Molecular Human Reproduction, Vol.17, No.8 pp. 457– 465, 2011
Advanced Access publication on May 24, 2011 doi:10.1093/molehr/gar041
NEW RESEARCH HORIZON Review
Sperm chemotaxis and regulation
of flagellar movement by Ca21
Manabu Yoshida 1,* and Kaoru Yoshida 2
1
Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, Miura, Kanagawa 238-0225, Japan 2Biomedical
Engineering Center, Toin University of Yokohama, Yokohama 225-8502, Japan
*Correspondence address. Tel: +81-46-827-6543; Fax: +81-46-881-7944; E-mail: [email protected]
Submitted on November 30, 2010; resubmitted on May 17, 2011; accepted on May 18, 2011
abstract: The chemotaxis of sperm towards eggs is a widespread phenomenon that occurs in most forms of life from lower plants
to mammals and plays important roles in ensuring fertilization. In spermatozoa, the attractants act as beacons, indicating the path leading
to the eggs from the same species. The existence of species-specific sperm chemotaxis has been demonstrated in marine invertebrates;
thus, sperm chemotaxis may be involved in preventing crossbreeding, especially in marine invertebrates with external fertilization.
However, the mechanisms of sperm chemotaxis in mammalian species differ from those of marine invertebrates. In mammals, the attractant source is not the egg, but follicular fluids or cumulus cells and chemotactic behaviour is shown only in small populations of sperm.
Nevertheless, the fundamental mechanisms underlying sperm chemotaxis are likely to be common among all species. Among these mechanisms, intracellular Ca2+ concentration ([Ca2+]i) is an important factor for the regulation of chemotactic behaviour in spermatozoa.
Sperm attractants induce the entry of extracellular Ca2+, resulting in [Ca2+]i increase in the sperm cells. Furthermore, [Ca2+]i modulates
sperm flagellar movement. However, the relationship between [Ca2+]i and the chemotactic response of a sperm flagellum is not well
known. Investigation of the dynamic responses of sperm cells to their attractants is important for our understanding of the regulation
of fertilization. Here, we reviewed sperm chemotaxis focusing on the mechanisms that regulate sperm flagellar beating during the chemotactic response.
Key words: Ca2+ / chemotaxis / fertilization / sperm flagella
Introduction
From plants to humans, the spermatozoa of many species exhibit chemotactic behaviour towards eggs. Sperm chemotaxis has been extensively studied and is considered a species-specific phenomenon in
marine invertebrates. Although this phenomenon is well understood
in many phyla of animals and plants, sperm attractants have been identified only in some invertebrate species (Fig. 1). Furthermore, despite the
importance of the dynamic changes that occur in spermatozoa in
response to specific attractants, the molecular mechanism of sperm
chemotactic responses has not been fully elucidated, and our understanding of the regulation of sperm chemotaxis, especially in
mammals. The present study reviews the mechanisms that regulate
attractant-induced sperm chemotaxis. A summary of sperm behaviour
during chemotaxis and the nature of sperm attractants are followed by a
description of the known mechanisms of regulation of flagellar movement induced by intracellular Ca2+. In addition, the putative signalling
mechanisms mediated by sperm attractants in spermatozoa are
discussed.
Chemical nature of sperm
attractants
The first sperm attractants were identified in plants (Brokaw, 1958).
