Article

Ion channels in sperm motility and capacitation

Abstract and Figures

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.
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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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... Specific mechanisms identified during capacitation include the removal of decapacitation factors (Bedford and Chang, 1962;Fraser, 1992); the loss of cholesterol to cholesterol acceptors (Visconti et al., 1995a, b); a subsequent increase in membrane fluidity (Wolf et al, 1986;Baumber and Meyers 2006;Jones et al., 2007;Girouard et al, 2008); redistribution or dissolution of lipid rafts (Sleight et al, 2005;Nixon et al, 2009); hyperpolarization of the plasma membrane and activation of adenylyl cyclase by HCO3 (Breitbart, 2002;Lefievre et al., 2002;Demarco et al., 2003;Beltran et al., 2007); increased outward K permeability and the activation of voltage gated and ligand gated Ca 2 + channels (Darszon et al., 2006;Darszon et al, 2007;Bedu-Addo et al, 2008). However, the ability to undergo the acrosome reaction and to bind to the oocyte is the end-points of sperm capacitation. ...
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The present study was designed to investigate the effects of toluene and formaldehyde inhalation on the testes and ovaries of adult mice. The mice divided into three groups, control group, toluene exposed and formaldehyde exposed groups. The male and female mice exposed to (3ml = 300ppm toluene) and (2.5ml = 300ppm formaldehyde) 3 hours daily for 21 days. In this study a histological, glycohistochemical and immunohistochemical techniques were used. Male exposed to both solvents showed a significant decrease in their body weights at (p<0.05). Histological examination of the testes of exposed mice (either to toluene or formaldehyde) revealed an increase in the thickness of basement membrane of semeniferous tubules and a significant decrease in diameter of seminiferous tubules, number of spermatogenic layers , number of leydig cells and decline in the number of spermatogenic cells after inhalation in comparisons with the normal groups. Regarding the Females, exposed animals also showed a significant decrease in their body weights at (p<0.05). Histological examination of the ovaries of exposed mice (either to toluene or formaldehyde) revealed an increase in the thickness of zona pellucida of ovarian follicles , a significant increase in the number of primary , secondary and graffian follicles with decrease in the number of primordial follicles after inhalation in comparisons to the normal groups. Also a significant increase was obtained in the localization of (proliferating cell nuclear antigen) PCNA protein in the testes and ovaries of mice exposed either to toluene or formaldehyde in comparisons with the normal groups. In this study, glycohistochemical technique applied by using different lectins conjugated with fluorescence isothiacynate in order to detect carbohydrates or sugars in testes and ovaries tissues before and after inhalation of toluene and formaldehyde. These lectins are : Wheat germ agglutinin WGA, Lotus tetragonolobus agglutinin LTA, Ulex europhors agglutinin UEA and Lens culinaris agglutinin LCA with different specificities was studied to demonstrate structural differences in the glycoprotein composition of various tissues of the testes and ovaries before and after inhalation of both toluene and formaldehyde in the mice. The results showed that all lectins reacted differentially with the various components of the testes and ovaries either before or after inhalation of toluene and formaldehyde. The highest reaction was shown with Wheat germ agglutinin.
... Specific mechanisms identified during capacitation include the removal of decapacitation factors (Bedford and Chang, 1962;Fraser, 1992); the loss of cholesterol to cholesterol acceptors (Visconti et al., 1995a, b); a subsequent increase in membrane fluidity (Wolf et al, 1986;Baumber and Meyers 2006;Jones et al., 2007;Girouard et al, 2008); redistribution or dissolution of lipid rafts (Sleight et al, 2005;Nixon et al, 2009); hyperpolarization of the plasma membrane and activation of adenylyl cyclase by HCO3 (Breitbart, 2002;Lefievre et al., 2002;Demarco et al., 2003;Beltran et al., 2007); increased outward K permeability and the activation of voltage gated and ligand gated Ca 2 + channels (Darszon et al., 2006;Darszon et al, 2007;Bedu-Addo et al, 2008). However, the ability to undergo the acrosome reaction and to bind to the oocyte is the end-points of sperm capacitation. ...
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... Sperm motility is a key feature in the fertilization process in various species (Freitas et al., 2017;Kho et al., 2005). This functionality is highly regulated by various physiological and molecular factors (Pereira et al., 2017), where the concentrations of various ions in conjunction with transporter proteins such as ion channels and transporters play a key role in the activation of the sperm cell (Darszon et al., 2007). Unlike mammals, some fish reproduce through external fertilization, where sperm are released into the aquatic environment (Cosson et al., 2008;Dzyuba and Cosson, 2014). ...
