Ion channels in sperm motility and capacitation

Abstract

Spermatozoa depend upon ion channels to rapidly exchange information with the outside world and to fertilise the egg. These efficient ion transporters participate in many of the most important sperm processes, such as motility and capacitation. It is well known that sperm swimming is regulated by [Ca2+]i. In the sea urchin sperm speract, a decapeptide isolated from egg outer envelope, induces changes in intracellular Ca2+ ([Ca2+]i), Na+, cAMP and cGMP, membrane potential (Em) and pH (pHi). Photoactivation of a speract analogue induces Ca2+ fluctuations that generate turns that are followed by straighter swimming paths. A fast component of the [Ca2+], increase that most likely occurs through voltage dependent Ca2+ channels (Ca(v)s) is essential for these turns. The Ca(v)s involved are modulated by the Em changes triggered by speract. On the other hand, mammalian sperm gain the ability to fertilise the egg after undergoing a series of physiological changes in the female tract. This maturational process, known as capacitation, encompasses increases in [Ca2+]i and pHi, as well as an Em hyperpolarization in mouse sperm. Our electrophysiological, immunological and molecular-biological experiments indicate that inwardly rectifying K+ channels regulated by ATP (KATP channels) and epithelial Na+ channels (ENaCs) are functionally present in mouse spermatogenic cells and sperm. Notably, pharmacological experiments indicate that the opening of KATP channels and closure of ENaCs may contribute to the hyperpolarization that accompanies mouse sperm capacitation. Remarkably, both in the sea urchin sperm speract response and in the mouse sperm capacitation, Em hyperpolarization seems necessary to remove inactivation from Ca(v) channels so they can then open.

Full text

229Ion channels in sperm motility and capacitation
Corresponding author E-mail: darszon@ibt.unam.mx
Ion channels in sperm motility and capacitation
A. Darszon,1 C.L. Treviño,1 C. Wood,1 B. Galindo,1,2 E. Rodríguez-Miranda,1,3
J.J. Acevedo,4 E.O. Hernandez-González,5 C. Beltrán,1 P. Martínez-López1 and
T. Nishigaki1
1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología,
Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México; 2 Centro de
Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Unidad
Monterrey, Monterrey, Nuevo León, México; 3Instituto Tecnológico Superior de Irapuato (ITESI).
Carretera Irapuato, Guanajuato, México; 4Departamento de Fisiología y Farmacología, Facultad de
Medicina, Universidad Autónoma del Estado de Morelos (UAEM), Cuernavaca, México;
5Departmento de Biología Celular, CINVESTAV-IPN, México D.F., México
Spermatozoa depend upon ion channels to rapidly exchange information
with the outside world and to fertilise the egg. These efficient ion
transporters participate in many of the most important sperm processes,
such as motility and capacitation. It is well known that sperm swimming
is regulated by [Ca2+]i. In the sea urchin sperm speract, a decapeptide
isolated from egg outer envelope, induces changes in intracellular Ca2+
([Ca2+]i), Na+, cAMP and cGMP, membrane potential (Em) and pH (pHi).
Photoactivation of a speract analogue induces Ca2+ fluctuations that
generate turns that are followed by straighter swimming paths. A fast
component of the [Ca2+]i increase that most likely occurs through voltage
dependent Ca2+ channels (Cavs) is essential for these turns. The Cavs
involved are modulated by the Em changes triggered by speract. On the
other hand, mammalian sperm gain the ability to fertilise the egg after
undergoing a series of physiological changes in the female tract. This
maturational process, known as capacitation, encompasses increases in
[Ca2+]i and pHi, as well as an Em hyperpolarization in mouse sperm. Our
electrophysiological, immunological and molecular-biological
experiments indicate that inwardly rectifying K+ channels regulated by
ATP (KATP channels) and epithelial Na+ channels (ENaCs) are functionally
present in mouse spermatogenic cells and sperm. Notably,
pharmacological experiments indicate that the opening of KATP channels
and closure of ENaCs may contribute to the hyperpolarization that
accompanies mouse sperm capacitation. Remarkably, both in the sea
urchin sperm speract response and in the mouse sperm capacitation, Em
hyperpolarization seems necessary to remove inactivation from Cav
channels so they can then open.
Spermatology. SRF Vol. 65. ERS Roldan and M Gomendio (eds) Nottingham University Press, Nottingham
16-Darszon.p65 3/19/2007, 11:21 AM229

230 A. Darszon et al.
Introduction
Fertilisation requires mature and competent male and female gametes. Sperm and egg must
find each other and fuse to generate a new individual. The codes that govern how sperm gather
information from the external world and translate signals coming from the egg are yet to be
fully understood. Ion transport regulation lies at the heart of the signalling events that allow
gametes to respond and interpret information from their vicinity (Darszon, Nishigaki, Wood,
Trevino, Felix and Beltran, 2005; Quill, Wang and Garbers, 2006). This review will focus on
the work we have carried out studying the role of ion channels in two of the most important
sperm processes, motility and capacitation.
Sea urchins release billions of sperm, whose motility, powered by a microscopic flagellar
engine, is exquisitely regulated by chemical signals from the environment and the egg that
alter their ion permeability (Darszon et al., 2005). On the other hand, sperm from internal
fertilisers (reptiles, birds and mammals) develop the potential for motility, probably also under-
going ion transport modulation, as they travel across the vas deferens (a duct connecting the
epididymis to the urethra) and the epididymis (Yanagimachi, 1994; Morisawa, 1994). Ejacula-
tion into the female reproductive tract activates sperm that need to further mature through a
process named capacitation which includes membrane permeability changes (Visconti,
Westbrook, Chertihin, Demarco, Sleight and Diekman, 2002). In mammals only few sperm,
from the millions released, reach their destination in the oviduct (Suarez and Ho, 2003). In
recent years it has become clear that sperm chemotaxis is not only important for marine organ-
isms but for mammals too (Eisenbach and Giojalas, 2006).
