BIOLOGY OF REPRODUCTION 80, 1092–1098 (2009)
Published online before print 11 February 2009.
Egg Coat Proteins Activate Calcium Entry into Mouse Sperm via CATSPER Channels1
Jingsheng Xia and Dejian Ren2
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania
During mammalian fertilization, the contact between sperm
and egg triggers increases in intracellular Ca2þconcentration
([Ca2+]i) in sperm. Voltage-gated Ca2þchannels (CaVs) are
believed to mediate the initial phase of [Ca2þ]iincreases in
sperm induced by egg coat (zona pellucida [ZP]) glycoproteins,
while store depletion-activated Ca2þentry is thought to mediate
the sustained phase. Using patch-clamp recording and Ca2þ
imaging, we show herein that CaVchannel currents, while found
in spermatogenic cells, are not detectable in epididymal sperm
and are not essential for the ZP-induced [Ca2þ]ichanges.
Instead, CATSPER channels localized in the distal portion of
sperm (the principal piece) are required for the ZP-induced
[Ca2þ]iincreases. Furthermore, the ZP-induced [Ca2þ]iincrease
starts from the sperm tail and propagates toward the head.
acrosome reaction, calcium, fertilization, signal transduction,
sperm motility, transport
Increases in intracellular Ca2þconcentration ([Ca2þ]i) have
fundamental roles in sperm function such as capacitation,
motility change, the acrosome reaction (AR), and egg
penetration [1, 2]. Proteins of several Ca2þ-permeable ion
channels have been found in mammalian sperm. These include
voltage-gated Ca2þchannels (CaVs), transient receptor poten-
tial channels, cyclic nucleic gated channels, and CATSPER
channels . A major pathway for Ca2þentry into sperm is
believed to be through CaVchannels, similar to that in neurons
and other excitable cells [3–5]. Following the interaction
between egg coat proteins (zona pellucida [ZP]) and sperm, for
example, sperm CaVs are believed to be activated by membrane
depolarization; the subsequent Ca2þinflux through the
channels is proposed to lead to an initial [Ca2þ]irise and a
sustained one later [1–6]. Consistent with a role of CaVs in
sperm, a low voltage-activated (T type) CaVcurrent has been
recorded from spermatogenic cells (spermatocytes and sper-
matids, the precursors of sperm) [7–9] and from early-stage
testis sperm . Because sperm do not normally have the
capability to synthesize new proteins, the T-type CaVchannels
are believed to be responsible for the CaV-dependent Ca2þ
influx in mature sperm .
Despite the seemingly overwhelming evidence supporting
the roles of CaVs in the Ca2þinflux, functional CaVhas not
been directly analyzed from epididymal sperm, partially
because of the difficulties in applying whole-cell patch-clamp
technique to sperm until recently . In addition, the in vivo
role of CaVs in sperm Ca2þsignaling and mammalian
fertilization is questioned recently by the lack of obvious
fertilization-specific phenotype in more than a dozen CaVgene
knockouts, despite profound phenotypes in the nervous,
muscle, and other systems [12, 13]. In this study, we directly
test the existence of CaVcurrents and their role in ZP-induced
Ca2þentry in epididymal mouse sperm using whole-cell patch-
clamp recording and Ca2þimaging. Surprisingly, CaVcurrent
is not detectable in epididymal sperm, nor are the CaVchannels
essential for the ZP-induced Ca2þinflux. Instead, CATSPER
channels are required for the ZP-induced [Ca2þ]iincreases.
MATERIALS AND METHODS
Reagents were from Sigma (St. Louis, MO) unless otherwise stated. Fluo-4
AM, Fura-2 AM, and pluronic F-127 were from Molecular Probes (Invitrogen,
Eugene, OR). Cell-Tak was from BD Biosciences (Bedford, MA). Protease
inhibitor cocktail and DNase were from Roche Diagnostics (Indianapolis, IN).
Pertussis toxin (PTX) and ionomycin were from Calbiochem (Gibbstown, NJ).