In animals, sperm attractants have been identified in eight nonmammalian species; an ascidian (Yoshida et al., 2002), a coral
(Coll et al., 1994), sea urchins (Ward et al., 1985; Guerrero
et al., 2010), a starfish (Böhmer et al., 2005), a cuttlefish (Zatylny
et al., 2002), an abalone (Riffell et al., 2002) and an amphibian
(Olson et al., 2001) (Fig. 1). Six of these attractants are proteins,
peptides or amino acids, and two of them are low-molecular-weight
compounds. Among mammals, sperm chemotaxis towards femalederived attractants was observed in humans (Ralt et al., 1991),
mice (Oliveira et al., 1999), rabbits (Fabro et al., 2002), horses
(Navarro et al., 1998), pigs (Serrano et al., 2001) and bulls (Gil
et al., 2008). Sperm chemotaxis towards factors in follicular fluid
has been demonstrated in humans, and several candidate sperm
attractants have been proposed in follicular fluid such as
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Yoshida and Yoshida
Figure 1 Identified sperm chemoattractants. (A) Attractants in invertebrate sperm. Invertebrate sperm attractants have been identified in eight
species: an ascidian (1) (Yoshida et al., 2002), a coral (2) (Coll et al., 1994), sea urchins (3,4) (Ward et al., 1985; Guerrero et al., 2010), a starfish
(5) (Böhmer et al., 2005), a cuttlefish (6) (Zatylny et al., 2002), an amphibian (7) (Olson et al., 2001) and an abalone (8) (Riffell et al., 2002). (B)
Candidate sperm attractants in mammals are N-formylated peptides (9) (Iqbal et al., 1980), atrial natriuretic peptide (10) (Zamir et al., 1993), progesterone (11) (Villanueva-Diaz et al., 1995; Teves et al., 2006; Guidobaldi et al., 2008) and RANTES (12) (Isobe et al., 2002), which were found in
follicular fluids. Certain artificial odorants act as mammalian sperm attractants: bourgeonal (13) (Spehr et al., 2003) and lyral (14) (Fukuda et al., 2004).
N-formylated peptides (Iqbal et al., 1980), atrial natriuretic peptide
(Zamir et al., 1993), progesterone (Villanueva-Diaz et al., 1995)
and regulated on activation, normal T cell expressed and secreted
(RANTES) (Isobe et al., 2002) (Fig. 1). Recently, progesterone
released from the cumulus oophorus was postulated as the mammalian sperm attractant (Teves et al., 2006; Guidobaldi et al., 2008),
although progesterone is present in other places in the female
reproductive tract. Mammalian spermatozoa are also known to
contain many G-protein-coupled odorant receptors (Parmentier
et al., 1992; Vanderhaeghen et al., 1993), and human olfactory
receptor 17-4 (hOR17-4), one of these receptors, seems to be
involved in the chemotaxis of human spermatozoa (Spehr et al.,
2003). Bourgeonal, an aromatic aldehyde used in perfumery, is a
potent ligand of hOR17-4 and acts as a chemoattractant
(Spehr et al., 2003) (Fig. 1). Similar results have been reported in
studies on murine sperm chemotaxis, and the odorant lyral was
found to act as an attractant of murine spermatozoa (Fukuda
et al., 2004). However, these odorants have not been identified in
the female reproductive tract yet, and native ligands for the
odorant receptors are as yet unknown.
There are several differences in sperm chemotaxis between
mammals and marine invertebrates. One of these differences is
species specificity. Sperm chemotaxis in marine invertebrates is a
species- or genera-specific phenomenon (Miller, 1985), but sperm
chemotaxis in mammals appears to have no species-specificity: the follicular fluids of humans, rabbits and cows attract both rabbit and
human spermatozoa (Sun et al., 2003). Moreover, progesterone can
act as an attractant for human and rabbit spermatozoa (Teves et al.,
2006; Guidobaldi et al., 2008). However, the potent human sperm
attractant bourgeonal (Spehr et al., 2003) does not attract murine
sperm (Fukuda et al., 2004). A more precise analysis of the speciesspecificity of mammalian sperm chemotaxis is necessary, although progesterone is a good candidate as the attractant in some mammalian
species.
