Pflugers Arch - Eur J Physiol (2014) 466:819831
DOI 10.1007/s00424-014-1478-2
INVITED REVIEW
T-type Ca2+ channels in spermatogenic cells and sperm
Alberto Darszon & Arturo Hernndez-Cruz
Received: 3 February 2014 / Accepted: 8 February 2014 / Published online: 6 March 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Cell function is importantly regulated by the intra-
cellular concentration of Ca2+ ([Ca2+]i). Sperm development
and function are deeply influenced by [Ca2+]i which is mod-
ulated amongst other ion transporters by plasma membrane
Ca2+ permeable channels. The presence and role of voltage-
dependent Ca2+ channels (CaV) of the T-type (CaV3) in sperm
physiology have become a matter of debate in recent years.
Though they are functionally present in later stages of devel-
opment in spermatogenic cells and testicular sperm and their
mRNAs and proteins detected from spermatogenic cells to
mature mammalian spermatozoa, their currents have not been
recorded in mature spermatozoa. This review critically sum-
marizes the evidence for the involvement of CaV3 channels in
sperm development and function.
Keywords Sperm acrosome reaction . Motility .
Capacitation . Voltage-dependent Ca2+ channels
Introduction
Generating a new individual requires a finely orchestrated
interaction between sperm and egg in organisms with sexual
reproduction. Within the gonads, sperm and egg are produced
This article is published as part of the Special Issue on T-type (CaV3)
calcium channels in health and disease.
A. Darszon (*)
Departamento de Gentica del Desarrollo y Fisiologa Molecular,
Instituto de Biotecnologa, Universidad Nacional Autnoma de
Mxico (UNAM, Campus Cuernavaca), Avenida Universidad
#2001, Col. Chamilpa, Cuernavaca, Morelos CP 62210, Mexico
e-mail: darszon@ibt.unam.mx
A. Hernndez-Cruz
Departamento de Neurociencia Cognitiva, Instituto de Fisiologa
Celular, UNAM (Campus Ciudad Universitaria), Mexico City,
Mexico
and initiate their maturation. Thereafter, in the female repro-
ductive tract, mammalian sperm continue maturing in a pro-
cess called capacitation. This later process is needed so that
sperm can respond to physiological inducers of the acrosome
reaction (AR) [71, 139]. In parallel or as a part of this matu-
rational process, the sperm flagellar beat changes from an
active, symmetric mode to a more vigorous and asymmetric
one. This motility change is known as hyperactivation [63].
Once capacitated, sperm are ready to undergo the AR, a one-
time exocytotic event. Where and when this reaction occurs
has recently become a matter of debate [69]. For many years,
it had been thought that the eggs extracellular matrix, the
zona pellucida (ZP), was the physiological inductor of the AR
but recent experiments in mice spermatozoa and earlier ones
in other species have questioned this notion [11, 69].
As in most cells, the concentration of intracellular Ca2+
([Ca2+]i) plays a key role in sperm signaling [19, 27, 67, 83,
128]. This divalent cation regulates motility, capacitation and
the AR. As CaV channels convert membrane potential (Em)
changes into [Ca2+]i signals, their involvement in sperm phys-
iology has been examined thoroughly. Two major functional
classes of CaVs are known: high-voltage-activated (HVA,
CaV1 and CaV2) and low-voltage-activated (LVA, CaV3)
channels. HVA channels require strong depolarizations to
open and inactivate slowly. Weaker depolarizations are need-
ed to open LVA channels. They inactivate faster and at more
negative potentials than HVA channels. CaV3 channels were
initially called T-type due to their transient nature [41]. The
reader is referred to excellent reviews containing the detailed
description of the biophysical, biochemical and molecular
characteristics of these channels [98, 112, 145].