Coomassie blue G-250 and mounting medium (Permount) were from Fisher
Scientific (Pittsburgh, PA).
All procedures described herein were reviewed and approved by the
University of Pennsylvania Institutional Animal Care and Use Committee
and were performed in accord with the Guiding Principles for the Care and
Use of Laboratory Animals. The Catsper1 knockout mutant strain had been
backcrossed to C57BL/6J for more than 10 generations . The GFP-
Catsper1 transgenic mice carry a transgene encoding a fusion protein
between GFP and CATSPER1 in the Catsper1-null background . Unlike
the Catsper1-null mice, the male transgenic mice are fertile, suggesting that
the fusion protein functionally replaced the wild-type CATSPER1.
Experiments involving mutant/wild-type paired preparations were done in a
blind manner in which the experimentalist did not know the genotype during
Mouse ZP Protein Preparation
The ZP were prepared using the Percoll gradient method . Ovaries from
25–35 mice (;3 wk old) were homogenized at room temperature in a glass
tissue grinder in 2 ml of homogenization buffer (HB) buffer containing 25 mM
triethanolamine,150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2(pH adjusted to 8.5
with 1 N HCl), 1 mM PMSF, DNase (0.2 mg/ml), and protease inhibitor
cocktail with 2 mM ethylene. The homogenate was mixed with detergents (0.2
ml of 10% Nonidet P-40 and sodium deoxycholate) loaded onto a
discontinuous three-step Percoll gradient in HB buffer with 2% (2 ml), 10%
(2 ml), and 22% (3 ml) Percoll in a siliconized 15-ml plastic tube and
centrifuged at 400 3 g for 2 h at 48C in a swinging bucket rotor. The 10%
Percoll layer, a major ZP-containing fraction, was diluted with 45 ml of HB
buffer and centrifuged to pellet the ZP at 200003g for 20 min at 48C. Pelleted
samples were washed with HB buffer followed by a wash with HS medium
 (135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM HEPES,
10 mM glucose, 10 mM lactic acid, and 1 mM pyruvic acid; pH adjusted to 7.4
with NaOH) or with divalent-free (DVF) buffer [11, 18] (150 mM Na-
gluconate, 20 mM HEPES, and 5 mM HEDTA [N-(2-hydroxyethyl)
1Supported by NIH grants 1R01HD047578 and 1R03HD045290.
2Correspondence: Dejian Ren, Department of Biology, University of
Pennsylvania, 415 S. University Ave., Philadelphia, PA 19104. FAX:
215 898 8780; e-mail: firstname.lastname@example.org
Received: 6 October 2008.
First decision: 22 October 2008.
Accepted: 23 January 2009.
? 2009 by the Society for the Study of Reproduction, Inc.
eISSN: 1259-7268 http://www.biolreprod.org
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ethylenediamine-N,N0,N0-triacetic acid], pH 7.4 with NaOH), each with a 20-
min centrifucation at 20000 3 g. The final ZP pellet was resuspended in 0.1–
0.2 ml cold HS medium or DVF buffer, and the number of ZP was counted
under the microscope. The ZP were solubilized by reducing the pH to 2.5 with
1 N HCl, followed by incubation at 378C for 15 min and centrifugation at
200003g for 10 min at room temperature. The supernatant was neutralized to
pH 7.4 with 1 N NaOH, stored in aliquots at?708C, and used within 3 months.
Negative controls included buffer alone (without the starting ovary materials)
that underwent similar initial procedures, preparation from the 22% Percoll
layer in the gradient spinning, and flow-through from solubilized ZP applied to
a concentrator with a 10-kDa molecular weight cutoff (YM-10; Millipore,
Bedford, MA) (the molecular weights of the mouse ZP are .60 kDa ). As
expected, these controls did not induce obvious [Ca2þ]ichanges.