Another significant difference in sperm chemotaxis between
mammals and marine invertebrates is the heterogeneity of the
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Sperm chemotaxis and calcium
sperm chemotactic response. In marine invertebrates, a very large
population of spermatozoa shows chemotactic behaviour in response
to the attractant. On the contrary, a chemotactic response was shown
in only a small subpopulation of mammalian spermatozoa. In humans,
a fraction of 2–12% of spermatozoa is chemotactically responsive to
progesterone, and this subpopulation is considered as the capacitated
spermatozoa (Cohen-Dayag et al., 1994, 1995). Furthermore, 36% of
human spermatozoa (Spehr et al., 2003) and ≈10% of murine spermatozoa (Fukuda et al., 2004) exhibited increases in Ca2+ levels in
response to bourgeonal and lyral, respectively. Murine olfactory
receptor 23, the odorant receptor for lyral, is expressed in only
≈30% of the seminiferous tubules in murine testes. Therefore, only
some ejaculated spermatozoon can respond to the attractant. In
humans, the diversity in genetic backgrounds could cause variation
in the proportion of fertile spermatozoa, which could therefore be
low in certain cases. On the other hand, experiments in mice are performed with inbred lines, and the genetic background of each mouse is
therefore the same. These results suggest that a subset of cells with
the complement of receptors required for fertilization is predetermined during spermatogenesis. This is surprising since predetermined
‘losing’ cells should be excluded during evolution. Thus, if the heterogeneous expression of chemotaxis-related molecules in the mammalian spermatozoa is a true reflection of chemotactic sensitivity,
sperm chemotaxis is not essential for mammalian fertilization. Alternatively, other molecules may be responsible for mammalian sperm chemotaxis. In human sperm, chemotaxis towards progesterone is
directly correlated to capacitation (Eisenbach and Giojalas, 2006),
and heterogeneity of sperm chemotaxis may be due to low levels of
capacitation rather than to the heterogeneous expression of
chemotaxis-related molecules. However, if the hypothesis is true,
we must consider heterogeneity of sperm capacitation. Furthermore,
in vitro capacitated mouse spermatozoa also show the heterogeneity in
their chemotactic response (Fukuda et al., 2004), and progesterone
seems not to attract mouse sperm. Further studies are required for
understanding the heterogeneous sperm response and the role of chemotaxis in selection of spermatozoa.
Analysis of the chemotactic
behaviour of spermatozoa
The analysis of sperm chemotaxis in invertebrates is commonly performed using the micropipette assay. In this activity assay for sperm
chemotaxis, the attractant is enclosed at the tip of a glass micropipette, the micropipette is placed in the sperm suspension and the
sperm trajectories around the micropipette tip are observed
(Miller, 1985; Yoshida, 2004). Some studies use photolysis of
caged attractants instead of the micropipette for applying sperm
attractant (Böhmer et al., 2005; Wood et al., 2007; Guerrero
et al., 2010). The advantage of using caged attractants is that the
timing of the release of the attractant to spermatozoa can be precisely controlled; however, in this method, the maintenance of a
continuous gradient field of attractants is difficult because uncaged
attractants by brief light flash are easily and quickly dispersed. The
quantitative evaluation of sperm chemotaxis has been performed
using various methods. As described later, during the chemotactic
response, spermatozoa often exhibit quick turning movements
with highly asymmetric flagellar waveforms (Miller, 1985). The curvature of sperm trajectories and/or an asymmetry in sperm flagellar
waveforms are used as indices for evaluating the chemotactic
response(Miller, 1982; Cosson et al., 1984; Yoshida et al., 2002;
Fukuda et al., 2004; Böhmer et al., 2005; Shiba et al., 2005;
Wood et al., 2007; Guerrero et al., 2010). The linear equation chemotaxis index has been proposed as an index for the measurement
of sperm chemotaxis based on sperm trajectories (Yoshida et al.,
2002; Yoshida, 2004). These methods for the evaluation of sperm
chemotaxis are linked to sperm flagellar movement and could be
applicable to the analyses of when and where a spermatozoon
responds to the attractant.