The regulation of sperm motility is controlled by several variables, including mainly ion concentrations. In fish, Ca2+ concentrations play an important role in the regulation of sperm motility, and several reports highlight the importance of certain Ca2+ channels in the regulation of this cell function. CatSper is a calcium channel scarcely studied in fish. In the species Salmo salar, it has been shown that it is key in the regulation of sperm motility. Taking into account the relevance of this channel in sperm activation in fish, in this study we evaluated the presence and probable functionality of this channel in the class Actinopterygii. For this purpose, a rational bioinformatic analysis was carried out, which had been previously validated using in vitro techniques by our group. The bioinformatic analysis of the present work revealed that the functionality of CatSper of the species of the class Actinopterygii could be exclusive to freshwater and anadromous fish species. The results of this study showed that only some anadromous and freshwater fish species contain 11 subunits of the CatSper channel, which are enough to trigger sperm motility. Consequently, this study provides new data for a better understanding of the sperm activation mechanism in fish.
... Membrane swelling is most probably caused by changes in the extracellular osmotic pressure during freezing and thawing, causing cells to accumulate or lose water. The sperm PM is known to mediate the exchange of sodium, potassium [38,39], and calcium [40], and these ion fluxes regulate motility and mitochondrial function as well as osmotic balance. An intact PM is also necessary for fusion with the outer acrosomal membrane and induction of the acrosome reaction [41]. ...
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This study evaluated the efficacy of Turraea fischeri leaf extract for maintaining the viability of cryopreserved goat sperm. Ejaculated semen was collected from 5 mature Baladi bucks (50-60 kg, 2-4 years of age) and those samples with mass motility ≥ 70% and sperm concentration ≥ 2.5 × 10 9 /mL were selected, pooled, and divided into 4 aliquots. Each aliquot was diluted in Tris-citric-soybean lecithin extender containing a different concentration of T. fischeri leaf extract (0, 125, 250, or 375 µg/mL). Treated semen samples were cooled to 5 • C, transferred to 0.25-mL French straws, and stored in liquid nitrogen (LN 2) at −196 • C. After thawing, membrane integrity was examined by transmission electron microscopy, apoptotic activity by Annexin/propidium iodide staining and flow cytometry, and both enzyme activities and antioxidant capacity by spectroscopic assays. The leaf extract at 375 µg/mL significantly improved semen quality as indicated by enhanced total antioxidant capacity, reduced H 2 O 2 concentration, a greater proportion of structurally intact motile sperm, and concomitant reductions in apoptosis and necrosis. The extract also significantly increased the proportion of sperm with a contiguous plasma membrane and intact acrosome (p < 0.05). Furthermore, LC-MS revealed numerous secondary metabolites in the extract that may contribute to sperm cryopreservation.
... Indeed, mitochondrial abnormalities found in PMCA 4-deficient spermatozoa lead to a calcium overload resulting in male infertility due to defective calcium extrusion (Okunade et al., 2004). Calcium is therefore not only required for the initiation of hyperactivation but also to its maintenance by directly regulating components of the axonemal machinery (Darszon et al., 2007). ...
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Calcium ions (Ca²⁺) are involved in nearly every aspect of cellular life. They are one of the most abundant elements in mammals and play a vital role in physiological and biochemical processes acting mainly as intracellular messengers. In spermatozoa, several key functions are regulated by cytoplasmic Ca²⁺ concentration such as sperm capacitation, chemotaxis, hyperactive motility, and acrosome reaction. The sperm-specific ion channel CatSper is the principal calcium channel in sperm mediating the calcium influx into the sperm flagellum and acting as an essential modulator of downstream mechanisms involved in fertilization. This review aims to provide insights into the structure, localization, and function of the mammalian CatSper channel, primarily human and mice. The activation of CatSper by progesterone and prostaglandins, as well as the ligand-independent regulation of the channel by a change in the membrane voltage and intracellular pH are going to be addressed. Finally, major questions, challenges, and perspectives are discussed.
... However, some peculiarities need to be taken into account for this cell: metabolism of nonhexose substrates, such as citrate, pyruvate, and lactate mentioned earlier; gluconeogenesis was found to be active and the environment inside the female genital tract would be mostly anaerobic. Ex-4 and GLP1 analogues act with the potential to significantly improve glucose homeostasis and glucose is provided to the sperm by seminal plasma and by female reproductive tract fluid in vivo (62), or by culture medium in vitro (63). Furthermore, several studies have indicated that stores of glycogen are endogenous sources of glucose in sperm, allowing sperm to survive in glucose-free conditions (64,65). ...