Caudal epididymides were excised and rinsed with HS medium. Sperm
were released from three small incisions at 378C, 5% CO2, for 15 min into HS
medium supplemented with 5 mg/ml of bovine serum albumin and 15 mM
NaHCO3. Released sperm were concentrated to 5 3 106to 13107/ml by
centrifugation for 4 min at 3003g, followed by capacitation in suspension for
90 min at 378C, 5% CO2in the same medium. During the last 25 min of
capacitation, cells were loaded with 10 lM Fluo-4 AM and 0.05% Pluronic F-
127, followed by two washes in imaging medium (HS supplemented with 15
mM NaHCO3), each with a 4-min spin at 3003g. Washed sperm were plated
onto coverslips coated with Cell-Tak. Small-volume imaging chambers (;1 cm
diameter [90 ll]) were formed with Sylgard (Dow Corning, Midland, MI) on
coverslips. Cells were allowed to attach for 10 min. A monochromator
(DeltaRAM V; PTI, Birmingham, NJ) with a 75-W xenon lamp was used to
generate the excitation at 488 nm. A 603 objective and a 1.63 adaptor on an
inverted microscope (IX-71; Olympus, Tokyo, Japan) were used for imaging.
Emissions (515–565 nm) were bandpass filtered (HQ540/50; Chroma,
Rockingham, VT) and collected with a cooled charge-coupled device camera
(CoolSNAP HQ; Roper Scientific, Tucson, AZ) for 25 milliseconds in every
0.5 sec for fast recording or for 100 milliseconds in every 6 sec for slow
recording. Online control, data collection, and image processing were done
using commercial software (ImageMaster 3; PTI). [Ca2þ]ichanges are
presented as DF:F0ratios after background subtraction, where DF is the
change in fluorescence signal intensity and F0is the baseline as calculated by
averaging the 10 frames before stimulus application. For imaging of sperm
from GFP-Catsper1 transgenic mice, the ratiometric measurement with Fura-2
AM (5 lM for loading) was used because of fluorescence from GFP. [Ca2þ]i
changes are presented as the ratio of F340:F380 after background subtraction.
Calcium imaging experiments were done at 378C unless otherwise stated. Cells
with uneven dye loading were excluded from analysis. Motile sperm (identified
by comparing multiple-image frames) that had one or two points attached to the
coverslip were used for analysis. Cells with peak changes of .50% in DF:F0
(for Fluo-4 AM) or .0.1 in F340:F380 (for Fura-2 AM) after application of ZP
were counted as responsive. To detect the Ca2þresponses at ‘‘clamped’’
membrane potential, Kþionophore valinomycin (1 lM) was added to the
imaging buffer, with additional Kþas indicated to replace equal molar
concentration of Naþ. The equilibrium (Nernst) potential for Kþ(EK) was
calculated based on the assumption of an intracellular Kþconcentration of 120
mM [19, 20].
All recordings were done at room temperature. Liquid junction potentials,
calculated using Clampex software (Molecular Devices, Sunnyvale, CA), were
corrected. Sperm were obtained from 4- to 8-mo-old mice and recorded without
capacitation. Whole-cell current recordings with seals between glass pipettes
(7–10 MX resistance) and the sperm cytoplasmic droplets followed previously
described methods . Unless otherwise stated, a modified HS solution (135
mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, 5 mM
Glucose, 10 mM lactic acid, 1 mM sodium pyruvate; pH adjusted to 7.4 with
NaOH) was used as bath . Pipette solution contained 135 mM Cs-
methanesulfonate, 5 mM CsCl, 5 mM Hepes, 10 mM ethyleneglycoltetracetic
acid, 5 mM Na2ATP, and 0.5 mM Na2-glutamyl transpeptidase (pH adjusted to
7.2 with CsOH). Pipette solutions containing CsF  were also used in some
recordings; no voltage-gated channel currents were detected in epididymal
sperm (n ¼ 9) (data not shown).
Patch-clamp recordings using spermatocytes and spermatids followed
previously described methods [7, 14]. Cells were extruded by manual
trituration from lightly dissociated seminiferous tubules with forceps and were
filtered through a 70-lm cell strainer. Data from pachytene spermatocytes and
round spermatids used for patch-clamp recordings were pooled.