As for HVA channels, the 1 subunit contains the pore-
forming region of CaV3 channels (CaV3.1, CaV3.2 and
CaV3.3; 1G, 1H and 1I, respectively) [97]. This subunit
consisted of four homologous domains symmetrically ar-
ranged around the pore in a single polypeptide. Six
820
transmembrane alpha helices (S1S6) form each of the four
domains. The pore or P loop connecting S5 and S6 has a
conserved alpha helix and a glutamate that form the selectivity
filter [98].
As stated earlier, sperm are tiny specialized cells of complex
morphology (Fig. 1) and whose plasma membrane is overall
rigid [26]. These characteristics precluded the in situ character-
ization of sperm ion channels by traditional electrophysiologi-
cal approaches [reviewed in 25]. Also, with the exception of a
few reports [55, 73], researchers in the field still believe that
spermatozoa are not able to synthesize proteins and thus mo-
lecular strategies such as interference RNA (RNAi) cannot be
used. As alternative strategies to circumvent these drawbacks,
sperm Ca2+ channels were studied in vivo using Ca2+-sensitive
and/or Em-sensitive fluorescent dyes and channel incorporation
into planar lipid bilayers. Furthermore, the presence of Ca2+
channel messengers and proteins, and specifically, CaV3s, was
examined using RT-PCR and specific antibodies.
Considering the above discussion, the ion channels present
in mature sperm are most likely synthesized during spermato-
genesis by their progenitor cells, the spermatogenic cells,
particularly during the more mature stages (pachytene sper-
matocytes and round spermatids). These later cells are ame-
nable for electrophysiology (Fig. 2) and molecular biology
[26, 39, 67]. The presence of several CaV channel messengers
and proteins has been reported and reviewed recently [27].
Here, we will focus on CaV3 channels, their presence and
function in spermatogenic cells and in spermatozoa. In the
following sections, we will describe the CaV3 messengers,
proteins and electrophysiological properties that have been
reported in these cells.
CaV3 channels in spermatogenic cells and sperm
Before their molecular identification, T-type Ca2+ channels
had been biophysically characterized [97, 98]. The main CaV
Fig. 1 Sperm morphology.
Schematics of human sperm. The
different cell regions are indicated
Pflugers Arch - Eur J Physiol (2014) 466:819831
whole-cell patch-clamp currents recorded from mouse sper-
matogenic cells have the most salient features of somatic cell
LVA currents [98] (see Fig. 2). They display low-voltage
thresholds for activation and inactivation and similar Ba2+
versus Ca2+ selectivity. Mibefradil, amiloride, pimozide,
kurtoxin and Ni2+, as well as nifedipine and related 1,4-
dihydropyridines, inhibit these currents at micromolar con-
centrations [3, 5, 93, 107].
CaV3.1 (1G) Northern blots revealed the presence of CaV3.1
mRNAs in mouse and human testis [76], and RT-PCR exper-
iments showed their mRNA in mouse and human spermato-
genic cells and sperm [34, 76, 103]. Thereafter, CaV3.1 pro-
tein was immunolocalized to the mouse and human sperm
head and principal flagellar piece and to the human midpiece
[125]. Using Ni2+ as a specific blocker, patch-clamp record-
ings on pachytene spermatocytes suggested that CaV3.2 is
mainly responsible for their T-type currents with CaV3.1 also
making a minor contribution [125]. Indeed, as currents re-
corded from spermatogenic cells of CaV3.1 null mice are
indistinguishable from those of wild-type spermatozoa,
CaV3.2 channels are likely responsible for most of the T-
type currents of these cells [118]. The presence of functional
CaV 3.1 or CaV 3.2 channels in epididymal mouse sperm is
still not fully settled [32, 125].
CaV3.2 (1H) mRNAs encoding CaV3.2 subunits were de-
tected in mouse testis by in situ hybridization and PCR exper-
iments [117], and CaV3.2 protein was immunolocalized to the
head region overlying the acrosome [32, 125]. As mentioned
above, CaV 3.1 and CaV 3.2 null mice experiments suggest
that CaV3.2 channels are mainly responsible for T-type cur-
rents of mouse spermatogenic cells [32, 118, 125]. In human
sperm, Jagannathan [66] found both CaV3.1 and CaV3.2 tran-
scripts, while Son [116] reported only CaV3.2 mRNA in testis
and Park [96] detected mRNAs encoding the three CaV3s.
