+ low in sea urchin spermatozoa. Proc Natl Acad Sci USA
99,6743–6748.
Suarez SS and Ho HC (2003) Hyperactivated motility in sperm. Reprod
Domest Anim 38,119–124.
Suarez SS, Varosi SM and Dai X (1993) Intracellular calcium increases with
hyperactivation in intact, moving hamster sperm and oscillates with the
flagellar beat cycle. Proc Natl Acad Sci USA 90,4660–4664.
Taylor CW, Genazzani AA and Morris SA (1999) Expression of inositol trisphosphate receptors. Cell Calcium 26,237–251.
Tovey SC, Godfrey RE, Hughes PJ, Mezna M, Minchin SD, Mikoshiba K and
Michelangeli F (1997) Identification and characterization of inositol
1,4,5-trisphosphate receptors in rat testis. Cell Calcium 21,311–319.
Toyoshima C and Inesi G (2004) Structural basis of ion pumping by
Ca2+-ATPase of the sarcoplasmic reticulum. Annu Rev Biochem
73,269–292.
Trevino CL, Santi CM, Beltran C, Hernandez-Cruz A, Darszon A and Lomeli H
(1998) Localisation of inositol trisphosphate and ryanodine receptors
during mouse spermatogenesis: possible functional implications. Zygote
6,159–172.
Trevino CL, Serrano CJ, Beltran C, Felix R and Darszon A (2001) Identification of mouse trp homologs and lipid rafts from spermatogenic cells and
sperm. FEBS Lett 509,119–125.
Trevino CL, Felix R, Castellano LE, Gutierrez C, Rodriguez D, Pacheco J,
Lopez-Gonzalez I, Gomora JC, Tsutsumi V, Hernandez-Cruz A et al.
(2004) Expression and differential cell distribution of low-threshold Ca2+
channels in mammalian male germ cells and sperm. FEBS Lett 563,87–92.
Vannier B, Peyton M, Boulay G, Brown D, Qin N, Jiang M, Zhu X and
Birnbaumer L (1999) Mouse trp2, the homologue of the human trpc2
pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+
entry channel. Proc Natl Acad Sci USA 96,2060–2064.
Vermassen E, Parys JB and Mauger JP (2004) Subcellular distribution of the
inositol 1,4,5-trisphosphate receptors: functional relevance and molecular
determinants. Biol Cell 96,3–17.
Vijayaraghavan S and Hoskins DD (1990) Changes in the mitochondrial
calcium influx and efflux properties are responsible for the decline in
sperm calcium during epididymal maturation. Mol Reprod Dev 25,
186–194.
Calcium signalling in human spermatozoa
Walensky LD and Snyder SH (1995) Inositol 1,4,5-trisphosphate receptors
selectively localized to the acrosomes of mammalian sperm. J Cell Biol
130,857–869.
Wennemuth G, Westenbroek RE, Xu T, Hille B and Babcock DF (2000)
CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarizationevoked entry of Ca2+ into mouse sperm. J Biol Chem 275,21210–21217.
Wennemuth G, Babcock DF and Hille B (2003) Calcium clearance mechanisms of mouse sperm. J Gen Physiol 122,115–128. Erratum in J Gen
Physiol (2003) 122, 375.
Wes PD, Chevesich J, Jeromin A, Rosenberg C, Stetten G and Montell C
(1995) TRPC1, a human homolog of a Drosophila store-operated channel.
Proc Natl Acad Sci USA 92,9652–9656.
Westenbroek RE and Babcock DF (1999) Discrete regional distributions suggest diverse functional roles of calcium channel alpha1 subunits in sperm.
Dev Biol 207,457–469.
Weyard I, Godde M, Frings S, Weiner J, Muller F, Alkenhofen W, Hatt H and
Kaup UB (1994) Cloning and functional expression of a cyclic nucleotidegated channel from mammalian sperm. Nature 368,859–863.
Weisner B, Weiner J, Middendorf R, Hagen V, Kaup UB and Weynard I
(1998) Cyclic nucleotide-gated channels on the flagellum control Ca2+
entry into sperm. J Cell Biol 142,473–484.
Wictome M, Henderson I, Lee AG and East JM (1992) Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin. Biochem J 283,525–529.
Williams KM and Ford WC (2003) Effects of Ca-ATPase inhibitors on the
intracellular calcium activity and motility of human spermatozoa. Int J
Androl 26,366–375.
Williams M, Hill CJ, Scudamore I, Dunphy B, Cooke ID and Barratt CL
(1993) Sperm numbers and distribution within the human female fallopian
tube around ovulation. Hum Reprod 8,2019–2026.
Wojcikiewicz RJ and Luo SG (1998) Differences among type I, II, and III
inositol-1,4,5-trisphosphate receptors in ligand-binding affinity influence
the sensitivity of calcium stores to inositol-1,4,5-trisphosphate. Mol Pharmacol 53,656–662.
Wootton LL, Argent CC, Wheatley M and Michelangeli F (2004) The expression, activity and localisation of the secretory pathway Ca2+-ATPase
(SPCA1) in different mammalian tissues. Biochim Biophys Acta
1664,189–197.
Wuytack F, Raeymaekers L and Missiaen L (2003) PMR1/SPCA Ca2+ pumps and
the role of the Golgi apparatus as a Ca2+ store. Pflugers Arch 446,148–153.
Yoshida M (2004) Fertilization and sperm chemotaxis in ascidians. Methods
Mol Biol 253,13–25.
Yoshida M, Ishikawa M, Izumi H, De Santis R and Morisawa M (2003) Storeoperated calcium channel regulates the chemotactic behavior of ascidian
sperm. Proc Natl Acad Sci USA 100,149–154.
Zeng Y, Clark EN and Florman HM (1995) Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. Dev Biol 171,554–563.
Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stephani E and Birnbaumer L
(1996) Trp, a novel mammalian gene family essential for agonistactivated capacitative Ca2+ entry. Cell 85,661–671.
Zucchi R and Ronca-Testoni S (1997) The sarcoplasmic reticulum Ca2+ channel/
ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol Rev 49,1–51.
Submitted on June 28, 2005; revised on October 24, 2005; accepted on
November 1, 2005
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