Detection of the AR with Coomassie Blue Staining
Detection of the AR with Coomassie blue G-250 was performed as
previously described [7, 21]. At least 300 sperm (.100 per sample) from three
pairs of mutant/wild-type mice were counted as acrosome reacted (no staining
in the acrosomal region) or as acrosome intact (dark blue staining over the
Data analyses were performed using ImageMaster3 (Photon Technology
International, Monmouth Junction, NJ), Excel (Microsoft, Redmond, WA), and
Origin (Microcal Software Inc., Northampton, MA). Student t-test and single-
factor ANOVA were used for statistical comparison between different
treatments. P , 0.05 was considered statistically significant.
CaVChannel Currents Are Undetectable in the
Ion channel currents have been difficult to characterize in
epididymal sperm because of technical difficulties of whole-cell
patch-clamp recordings in sperm until recently . To test
directly whether epididymal sperm have functional CaVs, we
recorded from mouse corpus epididymal sperm using the whole-
cell configuration . As a positive control for sperm channel
recording, the CATSPER channel current (ICATSPER) was readily
detectable (n ¼ 9) (Fig. 1A). However, we detected no CaV
currents during the depolarizing pulses or on repolarization to
?103 mV for tail current detection in any of the sperm cells (n¼
23) (Fig. 1B). Using the same bath and pipette solutions, T-type
CaVcurrents were detected in all 13 spermatocytes and
spermatids (peak current at ?53 mV; range, ?13 to ?137 pA
[mean 6 SEM,?55 6 11 pA]) (n¼13) (Fig. 1C). Similar to T-
type CaVchannels in somatic cells , the channels in
spermatogenic cells were easily inactivated even at hyperpolar-
ized membrane potentials (half-maximum inactivation,?93 mV)
(n¼5) (Fig. 1D). When spermatocytes and spermatids were held
at?33 mV, the channels became completely inactivated, and the
current was undetectable (n¼6) (Fig. 1E).
To maximize CaVcurrent detection sensitivity, we also
CaVs in other cells, the spermatogenic cell T-type CaVchannels
became nonselective and had a large peak inward current in the
DVF bath (range,?85 to?2582 pA [mean 6 SEM,?636 6 393
pA])(n¼6). SpermCaVcurrent recordingsin the DVFbathwere
performed using Catsper1-null sperm because large inward
current at the holding potential (?103 mV) through the wild-type
CATSPER channel in the absence of Ca2þmade the recordings
unstable (data not shown) (Fig. 1A) . Again, we detected no
significant CaVcurrents (n ¼ 9). These data suggest that
spermatogenic cells possess T-type CaVchannels, like neurons,
muscle cells, and fibroblasts, but functional CaVcurrents are
undetectable in the epididymal sperm.
The lack of detectable CaVcurrent in the epididymal sperm
was surprising because T-type CaVcurrents were clearly
present in spermatogenic cells and were recorded from some
testis sperm . When CaVcurrents recorded from spermato-
genic cells and different stages of sperm were analyzed, CaV
current sizes gradually decreased as testis sperm became more
mature; no current was detectable in sperm before leaving the
testis (Supplemental Fig. S1 and all Supplemental Data are
available online www.biolreprod.org).
Activation of CaVChannels Is Not Required for the
It is possible that small CaVcurrents are present in
epididymal sperm but that the sizes of the currents fall below
CATSPER CHANNELS MEDIATE ZP-ACTIVATED Ca2þENTRY
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our threshold for detection even with a DVF bath. Furthermore,
we recorded from incapacitated corpus sperm because the more
mature, capacitated caudal sperm used for fertilization are
inaccessible to robust whole-cell patch-clamp recording .