Motility
For many cells movement is fundamental and in many cases they are propelled by cilia and
flagella that have a conserved structure. Therefore it is important to understand how flagellar
beating is regulated. The axoneme, a complex machine lined by membrane and constituted by
microtubules, dynein ATPases and hundreds of other proteins, is responsible for flagellar move-
ment (Mitchell, 2004). Sperm are highly specialised flagellated cells evolved to deliver their
genetic material to the female of their species. Inherent in this fundamental function is the
need for sperm to be motile.
Though it is easy to observe sperm swimming, our understanding of the molecular mecha-
nisms involved in this essential function is far from being complete. Only recently, develop-
ments in our technical ability to record ion permeability changes in individual sperm are re-
vealing how ion channels participate in motility.
Early observations that the ionic composition of the outside media can have a determinate
influence on how sperm swim indicated the possible relevance of ion channels in the regula-
tion of this process. For example, sea urchin sperm and other marine organisms are immotile in
the testes prior to their release. This is mainly due to the high extracellular K+ concentration
([K+]e) (Darszon et al., 2005). Upon spawning sperm activate as [K+]e is reduced. On the other
hand, it has been known for many years that exposing permeabilised or demembranated inver-
tebrate or mammalian sperm to high [Ca2+] increases flagellar asymmetry and can even inhibit
flagellar movement (Brokaw, 1979; Tash and Means, 1982). Chemotaxis, the reorientation of
the sperm trajectory toward a chemoattractant gradient, such as may surround the egg, strictly
depends on the presence of external Ca2+ (Eisenbach and Giojalas, 2006). This phenomenon is
most widely observed and studied in sperm from invertebrate marine animals, but occurs also
in mammalian and plant sperm. Because chemotaxis requires external Ca2+, it is most likely
that Ca2+-permeant pathways are involved in this process.
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231Ion channels in sperm motility and capacitation
Ion channels and sperm motility regulation in sea urchin sperm
Not much is known about the participation of ion channels in the regulation of invertebrate
sperm motility. Sperm chemotaxis has been documented in many marine species and in all
cases it depends on [Ca2+]e (Miller, 1985; Morisawa, 1994; Darszon, Acevedo, Galindo,
Hernandez-Gonzalez, Nishigaki, Trevino, Wood and Beltran, 2006a). A chemotactic response
is often associated to a series of turns interspersed with periods of straighter swimming (Kaupp,
Hildebrand and Weyand, 2006). When Ca2+ is removed from the extracellular medium sperm
become unable to turn. Sea urchins are deuterostomes that share a common ancestor with the
vertebrates, thus studies of their sperm chemotaxis and motility regulation have general impli-
cations.
Sea urchin sperm are particularly suitable models for investigating motility because: (a) their
flagella beat in near-planar waves, confining them at water-glass and water-air interfaces (Cosson,
Huitorel and Gagnon, 2003), (b) they swim in circles due to an inherent asymmetry in the
flagellar planar waveform with respect to the long axis of the head. This circular swimming in
a two-dimensional plane above the cover slip surface persists for many minutes, facilitating
image analysis of their motile behaviour.
Small activating peptides (10-15 amino acids) (SAPs) that influence sperm motility have
been isolated from the egg jelly of sea urchins (Ward, Brokaw, Garbers and Vacquier, 1985;
Garbers, 1989). So far, resact, a SAP from Arbacia punctulata, is the only sea urchin egg
peptide demonstrated to be a chemoattractant (Ward et al., 1985). Photoactivation of ‘caged’
resact, induces a sequence of transient ‘chemotactic’ turns followed by longer periods of straighter
swimming that allow sperm to swim up the resact gradient (Kaupp, Solzin, Hildebrand, Brown,
Helbig, Hagen, Beyermann, Pampaloni and Weyand, 2003). Even though speract, a SAP iso-
lated from Strongylocentrotus purpuratus (Garbers, 1989), was shown to alter sperm motility
under certain conditions (Cook, Brokaw, Muller and Babcock, 1994), it has not been shown to
be a chemoattractant.
Speract binds to the sperm flagellum where it transiently activates a guanylyl cyclase (Garbers,
1989; Cardullo, Herrick, Peterson and Dangott, 1994). Subsequently, according to the canoni-
cal model (reviewed in Darszon, Beltran, Felix, Nishigaki and Trevino, 2001; Darszon et al.,
2006a), the cGMP elevation opens a K+-selective channel (Lee and Garbers, 1986, Babcock,
Bosma, Battaglia and Darszon, 1992) that allows K+ efflux and hyperpolarises sperm. Though it
has been proposed that cGMP directly activates the K+ channel (Galindo, Beltran, Cragoe and
Darszon, 2000), this has not been fully demonstrated. The speract-induced hyperpolarization
also activates an increase in pHi (Lee and Garbers, 1986; Babcock et al., 1992; Reynaud, De de
La Torre, Zapata, Lievano and Darszon, 1993) and a transient [Ca2+]i decrease (Nishigaki, Wood,
Tatsu, Yumoto, Furuta, Elias, Shiba, Baba and Darszon, 2004), the hyperpolarization has been
suggested to be required to remove inactivation from voltage-gated Ca2+ (Cav) channels (Lee
and Garbers, 1986; Granados-Gonzalez, Mendoza-Lujambio, Rodriguez, Galindo, Beltran and
Darszon, 2005). These Cav channels can then open as a consequence of a depolarization, such
as the speract-induced repolarization and trigger the complex [Ca2+]i changes essential for
motility regulation (Schackmann and Chock, 1986; Babcock et al., 1992; Darszon et al., 2006a).