In mammals, the most commonly used method for evaluating chemotaxis is the accumulation assay (see review, Eisenbach, 1999). In
this assay, the attractant is placed in one of two reservoirs of a
chemotaxis-analysing apparatus that are connected by a microporous filter or a connecting channel, which results in the formation
of a gradient of analyte by diffusion. Spermatozoa are introduced
into the other reservoir, and if they accumulate in the reservoir
that contains the analyte, the analyte is considered a sperm attractant. Although this method provides a simple and easily administered
assay, it cannot distinguish chemotaxis from sperm accumulation
caused by trapping or chemokinesis (Eisenbach, 1999) because the
method is based on sperm concentration. An improved method
based on the direction of sperm movement was therefore proposed
(Fabro et al., 2002). This method uses the Zigmond chamber to
measure cell accumulation (Zigmond, 1977), but the movement of
sperm in the channel between two reservoirs is recorded, and
the net distances that spermatozoa travel per unit time both parallel
to the attractant gradient (DX) and perpendicular to it (DY) are
measured. Although this method is more accurate than the accumulation assay, the analysis of the results cannot be linked to sperm
flagellar movement, and it is therefore difficult to establish when
and where the spermatozoon responds to the attractant.
Chemotactic behaviour
of spermatozoa
The chemotactic responses of spermatozoa have been precisely
determined in several marine invertebrate species. In the absence of
sperm attractants, spermatozoa of many marine invertebrates show
circular movement with low asymmetric flagellar beating, while mammalian spermatozoa exhibit linear movement with symmetric flagellar
beating. In the hydrozoan siphonophore Muggiaea kochi, the radius of
curvature of the sperm trajectory is reduced as the spermatozoon
approaches the cupule, which is the source of sperm attractant
(Cosson et al., 1984). In contrast, quick turning movements of spermatozoa exhibiting chemotactic behaviour are observed in other
hydrozoans. The turning movement of sperm is a typical phenotype
of the sperm during chemotactic behaviour, and during the turning
movements, the spermatozoa demonstrate a temporarily increased
asymmetrical flagellar beating (Miller, 1966, 1982; Miller and
Brokaw, 1970).
The spermatozoa of the ascidian Ciona intestinalis also exhibit
quick turning movements during chemotaxis towards sperm-activating
and -attracting factor (SAAF), the attractant for ascidian sperm
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Yoshida and Yoshida
Figure 2 Changes in the swimming paths and flagellar beating pattern of the ascidian spermatozoa in the presence of the sperm attractant. (A)
Traces of sperm flagellar waveforms of the ascidian C. intestinalis during chemotactic behaviour. The sperm attractant SAAF was set on an origin of
coordinates. There are three phases of flagellar beating during sperm chemotaxis: (1) The resting movement is similar to the swimming curvature of
the stable movement without attractant; (2) the chemotactic turn is observed when sperm swim away from the attractant; and (3) straight-swimming
after quick turning. The inset image shows the traces corresponding to the phases of flagellar beating (bold lines numbered 1 – 3). The series of ‘turn
and straight-swimming’ movements change the sperm swimming direction, and sperm again move towards the attractant. (B) Changes in the swimming path curvature (top) and asymmetric index (bottom) in (A). The asymmetric indices were calculated from the flagellar curvature to evaluate
flagellar asymmetry (Shiba et al., 2008). Open circles 1 – 3 correspond to events 1 – 3 in (A), respectively. Note that a straight swimming path results
in a value of 0 in the curvature and fully symmetric flagellar bending corresponds to a value of 1 in the asymmetric index. (Modified Figure from Shiba
et al., 2008).
(Miller, 1975, 1982; Yoshida et al., 1993, 2002). This movement has
been named the ‘chemotactic turn’. The activated Ciona spermatozoon
in seawater exhibits stable asymmetric flagellar beating, resulting in
circular movement with near-constant swimming path curvatures
(≈0.02 mm21). In contrast, the spermatozoon around the tip of the
micropipette containing SAAF often exhibits the chemotactic turn; the
flagellar waveform of the spermatozoon becomes highly asymmetric,
and the swimming path curvature suddenly increases (Yoshida et al.,
2002; Shiba et al., 2008) (Fig. 2). After the chemotactic turn, the spermatozoon changes its swimming pattern and swims in a straight line with
low path curvatures (≈0 mm21) and symmetric flagellar waveforms
(‘straight-swimming’ state). The spermatozoon then recovers its
stable circular movement, which indicates that it is again in a ‘resting’
state (Shiba et al., 2008) (Fig. 2). These dynamic changes in swimming
paths and asymmetry of flagellar waveforms are generally repeated,
and the spermatozoa finally approach the micropipette tip. In the presence of a gradient of attractant, the chemotactic behaviour of the ascidian spermatozoa is characterized by a transient switch in the
swimming patterns from ‘resting’ states to ‘turn-and-straight’ movements with quick regulation of flagellar asymmetry.