To further test whether T-type CaVchannels, if present, are
required for the ZP-induced Ca2þentry in mature sperm, we
imaged the [Ca2þ]ichanges in the head region of capacitated
caudal sperm using the Ca2þindicator Fluo-4 AM. Consistent
with other observations [23, 24], bath application of solubilized
ZP (2 ZP/ll) led to increases in [Ca2þ]iin the sperm head
(represented as normalized fluorescence changes DF:F0) in
66% (106 of 160) of the cells. Most of the responses appeared
within 20 sec of ZP application (Fig. 2A), and some (37%) also
had a delayed phase clearly separated from the initial phase
(Fig. 3A). The responses were disrupted by inhibiting the G
protein with PTX (100 ng/ml [18% responsive cells] [Fig. 2B])
or phospholipase C with neomycin (1 mM [15% responsive
cells] [Fig. 2C]), suggesting involvement of G proteins in the
ZP-induced [Ca2þ]ichanges [7, 23, 25]. As a control, cell-
permeable cGMP (8-Br-cGMP) induced an increase in [Ca2þ]i
 that was not blocked by PTX or neomycin treatment (Fig.
2, B and C).
We then increased the extracellular potassium concentration
([Kþ]o) from 5 mM to 18.5 mM and to 56.8 mM such that the
EKvalues were ?50 mV and ?20 mV, respectively (assuming
[Kþ]i¼120 mM [see Materials and Methods]). Because of the
prominent roles of Kþin determining the resting membrane
potential in sperm cells , increasing [Kþ]oshould
depolarize the cells and inactivate T-type CaVchannels.
Surprisingly, application of ZP still robustly led to increases
in [Ca2þ]i(Fig. 2, D and F). Neither the percentages of
responsive cells nor the response magnitudes were significantly
reduced (Fig. 2F). We then included the Kþionophore
valinomycin (1 lM) in the baths to clamp the membrane
potential to the EKat ?50 mV or ?20 mV. The T-type CaV
channels, if existing, would have been inactivated at those
holding potentials (Fig. 1). Again, application of ZP increased
[Ca2þ]i(Fig. 2, E and F). Taken together, these data suggest
that CaVchannel activation is not required for the ZP-induced
[Ca2þ]iincreases. Consistent with this notion, Zn2þ(10 lM)
effectively blocked the T-type channel current in spermatocytes
(n ¼ 4) (data not shown) but did not significantly inhibit the
ZP-induced Ca2þresponses (72% [18 of 25 cells] responsive;
mean 6 SEM DF:F0peak, 362% 6 57%) (n¼18). See Figure
3 (A and E) for a comparison with responses in the absence of
CATSPER Channels Are Required for the [Ca2þ]iRise
Induced by ZP
Another family of Ca2þ-permeable channels in sperm is the
CATSPER . All four CATSPER family members have
restricted expression in testis and sperm. The channel is
activated by intracellular alkalization and is weakly affected by
membrane potentials but, unlike CaVchannels, is not
inactivated by depolarization (Fig. 1A) . Sperm deficient
in CATSPER channels can fertilize zona-free eggs but not
zona-intact ones, presumably because of the mutant sperm’s
inability to penetrate the zona layer [14, 18, 26, 27]. We
compared the ZP-induced [Ca2þ]ichanges in sperm prepared
from wild-types vs. Casper1-null mutants, which have intact T-
type CaVchannel currents in spermatocytes . In contrast to
the wild-type (Fig. 3A), sperm deficient in CATSPER1 lacked
epididymal sperm. A) A representative
recording of epididymal sperm ICATSPERin
HS (black line) and DVF (red line) bath
solutions. The ramp voltage protocol is
shown above (HP, holding potential). B)
Recordings to detect CaVcurrent in the
same sperm cell as in A (HS bath [repre-
sentative of 23]). Step voltage control
protocol is shown above. A prepulse (P/4)
protocol was used for leak subtraction. C)
CaVcurrent recordings from a spermatocyte
(HS bath). D) Steady-state inactivation curve
reconstructed with recordings from five
spermatogenic cells. Current sizes (at ?53
mV) recorded after inactivation prepulses
(?113 to ?53 mV, 5 sec [see inset]) were
normalized to that recorded with the
prepulse of ?113 mV. E) Recording from a
spermatocyte showing complete inactiva-
tion of the T-type CaVchannel at a holding
potential of ?33 mV (representative of six).