Only CaV3.2 immunostaining was faintly detected in the
Pflugers Arch - Eur J Physiol (2014) 466:819831
Fig. 2 Recording of T-type Ca2+ currents from mouse pachytene sper-
matocytes. Left: Representative family of Ca2+ currents recorded from
pachytene spermatocytes obtained by the whole-cell patch-clamp tech-
nique. The ionic currents were elicited by 200-ms depolarizing steps
ranging from 90 to 10 mV from a holding potential of 90 mV with
steps increasing by 10 mV. The inward currents display the typical
voltage dependence and criss-cross pattern of T-type Ca2+ currents.
Inward current amplitude was measured at the peak and normalized. Cells
human sperm head and a stronger signal was found in the
flagellar midpiece [125]. Similar to rodent species, T-type
currents were recorded in spermatogenic cells from human
biopsies [66].
CaV3.3 (1I) CaV3.3 channel kinetics of activation, inactiva-
tion and recovery from inactivation are significantly slower
than those of CaV3.1 and CaV3.2 Ca2+ channels [97]. These
characteristics are dissimilar from those of the T-type currents
recorded from mouse spermatogenic cells. However, mRNAs
encoding the CaV3.3 subunits have been detected in mouse
sperm [125]. Likewise, while CaV3.3 protein was reported to
be present at the flagellar mouse midpiece, Stamboulian et al.
[118] were unable to find electrophysiological evidence for
their presence in mouse spermatogenic cells from either wild-
type or CaV3.1 null mice. In human testicular sperm, Son et al.
[117] did not find CaV3.3 mRNAs; however, they were de-
tected in spermatogenic cells [26]. In motile human sperm,
Park et al. [96] recognized small amounts of CaV3.3 mRNAs.
CaV3.3 protein was immunolocalized to the human flagellar
midpiece, similarly to mouse sperm [125].
Overall, the biophysical and pharmacological characteris-
tics of the voltage-dependent Ca2+ currents recorded from
mouse spermatogenic cells, including those obtained in the
CaV3.1 and CaV3.2 null mice, are consistent with the notion
that T-type Ca2+ currents result from the activity of channels
formed by the CaV3.2 isoform. These channels have been
reported to be regulated by protein kinases [4], albumin [35]
and cell adhesion molecule [86]. Even though the
involvement of CaV channels and specifically the CaV3s in
821
were bathed in a solution containing (in millomolars) CaCl2 (2), NaCl
(130), KCl (3), MgCl2 (2), NaHCO3 (1), NaH2PO4 (0.5), HEPES (5) and
glucose (10) (pH 7.3/NaOH). The internal (patch pipette) solution
contained (in millimolars) CsMeSO3 (110), CsF (10), CsCl (15), EGTA
(10), HEPES (5); ATP-Mg2 (4) and phosphocreatine (10) (pH 7.3/CsOH).
Right: Current-voltage (I-V) relationships of Ca2+ currents (n=11). The
values in the I-V plots are the meanS.E.M
sperm maturation, motility and the AR is under debate, T-type
currents from spermatogenic cells have been utilized to test
the effects of compounds of clinical and biological relevance
in male fertility and reproduction [i.e. 6, 8, 9, 36, 130, 131].
For instance, gossypol, a male antifertility compound iso-
lated from cotton, irreversibly inhibits T-type Ca2+ currents in
spermatogenic cells in a dose- and time-dependent manner.
Complete blockade occurs at 5 M gossypol [7]. Similarly,
the extract of the root xylem of Tripterygium wilfordii and one
of its monomers, demethylzeylasteral, inhibit T-type Ca2+
currents with an IC50 of 6.4 and 8.8 g/ml, respectively [6].