In sea urchin, on the other hand, a recent study showed that the
Lytechinus pictus spermatozoa displayed dissimilar motility responses
depending on their position relative to the source of the attractant
uncaged by the photolysis of caged speract, the sperm attractant of
L. pictus (Guerrero et al., 2010). The responses of spermatozoa in a
position proximal to stimulation are biphasic: the first phase of the
response of spermatozoa is the ‘turn-and-straight’ movement, swimming along negative speract gradients, and the second phase of the
response is characterized by the absence of turning events and by
increase in the size of the circular movements.
In contrast, mammalian spermatozoa usually exhibit straight swimming, and their flagellar beatings are almost symmetric (Spehr et al.,
2004). There are few studies on the sperm trajectories and flagellar
movements during the chemotactic responses of mammalian spermatozoa, mostly because many studies were performed by using the accumulation assay as described earlier. However, two studies investigated the
trajectories and flagellar beating of mammalian spermatozoa during chemotaxis (Fukuda et al., 2004; Spehr et al., 2004). Both studies demonstrated that the chemotactic behaviour of mammalian spermatozoa is
oriented towards the source of the attractant, and the cells sometimes
show highly asymmetrical flagellar beating similar to the ‘chemotactic
turn’. However, frequency of the ‘chemotactic turn’-like movement in
the mammalian spermatozoa is low: many sperm did not show the turnlike movement but simply orient to the attractant, and even in the sperm
showing the movement, it was observed only one time during their
pathway to the attractant. Thus, the mechanisms regulating the movement of mammalian spermatozoa during chemotaxis may be different
from those of marine invertebrates.
Ca21 changes in sperm during
chemotactic behaviour
Ca2+ plays a key role in the regulation of flagellar beating. In sea urchin
spermatozoa, Ca2+ concentration in the reactivation medium is correlated with the asymmetric flagellar beating of reactivated
Sperm chemotaxis and calcium
demembranated sperm models (Brokaw et al., 1974; Brokaw, 1979),
and sperm-activating peptides (SAPs) trigger increase in the intracellular Ca2+ concentration ([Ca2+]i) in the sperm (Cook and Babcock,
1993; Cook et al., 1994). The effect of extracellular Ca2+ concentration on flagellar asymmetry of the spermatozoon during chemotactic behaviour has been reported in hydrozoan (Miller and Brokaw,
1970; Cosson et al., 1984) and ascidian (Miller, 1982) spermatozoa.
During the last decade, Ca2+ imaging techniques have improved significantly, and Ca2+ concentration in moving spermatozoa has been
studied in sea urchins and ascidians. In sea urchins, [Ca2+]i fluctuations
in the sperm are induced by the sperm attractant resact (Arbacia punctulata and Arbacia lixula) and speract (L. pictus), and the [Ca2+]i
increases are closely related to the chemotactic turn and flagellar
461
asymmetry (Böhmer et al., 2005; Wood et al., 2005; Guerrero
et al., 2010). In the ascidian, the spermatozoa normally exhibit circular
movements as described earlier and maintain [Ca2+]i at very low
levels, while frequently showing transient [Ca2+]i increases in the flagella (Ca2+ bursts) during chemotactic behaviour (Shiba et al., 2008)
(Fig. 3). Interestingly, the Ca2+ bursts are consistently evoked at a
local minimal value for a given attractant concentration (Shiba et al.,
2008) (Fig. 3). Similar Ca2+ transients in the sperm during movement
down the attractant gradient are also observed in sea urchin (Böhmer
et al., 2005; Guerrero et al., 2010). Therefore, sperm attractants
appear to induce Ca2+ entry from extracellular spaces into the
sperm cell, and the increase in [Ca2+]i mediates the beating of
sperm flagella, resulting in the chemotactic turn and ‘turn-and-straight’
movements.