Lack of Cavchannel currents in
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any detectable [Ca2þ]ichanges within 2 min of ZP stimulation
(Fig. 3, B and E). Delayed responses were present in 18% of
sperm (Fig. 3C). ZP-induced [Ca2þ]ichanges were restored by
a transgene encoding a GFP-CATSPER1 fusion protein in the
Catsper1-null background (Fig. 3, D and E).
ZP Induce [Ca2þ]iRise in the Principal Piece
The dependence of the ZP-induced [Ca2þ]iincrease in
sperm head on CATSPER channels was unexpected, as
CATSPER proteins [14, 18, 26] and currents through
CATSPER channels  are localized selectively to the distal
portion of sperm flagella (the principal piece). Findings from
earlier studies using Ca2þ-sensitive dyes with lower sensitivity
(calcium green-1/rura-red ) or imaging at a slower frame
rate (0.5 frames/sec ) suggested that the ZP-induced
[Ca2þ]iincrease starts in the head. To further examine the
spatial-temporal kinetics of the [Ca2þ]iincreases in the cellular
subdomains along the sperm, we imaged the [Ca2þ]idynamics
with the highly sensitive Fluo-4 AM Ca2þindicator at a higher
frame rate (2 frames/sec). Similar to previous findings , the
absolute fluorescence changes (DF) observed in the tail regions
were small compared with those in the head, presumably
because of the smaller cellular volume in the tail regions. The
normalized changes (DF:F0), however, clearly started from the
principal piece (Figs. 4A and 5 and Supplemental Movie). At
378C, the delay between the principal piece and head was a
mean 6 SEM of 2.9 6 0.3 sec (n ¼ 20). Lowering the
temperature from 378C to 188C increased the interval to a mean
6 SEM of 4.4 6 0.3 sec (n ¼ 12). There was also a mean 6
SEM delay of 1.1 6 0.1 sec between the [Ca2þ]iincreases in
regions 5 lm apart within the midpiece (n¼20). In contrast, no
significant delay was detected between regions with similar
distance within the principal piece (Fig. 4B), the region where
CATSPER channels are localized [14, 18, 26]. Calcium
ionophore ionomycin (10 lM [Fig. 4A]) and A23187 (2 lM
[Supplemental Fig. S2]) led to [Ca2þ]iincreases simultaneous-
ly in all subregions.
In this study, we directly analyzed ion channel currents from
epididymal sperm using whole-cell patch-clamp recording. We
detected no CaVcurrents in the sperm cells, although
the ZP-induced [Ca2þ]irise in sperm head.
A) A representative recording of the [Ca2þ]i
changes (DF:F0; shown as DF/F0 (%) in
successive panels) induced by bath appli-
cation of ZP (2 ZP/ll, indicated by vertical
arrow). B and C) Representative [Ca2þ]i
changes (DF:F0) in sperm incubated with
PTX (B) (100 ng/ml, 30-min preincubation)
or neomycin (C) (1 mM, 5-min preincuba-
tion). Cell-permeable cGMP (8-Br-cGMP, 2
mM) was used as a control stimulus. D and
E) Representative recordings in increased
extracellular Kþconcentrations as indicated
without (D; shown as [K]o) or with (E)
valinomycin (1 lM) in the bath to clamp the
membrane potentials to ?20 mV ([Kþ]o¼
56.8 mM) or ?50 mV ([Kþ]o¼ 18.5 mM).
F) Averaged peak DF:F0in responsive cells
under various [Kþ]owith or without valin-
omycin in the baths. Numbers of analyzed
cells are in parentheses. There was no
statistically significant difference (P ¼ 0.47).