ZD7288, a blocker of hyperpolarization and cyclic nucleotide-
gated (HCN) channels, inhibits spermatogenic T-type Ca2+
currents with an IC50 of 100 M and it also inhibits CaV3
channels heterologously expressed in HEK cells [36].
Urocortin (UCN), a peptide related to the corticotropin-
releasing factor (CRF) and present in the seminal fluid, in-
hibits T-type Ca2+ currents with an IC50 of ~0.7 M. UCN
inhibition of Ca2+ currents does not involve binding to the
CRF receptor [123]. Fenvalerate, a type II pyrethroid insecti-
cide, blocks spermatogenic T-type Ca2+ currents with an
IC50 =0.25 M. Maximal inhibition (~40 %) is attained at a
concentration of 1 M. [137]. Fenvalerate effects are slow and
irreversible. 17-Estradiol (E2) significantly inhibits T-type
Ca2+ currents in a dose-dependent manner (IC50 =8.9 M). At
100 M, E2 produced maximal inhibition (53 %) and shifted
activation and inactivation curves by 5 mV in the negative
direction [87].
The protein tyrosine kinase inhibitor genistein, a phytoes-
trogen from soy, reversibly decreased spermatogenic T-type
822
Ca2+ currents in a concentration-dependent manner (IC50
22.7 M). Genistein inhibition does not involve TK activity
but rather acts directly, stabilizing the channel in the
inactivated state. In transfected HEK293 cells, 50 M genis-
tein inhibits CaV3.1 and CaV3.2 channels (by 43 and 52 %,
respectively), but not CaV3.3 channels [123]. Ghrelin, a newly
isolated peptide, also reversibly inhibits these currents in a
dose-dependent manner (IC50 =35 nM). Maximal inhibitory
effect of T-type Ca2+ currents is 38 %. Inhibition was blocked
by an antagonist of the growth hormone secretagogue receptor
1a (GHS-R1a), a G-protein coupled receptor expressed in
spermatogenic cells and mature spermatozoa [85]. Ghrelin-
induced T-type Ca2+ current inhibition does not involve inter-
action of G subunits with the channels. The chloride chan-
nel blocker niflumic acid (NA) blocks T-type Ca2+ currents
with an IC50 of 73.5 M. NA also blocks CaV3 channels
expressed in HEK2h3 cells. NA-estimated IC50 values are
78, 192 and 137 M for CaV3.1, CaV3.2 and CaV3.3, respec-
tively [8]. Finally, celecoxib (Cx), an anti-inflammatory drug
designed to inhibit cycloxygenase 2, has been shown to inhibit
T-type Ca2+ currents with an IC50 of 27 M. Surprisingly, Cx
also induces intracellular Ca2+ rise, depolarizes membrane
potential and triggers the acrosome reaction [9].
Sperm electrophysiology
Even though in principle the ion channels that will end up in
mature spermatozoa could be studied in spermatogenic cells
[3, 56, 81], we now know that they undergo drastic changes,
re-distribute and even functionally disappear during the last
stages of spermatogenesis [27, 114, 138]. Early on, electro-
physiological recording of ion channel activity in the sperm
cell membrane was extremely difficult. Only 1 % of the
attempts were successful [33, 50]. Swelling of sea urchin
sperm, by making sealing slightly easier than in normal sperm,
allowed cell-attached patch-clamp recordings, but they were
still difficult to obtain and only lasted for a few minutes [106].
Fortunately, new strategies have been developed to study
sperm ion channels in mature cells in situ. Three possibilities
are now available:
1. Whole-sperm patch-clamp recordings can be obtained
sealing on the 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 in mouse and other species but retained in
human spermatozoa [73, 82, 84].
2. The smart patch-clamp allows the simultaneous record-
ing of the topography of the sperm surface and gigaseal
formation directly on mature sperm heads [23, 24, 47, 49,
68]. This approach combines scanning ion conductance
microscopy and patch-clamp recording through a single
Pflugers Arch - Eur J Physiol (2014) 466:819831
glass nanopipette. The patch-clamp electrode is scanned
and gathers topographic information of the cell surface
with greater spatial resolution than the optical microscope.