Increases in cAMP and intracellular Na+ also follow the hyperpolarization (Garbers, 1989; Kaupp
et al., 2003; Rodriguez and Darszon, 2003).
The canonical model is being questioned by recent findings. Fast stopped-flow population
measurements using A. punctulata sperm reveal that low physiological resact concentrations
(pM) increase cGMP 30-fold in ~50 ms, while cAMP increases more slowly and only four-fold
(Kaupp et al., 2003). The sperm [Ca2+]i increase induced by pM resact is biphasic showing an
early peak within 2 s, followed by a shoulder and, at the highest concentrations, a second peak
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232 A. Darszon et al.
at ~10-15 s. The early peak displays a ~0.2 s delay which the authors attribute to the time
taken to synthesise cGMP. We had also recorded ~0.2 s delay for the [Ca2+]i increase induced
by high speract concentrations in stopped-flow population experiments in S. purpuratus sperm
(Nishigaki, Zamudio, Possani and Darszon, 2001). Kaupp et al. (2003) did not see the early
decrease in [Ca2+]i postulated by Cook et al. (1994) even with a 10 ms resolution. Their results
suggest that a rise in cGMP triggers the early, rapid [Ca2+]i transient while a later rise in cAMP
generates the slower, second [Ca2+]i peak. In a following paper Kaupp’s group reported that pM
resact, in contrast to the canonical model, increases [Ca2+]i before pHi. These results and others
suggest that the resact induced pHi change is not necessary for the increases in [Ca2+]i (Solzin,
Helbig, Van, Brown, Hildebrand, Weyand and Kaupp, 2004), though they do not discard that it
may influence their kinetics.
However, simultaneously we obtained time resolved pHi and [Ca2+]i changes induced by
uncaging speract, cGMP and cAMP in S. purpuratus sperm populations that differ from those
obtained by Kaupp’s group. We find that the increase in pHi precedes the [Ca2+]i rise at all the
speract concentrations we have tested, and that indeed a fast [Ca2+]i decrease does occur (dura-
tion ~280 ms) when sperm are exposed to speract or cGMP but not to cAMP. This [Ca2+]i
decrease is inhibited by increasing external [K+] and stimulated by IBMX, which enhances
sperm hyperpolarization (Nishigaki et al., 2004). The alternative model thus far proposed by
Kaupp’s group fails to explain why high external K+ inhibits all responses to speract but the
increase in cGMP (Harumi, Hoshino and Suzuki, 1992; Darszon et al., 2005). At this time the
physiological relevance of the decrease in [Ca2+]i and increase in pHi are unknown, though
they could modulate the delay to the [Ca2+]i increase and in this manner influence the synchro-
nization between sperm swimming and the responses to the SAP gradient.
The time-resolved measurements discussed above represent the average [Ca2+]i changes of
the sperm population. Independently and contemporaneously we have used high resolution
Ca2+ imaging to measure [Ca2+]i increases in single S. purpuratus sperm attached to coverslips
(Wood, Darszon and Whittaker, 2003). We found that sperm responded to low, physiological
concentrations of speract with periodic fluctuations in [Ca2+]i that were particularly prominent
in the sperm flagella. Noticeably, ~85% of the fluorescent dye signal from the sperm came
from Ca2+ in the head. Thus [Ca2+]i measurements in sperm suspensions, even at low peptide
concentrations, are biased towards the sperm head [Ca2+]i. A full understanding of [Ca2+]i sig-
nalling and motility regulation in this highly polarised cell requires analysis of individual swim-
ming sperm.
[Ca2+]i changes in swimming sea urchin sperm
Typically sea urchin sperm perform a “chemotactic turn” in 50-100 ms, so the ionic changes
that regulate it occur during this time. We have been able for the first time to simultaneously
measure [Ca2+]i, flagellar form and trajectory in individual swimming sea urchin sperm using a
stroboscopic photodiode illumination in our fluorescence imaging system (Wood, Nishigaki,
Furuta, Baba and Darszon, 2005; Nishigaki, Wood, Shiba, Baba and Darszon, 2006)(see Fig. 1).
Uncaging cGMP induces Ca2+ entry via at least two distinct pathways: (1) a fast (ms) transitory
nimodipine-sensitive pathway compartmentalised in the flagella that is a key regulator of flagellar
bending and directed motility changes and (2) a sustained [Ca2+]i elevation in the head and tail
whose physiological role is yet to be defined. Contrary to the then-current models (Brokaw,
1979; Cook et al., 1994), we found that flagellar bending does not vary in proportion to overall
[Ca2+]i. Instead we proposed a new model whereby flagella bending is increased by Ca2+ flux
through the nimodipine-sensitive pathway but unaffected by [Ca2+]i elevation through alterna-
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233Ion channels in sperm motility and capacitation
tive pathways. An important question, probably relevant to all ciliated cells, is how flagella
distinguish between Ca2+ uptake pathways. Our findings revealed that cGMP uncaging changes
the S. purpuratus sperm swimming behaviour in a similar manner to that induced by resact or
cGMP in A. punctulata sperm (Kaupp et al., 2003). Sperm redirect their trajectory in the direc-
tion orthogonal and centrifugal to the arc that they were describing when cGMP was uncaged
(see Fig. 2), an observation consistent with a chemotactic-like response (Wood et al., 2005).