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Aim Glucagon-like peptide-1 (GLP1) produces pleiotropic effects binding to the GLP1 receptor (GLP1-R) potentiating insulin secretion in the pancreas. GLP1-R is expressed in peripheral tissues and evidence for its role in reproduction came from knockout mice, although the relationship between GLP1 and male fertility needs to be clarified. Given that human sperm is an insulin-sensitive and insulin-secreting cell, we hypothesized that GLP-1/GLP1-R axis may be expressed and functional in these cells. Results and discussion: We evidenced GLP1-R presence by Western blotting and immunofluorescence analyses. Since Exendin-4 (Ex-4) displays similar functional properties to native GLP-1 we used this agonist to perform a dose-response study on progressive motility and cholesterol efflux, showing that 300 pM Ex-4 was the most effective treatment. These actions are mediated by GLP1-R and independent from sperm-secreted insulin. The exposure to Ex-4 fueled the PI3K/AKT signaling and reversed by H89, indicating a PKA-dependence of GLP1/GLP1-R signaling. It emerged that in sperm, insulin secretion regulated by Ex-4 did not occur in a strictly glucose-dependent manner. A stimulatory action of Ex-4/GLP1-R on LDH and G6PDH activities was observed. The Ex-4/GLP1-R decreased triglycerides content concomitantly to an enhanced lipase and Acyl-CoA dehydrogenase activities, addressing a lipolytic effect. Conclusion Collectively, we discovered that human sperm is a new GLP1 incretin target broadening our knowledge about the effects of the GLP1-R agonist in male reproductive field. Further findings in humans should be conducted in the future to confirm it and to improve the translational aspect of this study.
... At this point it is important to mention, that collecting samples at only five different times during capacitation is not sufficient for detailed characterization of the molecular changes, on which physiological process of capacitation is based and sperm life imaging is more appropriate method to study this in detail. For example, fast changes in calcium concentration should be measured by methods other than CTC [32,33]. Similarly, changes in actin polymerization should be measured by multiple analytical methods, since staining with FITC-phall can reflect rather changes in accessibility of actin epitopes than actin polymerization and depolymerization itself. ...
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Background: Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. Results: All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). Conclusions: Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.
... Some aspects of sperm physiology depend on the maintenance and regulation of [Ca 2+ ] i , which involves a range of pumps and channels at the plasma membrane or intracellular stores that import, export, and/or sequester Ca 2+ ions [reviewed by (Jimenez-Gonzalez et al., 2006;Clapham, 2007;Darszon et al., 2007Darszon et al., , 2011Correia et al., 2015)]. ...
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In the early 1950s, Austin and Chang independently described the changes that are required for the sperm to fertilize oocytes in vivo. These changes were originally grouped under name of “capacitation” and were the first step in the development of in vitro fertilization (IVF) in humans. Following these initial and fundamental findings, a remarkable number of observations led to characterization of the molecular steps behind this process. The discovery of certain sperm-specific molecules and the possibility to record ion currents through patch-clamp approaches helped to integrate the initial biochemical observation with the activity of ion channels. This is of particular importance in the male gamete due to the fact that sperm are transcriptionally inactive. Therefore, sperm must control all these changes that occur during their transit through the male and female reproductive tracts by complex signaling cascades that include post-translational modifications. This review is focused on the principal molecular mechanisms that govern human sperm capacitation with particular emphasis on comparing all the reported pieces of evidence with the mouse model.
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Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells’ current kinetics and current–voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.
Thesis
Declaration I declare that the subject of my thesis " Studying the negative effects of traditional sperm cryopreservation methods in Ossimi and Finnish rams using advanced techniques " is new and there is no other researchers took it before.
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Calcium and cyclic nucleotides have crucial roles in mammalian fertilization, but the molecules comprising the Ca2+-permeation pathway in sperm motility are poorly understood. Here we describe a putative sperm cation channel, CatSper, whose amino-acid sequence most closely resembles a single, six-transmembrane-spanning repeat of the voltage-dependent Ca2+-channel four-repeat structure. CatSper is located specifically in the principal piece of the sperm tail. Targeted disruption of the gene results in male sterility in otherwise normal mice. Sperm motility is decreased markedly in CatSper-/- mice, and CatSper-/- sperm are unable to fertilize intact eggs. In addition, the cyclic-AMP-induced Ca2+ influx is abolished in the sperm of mutant mice. CatSper is thus vital to cAMP-mediated Ca2+ influx in sperm, sperm motility and fertilization. CatSper represents an excellent target for non-hormonal contraceptives for both men and women.
Article
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The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca2+]i mediated by Ca2+ channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca2+ channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca2+ currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC50 values of ∼10 and ∼12 μM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca2+]i transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC50 of ∼10 μM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca2+ channel. They are also consistent with the involvement of T-channels in the AR.