CaVchannels are not required for
CATSPER CHANNELS MEDIATE ZP-ACTIVATED Ca2þENTRY
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CATSPER channel currents were readily detectable. CaV
channels, even if present in sperm, did not seem to be required
for the ZP-induced [Ca2þ]ichanges, as the [Ca2þ]irises were
not blocked by raising [Kþ]osuch that the channels would have
been inactivated. Finally, mutation in the Catsper1 channel
gene disrupted a ZP-induced [Ca2þ]ichange, and such
disruption could be rescued by a GFP-Catsper1 transgene.
How do these data reconcile with previous findings
implicating CaVs in the Ca2þinflux in sperm? First, sperm
membrane depolarization and intracellular alkalization, which
can be induced by ZP proteins, were shown to lead to Ca2þ
influx that can be inhibited by CaVchannel blockers with
variable efficacies [7, 29–32]. However, later findings revealed
that the alkaline depolarization-induced Ca2þinflux required
CATSPER channels . It is possible that some of the
inhibition on the ZP-induced Ca2þresponses by nonspecific T-
type CaVchannel blockers such as Cd2þand Ni2þwas due to
the drug’s blocking effects on CATSPER channels .
Second, mRNAs of several CaVpore-forming subunits (a1) and
the associated auxiliary subunits have been detected in testis
and proteins detected in sperm [3, 9, 17, 34, 35]. However, the
functional significance of these proteins in sperm physiology is
not clear, as none of more than a dozen CaVchannel gene
knockouts seems to have significant defect in sperm function or
male fertility [12, 13]. In contrast, disruption in any of the four
Catsper genes leads to profound defect in sperm hyperactivated
motility and to complete male infertility [14, 18, 26, 27]. Third,
as for the possible role of the T-type CaVcurrent recorded in
spermatocytes and spermatids, a targeted disruption in a CaV
gene (CaV3.2, official symbol Cacna1h) eliminates all
detectable CaVcurrent in spermatocytes but does not lead to
defect in the ZP-induced [Ca2þ]ichanges or male fertility ,
despite pronounced smooth muscle phenotypes in the mutant
. Taken together, the simplest explanation of these data is
that the ZP-induced [Ca2þ]iincrease starts from Ca2þentry via
CATSPER channels in the tail instead of T-type CaVchannels
in the head.
the ZP-induced [Ca2þ]irise in the sperm
head. A) Recording from a wild-type sperm.
Note that there were two phases of [Ca2þ]i
changes in this cell. B and C) Recordings
from Catsper1-null sperm. The delayed
response was present in the cell in C. Ca2þ
ionophore ionomycin (10 lM) was applied
as a control stimulus. D) A representative
recording from a transgenic sperm express-
ing a GFP-CATSPER1 fusion protein in the
Catsper1-null background. E) Percentage of
cells responsive to ZP application within 2
min in wild-type (WT) (n ¼ 24 imaging
runs, 14 mice, 160 cells), Catsper1-null
(Mut) (n ¼ 11 imaging runs, five mice, 78
cells), and Catsper1-mutant mice rescued
with the GFP-Catsper1 transgene (Tg) (n ¼ 8
imaging runs, three mice, 86 cells). Cells
from two Catsper1-null and the three Tg
mice were imaged with Fura-2 AM, and the
others were imaged with Fluo-4 AM. DF:F0
shown as DF/F0 (%) and F340:F380 shown
CATSPER channels are required for
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The pH-sensitive CATSPER channels are presumed to be
tetramers formed by all four CATSPER proteins , but the
biological activators of the channels are unknown. Our data
suggest that the egg coat proteins are potential activators, most
likely through ZP-induced intracellular alkalization [11, 30].