FlashCube 40
Controller
Figure 1. Schematic diagram of the imaging equipment. The LED (Luxeon V Star Lambertian
Cyan LED, Lumileds Lighting LLC) is attached on a custom-built aluminum holder and
mounted on a Flash Cube 40 assembly (Rapp Opto Electronic, Hamburg) connected to
the rear epifluorescence illumination port of the microscope. The LED is controlled by
custom-built stroboscopic power supply that provides 3 A current pulses of 1 ms duration,
which is synchronously triggered by a CCD camera. Photolysis of the caged compounds
is performed using a UV flash lamp system, JML-C2 (Rapp Opto Electronic, Hamburg) with
a UV band-pass filter (300-400 nm). The flash light is introduced into the microscope by a
liquid light guide (2 mm diameter, NA 0.45) connected to the FlashCube 40 with a dich-
roic mirror (400 nm).
Soon after, Kaupp’s group reported [Ca2+]i responses to the uncaging of cGMP and resact in
swimming A. punctulata sperm (Bohmer, Van, Weyand, Hagen, Beyermann, Matsumoto, Hoshi,
Hildebrand and Kaupp, 2005). In both cases they recorded [Ca2+]i fluctuations that regulate
sperm swimming, as we had proposed earlier from our experiments in immobilised S. purpuratus
sperm (Wood et al., 2003). Their responses to a resact gradient allowed them to correlate
[Ca2+]i fluctuations to a chemotactic response and to propose a model, a very significant contri-
bution to the sperm motility field (Bohmer et al., 2005).
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234 A. Darszon et al.
Figure 2. (A) Releasing caged cGMP by pulsing sperm with UV light increases both [Ca2+]i
and flagellar asymmetry in a swimming S. purpuratus sperm. The upper panels are images
of fluo-4 (a Ca2+ indicator) fluorescence that show an increase in Ca2+ in the flagellum after
exposure to a UV pulse. Camera exposure time is set equivalent to LED excitation light
duration, in this case 1 ms. Total delay between successive images = 25 ms. Note that the
short exposure time possible due to the stroboscopic nature of LED illumination clearly
defines flagellar form. Below each fluorescence image the normalised position of the
sperm shows that the flagellar asymmetry (in relation to the long axis of the head) increases
in synchrony with the rise in [Ca2+]i, and that the flagellum subsequently returns to a more
symmetric form while [Ca2+]i remains relatively elevated. The graph shows the increase in
fluo-4 fluorescence as an average along the length of the flagellum, with the numbers
corresponding to the images above. F/F0 is the normalised flagellar fluorescence, where
F0 is the flagellar fluorescence in the first image in which the flagellum is visible post-UV
flash. (B) The increase in [Ca2+]i induced by a UV pulse in a sperm under mercury-lamp
epifluorescent illumination. Due to the necessary increase in exposure time to 18 ms the
flagellar form is poorly defined and of lesser temporal resolution (total time between suc-
cessive images 40 ms).
Our new unpublished results uncaging speract show that indeed the [Ca2+]i fluctuations that we
observed in tethered S. purpuratus sperm occur in swimming sperm. Each [Ca2+]i fluctuation in
the flagella induces a rapid increase in flagellar asymmetry that results in a sharp turn in the
sperm swimming trajectory (Fig. 2). Each sperm turn was most frequently superimposed on a
background trajectory of decreased curvature, resulting in a series of turns interspersed with
periods of straighter swimming. This motility pattern is not exclusive to sea urchin sperm but
occurs in sperm from other marine species undergoing chemotaxis, and has been termed the
‘turn-and-run’ model (Bohmer et al., 2005). In three dimensions this circular motion would
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235Ion channels in sperm motility and capacitation
translate into a helical trajectory which upon encounter with a SAP, would display turn-and run
responses that would deviate sperm from their original path and, in principle, increase their
probability of finding an egg.
cGMP, the first signalling intermediate generated upon binding of egg-derived SAP, induces
chemotaxis-like motility changes that involve Ca2+ entry into the flagellum through a specific
pathway sensitive to Cav channel inhibitors (Wood et al., 2005). Recently we immunolocalised
Cav1.2 and 2.3 to the sea urchin sperm flagella (Granados-Gonzalez et al., 2005), hinting at a
role for these channels in regulating chemotaxis. In this work we also demonstrated function-
ally that Cav channels are present in S. purpuratus sperm. The inositol 1,4,5-trisphosphate (IP3)
receptor, another Ca2+ channel, has been localised to the flagella of sea urchin sperm (Zapata,
Ralston, Beltran, Parys, Chen, Longo and Darszon, 1997). This receptor is usually associated
with liberation of Ca2+ from internal Ca2+ stores, of which none have been described in the
flagella of sea urchin sperm. Two hyperpolarization-activated and cyclic nucleotide-gated channels
(HCN) are also present in S. purpuratus sperm flagella, SpHCN1 (Gauss, Seifert and Kaupp,
1998) and SpHCN2 (Galindo, Neill and Vacquier, 2005). These channels could be activated by
the hyperpolarization and/or increases in cyclic nucleotides induced by SAP. An inhibitor of
HCN channels alters the kinetics of the early [Ca2+]i changes induced by speract (Nishigaki et
al., 2004). Figure 3 presents our presently preferred model.
Figure 3. Speract signalling model. Proteins involved in speract signalling and their rela-
tionship are shown: 1, speract receptor; 2, guanylate cyclase (GC); 3, cGMP-regulated K+
channel; 4, K+-dependent Na+/Ca2+ exchanger (NCKX); 5, Na+/H+ exchanger (NHE); 6,
adenylate cyclase (AC); 7, sperm HCN channel (SpHCN); 8, voltage-gated Ca2+ (Cav) chan-
nel. Since GC is inactivated by pHi-dependent dephosphorylation and cGMP is rapidly
hydrolysed by phosphodiesterases, the opening of the cGMP-regulated K+ channel is
transient. Em hyperpolarization facilitates Ca2+ extrusion activity of NCKX and removes
inactivation from Cav channels which can then open upon repolarization and depolariza-
tion. Depolarization and Ca2+ influx may activate Cl- and/or K+ channels which can re-
hyperpolarise sperm Em and sustain [Ca2+]i oscillations.