This information allows positioning of the pipette onto the
cell surface with nanometer precision to obtain single-
channel recordings from small cells and submicron cellu-
lar structures inaccessible by conventional methods [77].
The spatial resolution of the smart patch-clamp is now
able to resolve plasma membrane protein complexes
[115].
3. The perforated-cell patch-clamp technique to record ionic
currents in the whole-cell mode from the mature human
sperm head was derived from work on cardiac myocytes
and neurons [104]. A saponin/-escin mixture is used in
the pipette tip to induce controlled patch permeabilization.
Stable whole-cell currents can be achieved after patch
perforation from dialyzed spermatozoa of several species
with the advantage of not requiring the presence of a
cytoplasmic droplet. Using this strategy, a Ca2+-regulated
Cl channel was first characterized in human spermatozoa
[94].
Patch-clamp recording on the cytoplasmic droplet of tes-
ticular and epididymal mouse spermatozoa has revealed the
activity of several ion channels [27, 82, 88, 91, 100, 108, 138].
Examples are mouse and human CatSper [73] and mouse and
now human Slo3 channels [80, 90, 108, 111, 143]. Until now,
these are the only two sperm-specific channels whose genetic
removal results in infertility. The CatSper cation channel has a
tetrameric organization of four subunits (CatSper 14) plus
three auxiliary subunits till now (CatSper, CatSper,
CatSper). It is Ca2+-permeable and potently regulated by
pHi [83]. The Slo3 channel belongs to the Slo K+ channel
family. Slo3 currents in mouse testicular and epididymal
sperm are activated by intracellular alkalinization, depolariza-
tion and cAMP (testicular); blocked by 1 mM Ba2+ and high
[TEA] (60 mM); and poorly K+-selective (PK+/PNa+ ~8), in a
similar fashion as they are when heterologously expressed in
oocytes. These currents are absent in testicular and epididymal
sperm from Slo3 null mice, confirming their identity [108,
140, 143]. Whole-sperm recordings have also revealed the
presence of a voltage-sensitive H+ channel involved in pHi
regulation, more prevalent in human than in mouse sperm [74]
and of ATP-gated channels of the purinergic family, P2X2, in
mouse epididymal sperm [92]. TRPM8-like currents, absent
in sperm from TRPM8 null mice, have been recorded in
testicular sperm [44, 89]. Pharmacological and functional
experiments are consistent with the presence of a similar
channel in human spermatozoa [29].
The T-type Ca2+ currents were recorded from testicular
mouse sperm sensitive to micromolar concentrations of
mibefradil, nifedipine and Ni2+. These results confirmed pre-
dictions from expression patterns of CaV3 channels detected
Pflugers Arch - Eur J Physiol (2014) 466:819831
with specific antibodies with the presence of at least some
CaVs in mature mouse sperm [27, 32, 125]. In addition, they
are consistent with earlier experimental work using pharma-
cological tools, [Ca2+]i imaging and AR determinations, sug-
gesting the participation of CaV channels in the sperm AR.
In spite of the fact that CaV3 currents similar to those
recorded in spermatogenic cells were detected in testicular
mouse spermatozoa, they have not been detected in epididymal
sperm [27, 83, 138]. Even examination of the currents in sperm
from CatSper and Slo3 null mice has not revealed the functional
presence of any CaVs [144]. These results question previous
functional, molecular and pharmacological data indicating the
involvement of CaVs in diverse sperm functions as they indicate
that the CatSper channel is the principal Ca2+-conducting chan-
nel in mammalian spermatozoa [83, 100, 144].