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Transduction by sperm of the instructive signal provided by the egg peptide speract involves rapid, complex changes in internal ion and cyclic nucleotide content. Here, investigations of hypotonically swollen sperm provide insight into the underlying processes and identify K+ channel activation as an initial ionic event in gamete recognition. A sustained hyperpolarization of swollen sperm is promoted by less than 2.5 pM speract and is followed (with greater than 100 pM speract) by transient repolarization and (with greater than 10 nM speract) by depolarization that is dependent on external Ca2+. Monophasic increases in pHi are produced only by greater than 25 pM speract, indicating that hyperpolarization may not directly promote alkalinization. Increased K(+)-selective (K+ greater than Rb+ greater than Cs+ greater than Na+) membrane permeability is found after all speract greater than 2.5 pM, suggesting that hyperpolarization results from persistent activation of K+ channels and that repolarization has a different ionic basis. Supporting this contention, the K+ channel blocker tetraethylammonium (20 mM) inhibits the increased K+ permeability that follows treatment of swollen sperm (and of sperm in seawater) with 2.5 pM speract. Such induced activation of K+ channels is observed in patch-clamped swollen sperm examined in the cell-attached configuration, upon application of 5-50 pM speract to the bath medium. The efficacy of externally applied speract and its potency indicate that activation is indirect and probably involves an as yet unidentified diffusible mediator whose production is promoted by speract at concentrations 0.01-0.001 times those predicted from reported estimates of the Kd for the known speract receptor.
Article
The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K+] sea water were examined. In high [K+] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K+ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K+ sea water. However, in high [K+] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca2+ concentration of spermatozoa in normal sea water but not in 100mM K+ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K+] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K+ channels.
Article
Speract, a decapeptide from Strongylocentrotus purpuratus sea urchin eggs, transiently stimulates a membrane guanylyl cyclase and activates a K+-selective channel that hyperpolarizes sperm. However, previous studies of sperm and of sperm membrane vesicles reached conflicting conclusions about the mechanisms that open these channels. We find that speract hyperpolarizes and increases the cGMP content of flagellar vesicles. We confirm previous findings that intravesicular GTPγS and GTP enhance this hyperpolarization, but not GDPβS. The G protein activators AlF−4 and mastoparan also are ineffective. Thus, it is unlikely that a G protein participates in the speract response. In contrast, hyperpolarization responses to speract are increased by 3-isobutyl-1-methylxanthine, which preferentially inhibits cGMP-selective phosphodiesterases of sperm, and the 8Br-cGMP derivative hyperpolarizes vesicles in the absence of speract. The responses to speract and to 8Br-cGMP have similar ionic selectivities (K+ > Rb+ > > Li+ > Na+) and sensitivities to the channel blockers 4-aminopiridine and 3,4-dichlorobenzamil, indicating that they likely result from opening of the same K+ channel. Inhibitors that preferentially inhibit cAMP-selective phosphodiesterases do not alter responses to speract, and permeant cAMP analogs do not hyperpolarize vesicles. In addition, inhibitors of protein kinases and phosphatases fail to alter vesicle hyperpolarization by speract. The increase in vesicular cGMP content produced by speract therefore may directly mediate opening of the channel that hyperpolarizes sperm membrane vesicles. Similar mechanisms presumably operate in intact sperm.
Article
Speract, a decapeptide from sea urchin egg jelly, induces various sperm responses. Stopped-flow fluorometry was used to examine the binding of labeled speract and the intracellular changes in pH (pHi) and Ca2+ ([Ca2+]i) it induces in sperm. We observed significant time delays for the increase in pHi and [Ca2+]i induced by 200 nM speract (69 and 190 ms, respectively). Also, we found that the receptor undergoes a pHi-dependent affinity change at around 129 ms. These time delays probably reflect biochemical processes underlying each sperm response to speract that circumscribe the time sequence of the signaling events.
Article
Do not touch! The surface of a living cell is soft and responsive and therefore high-resolution imaging of the cell membrane has not been possible to date. Now noncontact imaging of protein complexes in the plasma membrane of living cells has been demonstrated (see picture) and has been used to follow the cells' structural reorganization. This breakthrough opens up a wealth of new experiments in membrane and cell biology.
Article
Asymmetrical bending waves can be obtained by reactivating demembranated sea urchin spermatozoa at high Ca2+ concentrations. Moving-film flash photography shows that asymmetrical flagellar bending waves are associated with premature termination of the growth of the bends in one direction (the reverse bends) while the bends in the opposite direction (the principal bends) grow for one full beat cycle, and with unequal rates of growth of principal and reverse bends. The relative proportions of these two components of asymmetry are highly variable. The increased angle in the principal bend is compensated by a decreased angle in the reverse bend, so that there is no change in mean bend angle; the wavelength and beat frequency are also independent of the degree of asymmetry. This new information is still insufficient to identify a particular mechanism for Ca2+-induced asymmetry. When a developing bend stops growing before initiation of growth of a new bend in the same direction, a modification of the sliding between tubules in the distal portion of the flagellum is required. This modification can be described as a superposition of synchronous sliding on the metachronous sliding associated with propagating bending waves. Synchronous sliding is particularly evident in highly asymmetrical flagella, but is probably not the cause of asymmetry. The control of metachronous sliding appears to be unaffected by the superposition of synchronous sliding.