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236 A. Darszon et al.
Ion channels and sperm motility regulation in mammalian sperm
Mammalian sperm can display two main motility modes, activated and hyperactivated. Freshly
ejaculated sperm swim with a relatively low-amplitude flagellar beat that propels them across the
entrance of the female reproductive tract. This activated motility is stimulated by Ser/Thr and Tyr
phosphorylation of flagellar proteins (reviewed in Turner, 2006). The phosphorylation state of
these proteins is regulated by cAMP, protein kinase A (PKA) and a soluble adenylyl cyclase (sAC)
(Litvin, Kamenetsky, Zarifyan, Buck and Levin, 2003; Esposito, Jaiswal, Xie, Krajnc-Franken, Robben,
Strik, Kuil, Philipsen, van Duin, Conti and Gossen, 2004). The activity of sAC depends on HCO3
-
and Ca2+ (Litvin et al., 2003), therefore Ca2+ transport influences its operation.
Hyperactivated sperm swim with exaggerated, large-amplitude flagellar beats that result in
characteristic “figure-of-eight” trajectories in normal medium, while in high-viscosity medium,
they are more progressive (Suarez and Ho, 2003). This motility mode often coincides with the
initiation of capacitation, although the two processes are now considered independent by some
authors (Ho and Suarez, 2003). Sperm isolated from the upper regions of the female reproductive
tract display hyperactivated motility. Advancement of sperm through the viscous environment of
the oviduct, and possibly penetration of the egg cumulus may rely on hyperactivated motility.
Sperm hyperactivation requires Ca2+ to regulate components of the axoneme. [Ca2+]i fluctua-
tions occurring in hyperactivated hamster sperm correlate with their flagellar beat frequency (Suarez,
Varosi and Dai, 1993). The Ca2+ required for this process seems to originate from a reticular
structure at the flagellum neck, the redundant nuclear envelope (RNE) which contains IP3 receptors
(IP3Rs) (Ho and Suarez, 2003). It is interesting that a progesterone gradient induces long-lasting
[Ca2+]i oscillations in human sperm originating from the RNE that are comparable to those occur-
ring during hyperactivation and correlate with increases in flagellar activity. Apparently, ryanodine
receptors (RyRs) and not IP3Rs are involved in these oscillations (Harper, Barratt and Publicover,
2004). In contrast, in bull sperm RyRs have not been detected (Suarez and Ho, 2003; Jimenez-
Gonzalez , Michelangeli, Harper, Barratt and Publicover, 2006). We have detected both IP3Rs and
RyRs in mouse sperm (Trevino, Santi, Beltran, Hernandez-Cruz, Darszon and Lomeli, 1998). Very
recent evidence indicates that, at pM concentrations, progesterone is chemotactic (Teves, Barbano,
Guidobaldi, Sanchez, Miska and Giojalas, 2006).
New findings related to a novel class of Ca2+ channels, named CatSpers, some of which are
expressed mainly in testes, have emphasised the important participation of ion channels in sperm
motility. Of the four members of this family that have been described so far, CatSpers 1 and 2
localise to the sperm flagella (Ren, Navarro, Perez, Jackson, Hsu, Shi, Tilly and Clapham, 2001;
Quill, Ren, Clapham and Garbers, 2001; Lobley, Pierron, Reynolds, Allen and Michalovich, 2003;
Jin, O’Doherty, Wang, Zheng, Sanders and Yan, 2005). Interestingly, sperm from catsper1 and
catsper2 null mice are unable to undergo hyperactivation and these animals are infertile (Ren et
al., 2001; Carlson, Westenbroek, Quill, Ren, Clapham, Hille, Garbers and Babcock, 2003). The
phenotypes of these null mice are identical since neither protein alone localises to the flagellum
without co-expression of the other.
Unfortunately it has been impossible to functionally express CatSpers in heterologous systems.
On the other hand, studying sperm ion channels by traditional patch-clamping has been hampered
by their small size and plasma membrane rigidity (reviewed in Darszon et al., 2006a). Though
spermatogenic cells have been used to study sperm ion channels electrophysiologically and using
the tools of molecular biology (Hagiwara and Kawa, 1984; Arnoult, Cardullo, Lemos and Florman,
1996; Lievano, Santi, Serrano, Trevino, Bellve, Hernandez-Cruz and Darszon, 1996), these cells
can re-distribute and dispose of their channels during the last stages of spermatogenesis (Serrano,
Trevino, Felix and Darszon, 1999). Two new strategies have recently emerged that allow ion
channel recordings directly on sperm: (1) the “smart patch-clamp” which, in addition to gigaseal
formation and electrophysiological characterization, permits determination of the position and
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237Ion channels in sperm motility and capacitation
regulation of sperm ion channels (Gorelik, Gu, Spohr, Shevchuk, Lab, Harding, Edwards, Whitaker,
Moss, Benton, Sanchez, Darszon, Vodyanoy, Klenerman and Korchev, 2002; Gu, Kirkman-Brown,
Korchev, Barratt and Publicover, 2004; Shevchuk, Frolenkov, Sanchez, James, Freedman, Lab,
Jones, Klenerman and Korchev, 2006), and (2) establishing a gigaseal on the sperm cytoplasmic
sperm droplet, a remnant of the precursor germ cell cytoplasm located along the midpiece of
maturing sperm that is shed around the time of ejaculation, to record whole sperm patch-clamp
currents (Kirichok, Navarro and Clapham, 2006). Using this approach Kirichok et al. recorded a
constitutively active, weakly voltage-dependent Ca2+ current which is strongly potentiated by
intracellular alkalinization. catsper1-null mice lack this current indicating that CatSper1 is a com-
ponent of a flagellar Ca2+ channel. The channel is permeable to Na+ in the absence of external
Ca2+.