There are puzzling and contrasting findings regarding the
presence of CaV channels in sperm (see Concluding re-
marks). Sound immunolocalization and functional evidence
recording [Ca2+]i increases triggered by K+-induced depolar-
ization and the AR from several laboratories indicate the
presence of at least CaV3.2 and/or other HVA CaV channels
such as CaV2.3 in mouse and human spermatozoa [32, 70,
103, 133, reviewed in 27]. Notably, the motility and [Ca2+]i
increase induced by K+ depolarization are altered in sperm
from CaV2.3 null mice [103, 109]. However, whole-cell
patch-clamp recording at the cytoplasmic droplet does not
reveal any CaV currents. Single-channel recordings on the
mammalian sperm head will be necessary to determine if
and which type of CaVs are present.
Possible roles of T-type Ca2+ (CaV3) channels
in spermatogenesis and sperm function
Mammalian spermatogenesis
Spermatogenesis occurs inside the seminiferous tubules of the
testicle and involves the transformation of diploid
spematogonial stem cells into haploid spermatozoa. Commit-
ted spermatogonia proliferate (mitosis) and develop into sper-
matocytes which through meiosis produce round spermatids.
After undergoing a morphological transformation called sper-
miogenesis, spermatids mature into spermatozoa. Spermato-
genesis is a highly conserved process throughout vertebrate
species that is hormonally controlled mainly by the hypothal-
amus and the pituitary gland. However, as spermatogenesis is
a complex stage-specific, multifactorial process, a variety of
endocrine, paracrine and autocrine factors converge to control
it [62].
During spermiogenesis, the terminal differentiation process
of spermatozoa, haploid round spermatids go through exten-
sive biochemical and morphological changes that include
acrosome formation, flagellar development, chromatin
823
condensation and a dramatic nuclear and cellular reorganiza-
tion. Morphological considerations have led to a classification
of mouse spermiogenesis that involves 16 developmental
steps. They can be summarized as follows: round (steps 5
8], elongating (steps 911), condensing (steps 1214) and
condensed (steps 1416) spermatids. Early on, round sperma-
tids are transcriptionally active and this activity decreases in
elongating spermatids to finally turn off [120, 134].
Spermatogenic cells are unable to differentiate alone; they
are nursed by Sertoli cells inside the tubules. Leydig cells from
the interstitial compartment also provide hormonal stimula-
tion [43]. Intriguingly, not much is known about the role of
Ca2+ during spermatogenesis. Spermatogenic cells, as all
cells, finely regulate their [Ca2+]i. For example, in rat sper-
matogenic cells, high temperatures can induce programmed
cell death which involves [Ca2+]i regulation [60]. On the other
hand, an external supply of oxidative glycolytic substrates
such as glucose and lactate differentially regulates [Ca2+]i in
round spermatids and pachytene spermatocytes [101].
Our group recently reported that mice spermatogenic cells
undergo spontaneous [Ca2+]i oscillations that depend on extra-
cellular Ca2+. These oscillations are partially inhibited by CaV3
Ca2+ channel blockers and Ni2+ [105]. It would be very impor-
tant to fully characterize these spontaneous [Ca2+]i oscillations
to determine if and how they influence spermatogenesis.
The physiological importance of CaVs in spermatogenesis
and steroidogenesis is underscored by the finding that CaV3
channels are the main voltage-gated Ca2+ channels functional-
ly expressed in meiotic and post-meiotic male germ cells.
Steroidogenesis is a complex multienzyme process by which
cholesterol is converted to biologically active steroid hormones
not only in the adrenal cortex but also in the testis. Moreover,
functional CaVs are required for normal germ cell develop-
ment: blocking of either CaV3 or CaV1 channels by their
respective blockers during mice prepubertal testicular matura-
tion results in abnormal spermatogenesis and steroidogenesis.
This alteration leads to male sterility, by arresting developing
spermatids and diminishing the Leydig cell population [78].
Motility
In many species, flagellar propulsion is a key feature that
allows spermatozoa to reach the egg and achieve fertilization.
The ionic composition of the sperm environment and the
regulation of its ion plasma membrane permeability are funda-
mental for motility regulation. The flagellar propulsion ma-
chinery, the axoneme, is the site of Ca2+ regulation and thus
[Ca2+]i influences flagellar beat symmetry [16, 17]. Sperm
from many marine species display chemotaxis, a motility
response that is dependent on external Ca2+ and encompasses
a series of turns interspersed with periods of straight swimming
[27, 54, 72, 135]. In swimming sea urchin sperm, [Ca2+]i
transients regulate chemotaxis [13, 136, reviewed in 27, 72].