In mammals, transient [Ca2+]i increases were also induced by the
candidate attractants: bourgeonal and progesterone induce [Ca2+]i
increases in human spermatozoa (Spehr et al., 2003, 2004; Teves
et al., 2009), while lyral affects mouse spermatozoa (Fukuda et al.,
2004). However, a relationship between the [Ca2+]i increases in the
sperm cells and flagellar beating has not yet been found, possibly
because there are few studies on sperm movement during chemotactic behaviour as described already.
Signalling mechanisms of sperm
chemotaxis: from attractant
to [Ca21]i increase
Despite the identification of eight sperm attractants, the receptors for
these attractants have only been unequivocally identified in echinoderms among marine invertebrates. In the sea urchin, many SAPs
have been identified (Suzuki and Yoshino, 1992), and among them,
the peptides resact and speract have been found to act as sperm
attractants (Ward et al., 1985; Guerrero et al., 2010). The receptor
for resact was identified and characterized as a transmembrane-type
guanylylcyclase (GC) (Singh et al., 1988). Cyclic GMP (cGMP)
Figure 3 The [Ca2+]i signal and flagellar bending in the sperm of
the ascidian C. intestinalis during chemotactic behaviour. (A) Timecourse of Ca2+ changes in the ascidian spermatozoa during chemotactic behaviour. The pseudo-coloured [Ca2+]i signal images of the
sperm around the tip of a capillary containing egg-derived attractant
at 20-ms intervals are shown. Bar; 10 mm. (B) Data points taken
from (A). Dots show the sperm head points from the image, and
pseudo-colours show the average intensity of the [Ca2+]i signal in
the flagellum. Arrowheads 1, 2 and 3 indicate the signals obtained
at instances 1, 2 and 3 shown in (A), respectively. The arrows indicate
the swimming direction. Interestingly, Ca2+ levels in spermatozoa
increased during turns and leading into straight swimming. (C) Top:
Changes in [Ca2+]i signals derived from the head (blue line) and flagellum (orange line). Bottom: Changes in the swimming-path curvature (black line) and the asymmetric index (red dots) of the
spermatozoon shown in (A). The grey bar indicates the period
during which the images shown in (A) were captured. The numbered
dots indicate signals obtained from the corresponding images in (A).
[Ca2+]i in the flagellum of the ascidian sperm is poorly correlated with
flagellar asymmetry. (Modified Figure from; Shiba et al., 2008).
462
synthesized by the receptor GC induces the hyperpolarization of
membrane potential by K+ efflux through cGMP-activated K+ channels (Galindo et al., 2000; Darszon et al., 2001). The change in the
membrane potential in turn increases the intracellular pH and Ca2+
and cAMP levels (Darszon et al., 2001; Kaupp et al., 2003; Böhmer
et al., 2005; Strunker et al., 2006). The receptor for another SAP,
speract, is not a GC (Dangott et al., 1989), but the receptor associates
with GC (Bentley et al., 1988). The SAP of the starfish Asteria samurensis, asterosap, can also act as a sperm attractant (Böhmer et al.,
2005), and its receptor is also a GC (Nishigaki et al., 2000).
Asterosap-induced cGMP increases result in [Ca2+]i increases (Matsumoto et al., 2003; Böhmer et al., 2005).