Various Cav channel subunits have been found in mammalian sperm (reviewed in Darszon,
López-Martínez, Acevedo, Hernández-Cruz and Trevino, 2006b). In this section we will discuss
those found in the flagella (Fig. 4). Cav1.2, 2.1 and 2.3 subunits were localised to the proximal
piece of mouse sperm flagella (Westenbroek and Babcock, 1999), as was the auxiliary ß3 subunit
(Serrano et al., 1999). Though fertile, sperm from Cav2.3 null mice swim with increased linearity
(Sakata, Saegusa, Zong, Osanai, Murakoshi, Shimizu, Noda, Aso and Tanabe, 2002). The three T-
type Cav3 channel isoforms are also present in the mouse and human sperm flagellum (Trevino,
Felix, Castellano, Gutierrez, Rodriguez, Pacheco, Lopez-Gonzalez, Gomora, Tsutsumi, Hernandez-
Cruz, Fiordelisio, Scaling and Darszon, 2004). Very recently CatSper1 and 2 have been shown to
interact and down-regulate Cav3.3 in the principal piece of human sperm, a notable finding which
could have functional implications related to motility (Zhang , Chen, Saraf, Cassar, Han, Rogers,
Brioni, Sullivan and Gopalakrishnan, 2006).
Channels possibly involved in Chemotaxis/Motility
RyR3, Catsper1, Catsper2,
Ca
v
2.2, 3, Ca
v
3.1, Ca
v
3.2,
Ca
v
3.3, CNGA1, CNGB1
Flagelar principal piece
Mammalian sperm
IP3R, TRPC6Nuclear redundant envelope
Ca
v
1.2, Ca
v
2.3, Cav3.1, Ca
v
3.2,
Ca
v
3.3, TRPC1, CNGA3
Flagellar midpiece
Channels detectedLocalization
Ca
v
1.2, Ca
v
2.3, IP3R
spHCN1, spHCN2
Flagella
Channels detectedLocalization
Sea urchin sperm
RyR3, Catsper1, Catsper2,
Ca
v
2.2, ß3, Ca
v
3.1, Ca
v
3.2,
Ca
v
3.3, CNGA1, CNGB1
Flagelar principal piece
Mammalian sperm
IP3R, TRPC6Nuclear redundant envelope
Ca
v
1.2, Ca
v
2.3, Cav3.1, Ca
v
3.2,
Ca
v
3.3, TRPC1, CNGA3
Flagellar midpiece
Channels detectedLocalization
Ca
v
1.2, Ca
v
2.3, IP3R
spHCN1, spHCN2
Flagella
Channels detectedLocalization
Sea urchin sperm
ENaCAcrosome
Ca
v
1.2, Ca
v
2.2, Ca
v
2.3, Ca
v
3.1, Ca
v
3.2, Ca
v
3.3,
TRPC1, TRPC3, TRPC4, TRPC6, Kv1.2, Kv3.1,
K2p5.1, SUR2
Flagellar
principal
piece
Ca
v
1.2, Ca
v
2.1, Ca
v
2.3, Ca
v
3.1, Ca
v
3.2, Ca
v
3.3,
TRPC1, TRPC3, TRPC4, TRPC5, Kir6.1, Kir6.2,
Kv1.1, Kv1.5, SUR1, ENaC
Flagellar
midpiece
Ca
v
1.2, Ca
v
2.1, Ca
v
2.2, Ca
v
2.3, Ca
v
3.1, Ca
v
3.2
TRPC1,TRPC2, TRPC3, TRPC4, TRPC5, TRPC6
Kv1.1, Kv1.2, Kir6.2, SUR1
Head
Channels detectedLocalization
ENaCAcrosome
Ca
v
1.2, Ca
v
2.2, Ca
v
2.3, Ca
v
3.1, Ca
v
3.2, Ca
v
3.3,
TRPC1, TRPC3, TRPC4, TRPC6, Kv1.2, Kv3.1,
K2p5.1, SUR2
Flagellar
principal
piece
Ca
v
1.2, Ca
v
2.1, Ca
v
2.3, Ca
v
3.1, Ca
v
3.2, Ca
v
3.3,
TRPC1, TRPC3, TRPC4, TRPC5, Kir6.1, Kir6.2,
Flagellar
midpiece
Ca
v
1.2, Ca
v
2.1, Ca
v
2.2, Ca
v
2.3, Ca
v
3.1, Ca
v
3.2
TRPC1,TRPC2, TRPC3, TRPC4, TRPC5, TRPC6
Kv1.1, Kv1.2, Kir6.2, SUR1
Head
Channels detectedLocalization
Channels possibly involved in Capacitation
Flagella
Sea urchin Sperm
Head
Mitochondria
Acrosome
A
Flagella
Sea urchin Sperm
Head
Mitochondria
Acrosome
A
Nuclear redundant
envelope
Flagellar principal
piece
Flagellar midpiece
Mammalian Sperm
Acrosome
Head
Flagellar endpiece
B
Nuclear redundant
envelope
Flagellar principal
piece
Flagellar midpiece
Mammalian Sperm
Acrosome
Head
Flagellar endpiece
B
C
D
Figure 4. Ion channels present in sperm cells. Diagram of sea urchin (A) and mammalian
(B) sperm illustrating the principal cell components. Summary of the channels that have
been immunolocalised to distinct sperm section and may participate during the process of
chemotaxis and/or motility (C) and capacitation (D).