Consistent with indirect activation, we did not observe direct
potentiation of ICATSPERby ZP application during whole-cell
patch-clamp recordings (n¼6) (Supplemental Fig. S3). Similar
indirect activation of CATSPER channels was observed with
cyclic nucleotides, which induce CATSPER-dependent [Ca2þ]i
increases  but do not seem to activate the channel directly
. Given the localization of CATSPER proteins in the
principal piece [14, 18], Ca2þions entering through the
channels may diffuse toward the head but perhaps more likely
are used as a signal to trigger the eventual [Ca2þ]irise in the
midpiece and head . A ‘‘tail-to-head’’ sequence has also
been described in the sperm [Ca2þ]iincreases induced by
odorant bourgeonal in human , by cyclic nucleotides in
mouse , and by egg peptide speract in sea urchin . How
activation of the ZP receptors, presumably located in sperm
head, is coupled to channel opening in the principal piece
remains to be further examined, along with how Ca2þentry
through the channel in the tail leads to the eventual [Ca2þ]i
rises in the midpiece and head.
The [Ca2þ]iincreases seen in sperm can be divided into two
temporal phases: a CATSPER-dependent initial phase that lasts
minutes and a delayed phase that apparently does not have an
absolute requirement for CATSPER but may occur via a store
depletion-activated mechanism . The ZP-induced [Ca2þ]i
increases are believed to be important for the AR, a Ca2þ-
dependent exocytotic process by which the sperm cell releases
its acidic acrosomal contents and prepares for its final fusion
with the egg. We assayed the ZP-induced AR rates and found
no significant difference between the Catsper1-null and wild-
type sperm (Supplemental Fig. S4). The Ca2þrequirement for
the AR may be from the delayed phase of the [Ca2þ]irises that
is present in the mutant sperm (Fig. 3), consistent with the slow
time course of the AR (lasting minutes). The Catsper1
mutant’s ability to undergo the AR and inability to penetrate
ZP-intact eggs suggest that the [Ca2þ]iincrease initiated in the
principal piece and propagating along the whole sperm may be
involved specifically in egg penetration. Future studies with
freely moving sperm will be needed to further dissect the role
of the CATSPER channels under physiological conditions.
We thank Drs. Mariano G. Buffone and George Gerton for performing
pilot studies; Bayard Storey for showing us ZP preparation and AR
detection; Betsy Navarro, Yuriy Kirichok, David Clapham, and George
Gerton for critically reading early versions of the manuscript; and Alberto
Darszon, Pablo Martinez Lopez, and Harvey Florman for useful
piece. A) Representative time courses of the [Ca2þ]ichanges in the
principal piece (PP1), midpiece (MP1), and head. Increases in [Ca2þ]i
started in the principal piece and propagated to the head, as reflected by
the time differences between the application of ZP and the onset of
fluorescence changes (defined as the time point when DF:F0[shown as DF/
F0 (%)] started to have a steep rise) (B) (n ¼ 20). Ca2þionophore
ionomycin induced [Ca2þ]iincreases simultaneously in all subregions (A).
The locations of the subregions within the principal piece (PP1 and PP2),
midpiece (MP1 and MP2), and head chosen for analysis are illustrated in
the inset (B, original magnification396). PP1 and MP1 are 5 lm from the
annulus. There is a 5-lm distance from PP1 to PP2 and from MP1 to MP2.
CATSPER1 protein is localized to the principal piece . See also the
Supplemental Movie showing the wavelike propagation of [Ca2þ]i
increase along the sperm after the application of ZP. Wild-type sperm
were used. ns, not statistically significant compared with PP2 (P . 0.05);
*, statistically significant (P , 0.05).
Initiation of the ZP-induced [Ca2þ]iincreases in the principal
propagation of the [Ca2þ]iincreasing along sperm flagella after ZP
application. Images are presented in a pseudocolor format (processed with
ImageJ; National Institutes of Health, Bethesda, MD). Blue and red
represent the low and high fluorescence signal levels, respectively. For
clarity, a cell with little movement is shown. The movie is three times as
fast as real time (6 frames/sec; each frame represents 0.5 sec of real time).
On application of ZP (2 ZP/ll), [Ca2þ]iincreases began in the principal
piece and reached the head a few seconds later. Original magnification
Still image from the Supplemental Movie showing the
CATSPER CHANNELS MEDIATE ZP-ACTIVATED Ca2þENTRY
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