The signalling of mammalian sperm chemotaxis stimulated by odorants is mediated by G-protein-coupled odorant receptors (Spehr
et al., 2003; Fukuda et al., 2004). The human sperm odorant receptor
hOR17-4, which is a potential candidate for mediating human sperm
chemotaxis, activates membrane-associated adenylyl cyclases (mACs)
(Spehr et al., 2004) and may result in increases in the [Ca2+]i and
cAMP concentrations. Furthermore, a recent study suggested that the
cAMP-dependent protein kinase and cGMP-dependent protein kinase
pathways are involved in human sperm chemotaxis induced by progesterone (Teves et al., 2009), although the receptor for progesterone
is still unknown. In both echinoderm and mammalian cases, the cyclic
nucleotide is the first signal, and [Ca2+]i increases follow. On the
other hand, other recent studies showed that progesterone directly activates the sperm-specific cation channel, CatSper, without the increase
of cAMP levels (Lishko et al., 2011; Strünker et al., 2011). They also proposed that CatSper is a receptor for progesterone, though there is currently no direct evidence of interaction between progesterone and
CatSper. Progress of the studies should lead breakthrough of the
study on mechanism of mammalian sperm chemotaxis.
The increase in [Ca2+]i in spermatozoa is mediated by Ca2+ channels
on the sperm plasmamembrane. In the sea urchin A. punctulata, resact
induces an increase in [Ca2+]i through cGMPand cAMP (Cook et al.,
1994), and the [Ca2+]i increase is regulated by K+-selective cyclic
nucleotide-gated cation channels (Strunker et al., 2006; Galindo et al.,
2007; Bönigk et al., 2009). Fluctuations in [Ca2+]i have been observed
in the swimming spermatozoa of sea urchins, and these [Ca2+]i fluctuations could be induced by caged cGMP (Böhmer et al., 2005; Wood
et al., 2005). In the ascidian, store-operated Ca2+ channels mediate
the asymmetric flagellar waveform of spermatozoa and result in chemotactic behaviour (Yoshida et al., 2003). In mammalian sperm, although
the Ca2+ channels that mediate sperm chemotaxis have not been identified, many Ca2+ channels are found in the sperm head and flagella (see
reviews, Darszon et al., 2005, 2006). In particular, the voltage-gated
Ca2+ channel Cav2.3 (Sakata et al., 2002), transient receptor potential
channels (Castellano et al., 2003) and CatSper1 –4 (Ren et al., 2001;
Carlson et al., 2003, 2005; Quill et al., 2003) are thought to play a role
in the swimming behaviour of mammalian sperm and may be involved
in sperm chemotaxis.
How do Ca21 levels mediate
flagellar beating?
As described already, sperm flagellar beating modulates the chemotactic response of spermatozoa. The asymmetry of sperm flagellar
Yoshida and Yoshida
bending is generally believed to be closely correlated with [Ca2+]i
because the pattern of microtubule sliding and flagellar bending in
demembranated sperm models have been shown to be correlated
with Ca2+ concentrations in the medium. In sea urchins (Brokaw
et al., 1974; Brokaw, 1979; Bannai et al., 2000) and ascidians
(Brokaw, 1997), the Ca2+ levels in reactivated solutions are correlated
with the flagellar asymmetry of the reactivated models. Thus, it has
been postulated that [Ca2+]i fluctuates during sperm chemotaxis
and that high and low [Ca2+]i induce turning and straight swimming
with asymmetric and symmetric flagellar bending, respectively (Cook
et al., 1994; Nishigaki et al., 2004). However, in the flagellum of the
ascidian sperm, [Ca2+]i is poorly correlated with flagellar asymmetry,
despite the observation that stereotypic chemotactic responses comprising turning and straight swimming always occur during Ca2+ bursts
(Shiba et al., 2008) (Fig. 3). Poor correlation between [Ca2+]i and flagellar asymmetry was also observed in the sea urchin sperm (Böhmer
et al., 2005; Wood et al., 2005), and the magnitude and duration of
the SAP-induced Ca2+ fluctuations in the sea urchin spermatozoa
affect the properties of sperm turning (Wood et al., 2007). Thus,
[Ca2+]i in the flagellum alone does not determine the magnitude of
beat asymmetry of sperm flagella, but some unknown mechanisms
activated by Ca2+ are likely involved in this case.