16-Darszon.p65 3/19/2007, 11:21 AM237

238 A. Darszon et al.
Using the new strategy to obtain a whole-cell configuration on the citoplasmic droplet, we
have reproducibly obtained patch-clamp recordings from individual mouse sperm isolated from
testicle and caput epididymus. Under conditions to record CaV channels (Lopez-Gonzalez, De
La Vega-Beltran, Santi, Florman, Felix and Darszon, 2001), we have corroborated, for the first
time, that T-type Ca2+ currents are present in mouse sperm. As anticipated, these currents are
blocked by micromolar concentrations of mibefradil and nifedipine (Fig. 5). As in mouse sper-
matogenic cells, the T-type Ca2+ currents are also blocked by 50 µm Ni2+ (unpublished results).
These findings are consistent with early work indicating the participation of Cav channels in
mammalian sperm physiology, with the presence of their mRNAs both in spermatogenic cells
and sperm and with their immunolocalization (reviewed in Jimenez-Gonzalez, Michelangeli,
Harper, Barratt and Publicover, 2006; Darszon et al., 2006a) (see Fig. 4).
Figure 5. Whole cell patch-clamp recordings on testicular mouse sperm. (A) Representa-
tive family of total ionic currents from testicular sperm sealing on the cytoplasmic droplet.
Ionic currents were evoked by 300 ms depolarising steps from -80 up to 40 mV from a
holding potential of -70 mV. Before the test pulses a hyperpolarising pre-pulse was applied
for 1 s. An inward and an outward ion current are apparent under control conditions in
these representative upper traces. Inward current amplitude was determined at the peak
(0-50 ms after activation), while outward current amplitude was measured as the mean of
the whole trace. Mibefradil (10 µM), a T-type channel blocker, inhibits the inward compo-
nent of the sperm ion currents (lower traces) but does not alter the outward component. (B)
I-V plot of the ion currents shown in panel A in the absence (control, closed circles; n =
20) and presence of mibefradil (10 µM, open circles; n = 4). (C) Inhibition of sperm inward
ion current by 10 µM nifedipine, another blocker of T-type currents in mouse spermatoge-
nic cells. A 300 ms test pulse from a HP of -70 mV to -30 mV was used to evoke the
currents. Around 80% of the inward current is inhibited by this nifedipine concentration
Figure 5 (contd). suggesting that indeed T-type Ca2+ channels are present in mouse sperm.
16-Darszon.p65 3/19/2007, 11:21 AM238

239Ion channels in sperm motility and capacitation
(D) Summary of the results with the two T-type channel blockers. Both mibefradil (10 µM;
n = 4) and nifedipine (10 µM; n = 4) inhibit 94% and 80% of the inward component,
respectively. Neither mibefradil nor nifedipine affect the outward component of the sperm
ion currents. In all plots, symbols represent the mean ± S.E.M.
Our perseverant interest in the sperm acrosome reaction led us to also study the role of TRP
channel family members in mouse and human sperm physiology. TRPC1, C3 and C6 were found
in the flagella of human sperm while TRPC1, C3, C4 and C6 were detected in mouse sperm tail
hinting for a role in motility. Interestingly, human sperm motility was decreased by some TRP
channel inhibitors (Castellano, Trevino, Rodriguez, Serrano, Pacheco, Tsutsumi, Felix and Darszon,
2003).
Cyclic nucleotide-gated (CNG) channels are expressed in the mammalian sperm flagella. Actu-
ally CNGA3, a member of this family was the first sperm ion channel cloned (Weyand, Godde,
Frings, Weiner, Muller, Altenhofen, Hatt and Kaupp, 1994). It is distributed along the flagellum.
CNGA3 knockout mice are fertile (Turner, 2006); however their motility has not been characterised
in detail. On the other hand, CNGB1 is confined to the principal piece in bovine sperm (Wiesner,
Weiner, Middendorff, Hagen, Kaupp and Weyan, 1998). Major efforts are still required to fully
understand sperm motility.
Mammalian sperm capacitation
While in transit through the female reproductive tract, mature epididymal sperm become compe-
tent to fertilise. This maturational process is named capacitation and involves complex sperm
changes which include plasma membrane reorganization, cholesterol removal, protein tyrosine
phosphorylation, Em hyperpolarization (in mouse sperm) and increases in pHi and [Ca2+]i (reviewed
in Visconti et al., 2002; Darszon et al., 2005). As indicated earlier, capacitation is associated with
the emergence of hyperactivation (Suarez and Ho, 2003), and encompasses changes that prime
sperm to respond to the zona pellucida (ZP), the extracellular egg coat that triggers the acrosome
reaction. The molecular mechanisms involved are not fully understood and include Tyr-phosphory-
lation regulated by a cAMP-dependent pathway involving PKA (Visconti et al., 2002). sAC ap-
pears to be the principal cyclase responsible for the cAMP changes that occur during capacitation
(Esposito et al., 2004; Hess, Jones, Marquez, Chen, Ord, Kamenetsky, Miyamoto, Zippin, Kopf,
Suarez, Levin, Williams, Buck and Moss, 2005), although transmembrane ACs may also contribute
(Fraser, Adeoya-Osiguwa, Baxendale, Mededovic and Osiguwa, 2005).