The molecular mechanisms connecting [Ca2+]i increases to sperm
flagellar asymmetry remain obscure. The light chain of the outer
arm dynein, which is the motor protein in the sperm flagellum, has
a Ca2+-binding site (King and Patel-King, 1995) and might regulatedynein motor activity in response to Ca2+-binding (Sakato et al., 2007) in
the Chlamydomonas flagellum. Recently, the sperm flagellar-specific
Ca2+-binding protein calaxin was found to regulate the inner arm
dynein during the chemotactic behaviour of ascidian sperm (Mizuno
et al., 2009). Further investigation is required to understand how
Ca2+ levels modulate flagellar beating.
Conclusions: problems and
future aspects of sperm
chemotaxis research
In this paper, the mechanisms involved in the regulation of sperm flagellar beating during the chemotactic response were reviewed. The
review was based mainly on studies of marine invertebrates because
although mammalian sperm chemotaxis has been studied extensively,
few studies have specifically addressed the mechanisms of flagellar
beating. Furthermore, the study of mammalian sperm chemotaxis is
complex and has been limited by discrepancies, in part because mammalian sperm movement is closely linked to other phenomena that
occur in the female reproductive tract, such as semen liquefaction,
capacitation, hyperactivation and acrosome reaction. One series of
studies reported that the chemotactic behaviour of human sperm is
observed only in capacitated spermatozoa (Eisenbach and Giojalas,
2006), while a different series of studies made no reference to capacitation (Spehr et al., 2003, 2004). Several candidate sources and
chemical compositions of sperm attractants exist, as described
already, and these different forms are disputed in the literature. The
integration and verification of the discrepant studies is necessary to
improve our understanding of mammalian sperm chemotaxis.
Sperm chemotaxis and calcium
Confusingly, most of the sperm attractants can induce phenomena
other than chemotaxis not only in mammalian but also in marine
invertebrate sperm, and most of these functions involve Ca2+ signalling. For example, SAAF and resact can induce the activation of
sperm motility in the ascidian and sea urchin spermatozoa, respectively; progesterone can induce capacitation, hyperactivation and acrosome reaction in the mammalian sperm. Therefore, it is inadequate to
use Ca2+ responses as the basis for evaluating attractant-induced chemotactic responses, although many studies have used this assay
because it is easier to observe the Ca2+ response than the chemotactic behaviour of sperm. Although the Ca2+ response plays an important role in the chemotactic response, it should be observed
concomitantly with sperm flagellar movement.
Another important question that remains unanswered is when and
where the spermatozoa detect the attractant. Furthermore, the wide
dynamic range of detection possessed by sperm is intriguing, as sperm
can respond to concentrations of attractants from 10212 to 1026 M.
Technical advances in research are necessary to shed light on some
of the issues associated with sperm chemotaxis and to overcome
the difficulties associated with its study, such as the fact that the chemotactic response is too fast to analyse biochemically, and the maintenance of the gradient field of attractant during experimentation
is difficult.
Finally, the species specificity of sperm chemotaxis is interesting
from the point of view of the evolution of fertilization. In marine
sperm, the reproductive season is reached simultaneously by many
animals, and species-specific sperm attractants ensure that the spermatozoa find eggs from the same species and may thus participate
in the prevention of crossbreeding. In mammals, sperm chemotaxis
has been reported to have no species specificity (Sun et al., 2003;
Teves et al., 2006; Guidobaldi et al., 2008), although another study
on odorous attractants reported species-specific sperm attraction
(Fukuda et al., 2004). The species-specificity of mammalian sperm chemotaxis should be examined more accurately, even though progesterone is a potent candidate as an attractant in some mammalian species.
Further studies would provide new insights into the diversity and generality of the mechanisms that regulate sperm chemotaxis.
Funding
This work was supported in part by Grants-in-Aid for Scientific
Research from MEXT and JSPS (KAKENHI) (21370030, 22112507
and 21026008) to M.Y.
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