Capacitation in vitro depends on the presence of Ca2+, K+, HCO3
- and Na+ in the medium
(Visconti et al., 2002; Darszon et al., 2005). In addition, bovine serum albumin (BSA) is also
required. How and which of the ion channels and transporters present in sperm regulate Em, pHi
and [Ca2+]i to achieve capacitation is not well established (Darszon et al., 2005). Non-capacitated
mouse sperm are relatively depolarised (Em~-35 to -45 mV) and hyperpolarise to ~-80 mV during
capacitation (Arnoult, Kazam, Visconti, Kopf, Villaz and Florman, 1999; Munoz-Garay, De la
Vega-Beltran, Delgado, Labarca, Felix and Darszon, 2001). It is thought that the hyperpolarization
is needed to remove inactivation from T-type Cav channels, recruiting them to a closed state from
which they can open in response to ZP to induce the acrosome reaction (Arnoult et al., 1999).
Sperm Cav channels other than T-type, as well as several members of the TRP family (see Fig. 4),
may influence capacitation.
It was proposed that in mouse sperm the K+ permeability participates in the capacitation process
since external K+ and K+-channel blockers affect the hyperpolarization that accompanies this
process (Arnoult et al., 1999; Munoz-Garay et al., 2001; Acevedo, Mendoza-Lujambio, de la
16-Darszon.p65 3/19/2007, 11:21 AM239

240 A. Darszon et al.
Vega-Beltran, Trevino, Felix and Darszon, 2006). Voltage-gated K+ channels (Salvatore, D’Adamo,
Polishchuk, Salmona and Pessia, 1999), Ca2+-activated K+ channels (Chan, Wu, Sun, Leung, Wong,
Chung, So, Zhou and Yan, 1998) and inwardly rectifying K+ (Kir) channels (Munoz-Garay et al.,
2001; Felix, Serrano, Trevino, Munoz-Garay, Bravo, Navarro, Pacheco, Tsutsumi and Darszon,
2002; Acevedo et al., 2006) have been reported to be present in spermatogenic and sperm cells.
Sperm pHi is relatively acidic before capacitation and may down-regulate sperm Kir channels. This
could maintain Em depolarised keeping Cav channels inactivated and unable to cause unregulated
Ca2+ entry and the acrosome reaction (Zeng, Oberdorf and Florman, 1996).
Using whole-cell patch clamp recordings in mouse spermatogenic cells, we found Kir currents
sensitive to Ba2+, glucose, tolbutamide and glibenclamide (Acevedo et al., 2006). These last two
compounds are sulfonylureas known to block Kir channels regulated by ATP (KATP channels).
Furthermore, we were able to detect transcripts for the KATP channel subunits SUR1, SUR2, Kir6.1
and Kir6.2 in total RNA from elongated spermatids in RT-PCR assays, and immunolocalised them
in mature mouse sperm (Fig. 4). Incubation of sperm under capacitating conditions with tolbuta-
mide abolishes the hyperpolarization and significantly inhibits the acrosome reaction. Since mouse
sperm capacitation is accompanied by a pHi increase and an ATP decrease (Baker and Aitken,
2004), it is feasible that KATP channels contribute to the capacitation-associated hyperpolarization
in mouse sperm.
Figure 6. Diagram of ion fluxes and signalling events of mammalian sperm capacitation.
Uptake of bicarbonate (HCO3
-) through the Na+–HCO3
- cotransporter and other transport-
ers stimulates sAC and lipid reorganization in the plasma membrane. Cholesterol accep-
tors such as albumin also promote the process of lipid reorganization. cAMP has several
targets such as CNG channels, PKA and ENaC possibly indirectly. Also, an increase in
intracellular Ca2+ may directly activate sAC and an increase in pHi can promote Ca2+ influx
through CatSper1 and 2 channels. Therefore, there is a cAMP, Ca2+ and pHi synergism.
Furthermore, the changes in these intracellular parameters lead to tyrosine phosphoryla-
tion involving PKA activation, hyperactivation of sperm motility and the hyperpolarization
by opening K+ channels (Kir) and closing ENaC. PTK, protein tyrosine kinase.
16-Darszon.p65 3/19/2007, 11:21 AM240

241Ion channels in sperm motility and capacitation
Recently we found that an electrogenic Na+ transporter, possibly involving an amiloride sensi-
tive Na+ channel, contributes to depolarise the sperm resting Em before capacitation. Indeed,
the α and δ subunits of epithelial Na+ channels (ENaCs) are present in the sperm flagellar
midpiece and acrosome region, respectively (Hernandez-Gonzalez, Sosnik, Edwards, Acevedo,
Mendoza-Lujambio, Lopez-Gonzalez, Demarco, Wertheimer, Darszon and Visconti, 2005).
These channels are amiloride-sensitive and contribute to the resting Em in cells by displacing it
towards the Na+ equilibrium potential (Awayda, Boudreaux, Reger and Hamm, 2000). Further-
more, spermatogenic cells display amiloride-sensitive inward Na+ currents whose characteris-
tics match those of ENaCs. Addition of cell-permeant cAMP analogues which lead to in vitro
capacitation, decrease the sperm depolarization induced by addition of external Na+. These
findings indicate that the increases in cAMP that accompany capacitation may inhibit ENaCs
and contribute to achieve the hyperpolarization required for this process to occur. Figure 6
illustrates our working model for the involvement of ion channels and transporters in mouse
sperm capacitation.
Acknowledgements
The authors would like to acknowledge José Luis de la Vega for technical assistance. The work
was supported by grants DGAPA-UNAM (to AD, CB, TN and CT), CONACYT (to AD and CB),
the Wellcome Trust (to AD) and FIRCA R03 TW 006121 (to AD).
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