824
Immunolocalization and western blots using mammalian CaV
antibodies suggested the presence of CaV1.2 and CaV12.3 in
the flagella and acrosomal region of sea urchin sperm, respec-
tively. Furthermore, CaV channel blockers, nifedipine and
nimodipine, that inhibit the AR in this sperm species decrease
Ca2+ uptake caused by a K+-induced depolarization in
valinomycin-treated cells [48] Such Ca V blockers as
nimodipine and micromolar Ni2+ also eliminate the [Ca2+]i
transients triggered by speract, a sperm motility regulator
[135]. The Strongylocentrotus purpuratus genome contains
CaV3 sequences; however, the presence of these proteins in
sea urchin sperm has not been demonstrated. On the other
hand, CaV channel blockers inhibit ascidian sperm motility
activation by an egg-derived factor which also induces chemo-
taxis that is sensitive to a store-operated Ca2+ channel (SOC)
inhibitor [141].
Ion channels have been implicated in the modulation of
sperm motility; however, only after sperm from CatSper null
mice were shown to be infertile and incapable of
hyperactivating was this established [20, 99]. Mammalian
sperm can swim in two modes, activated and hyperactivated.
Upon ejaculation, sperm display a relatively low-amplitude
flagellar beat called activated motility. In contrast, after capac-
itation, a fraction of sperm swims in figure of eight trajec-
tories in low-viscosity medium, due to large-amplitude asym-
metric flagellar beats. In the upper female reproductive tract,
which has a higher viscosity, hyperactivated sperm are more
progressive. Progression through the oviductal higher-
viscosity media and possibly egg cumulus penetration may
require hyperactivated motility which is Ca2+-dependent [22].
The flagellar beat frequency of hyperactivated hamster sperm
is correlated with transitory [Ca2+]i increases they display
[121]. The redundant nuclear envelope, a reticular structure
at the flagellum neck, is an internal sperm Ca2+ store appar-
ently involved in hyperactivation. Stimulation of InsP3 recep-
tors found in this store or of Ca2+ release from such internal
stores with thimerosal or 4-AP induces hyperactivation [2,
63]. At this time, the presence of functional CaV3 channels
in mammalian flagella is unlikely, as CaV3.1 immunolocal-
ization results obtained in the mouse flagella [125] were not
corroborated using sperm from CaV3.1 null mice [32], and no
Ca2+ currents have been detected in mouse epididymal sper-
matozoa [138], our unpublished recordings] nor in ejaculated
human sperm [83]. Single-channel recordings would corrob-
orate the absence of any CaV channels in the mammalian
sperm flagella.
Capacitation
After ejaculation, mammalian sperm need to mature within
the female tract to acquire the ability to fertilize the egg. This
complex maturational process is called capacitation [128].
This non-linear set of events allows spermatozoa to respond
Pflugers Arch - Eur J Physiol (2014) 466:819831
to stimuli that induce the AR and to penetrate the egg invest-
ments prior to fertilization [38, 127]. Cholesterol removal by
albumin, bicarbonate and Ca2+ are amongst the stringent
requirements of capacitation which lead to mammalian sperm
plasma membrane reorganization, extensive changes in pro-
tein phosphorylation state, increases in pHi and [Ca2+]i and
plasma membrane hyperpolarization. A cAMP- and PKA-
dependent non-linear pathway regulates the phosphorylation
changes associated to capacitation which encompasses hyper-
activation [reviewed in 26, 65, 127, 128].
In mouse sperm, it was proposed that plasma membrane
hyperpolarization removes CaV3 channel inactivation, leaving
Ca2+ channels in a state from which they could open upon
induction of the AR by ZP3 (see the The AR section) [54].
This proposal is based on the assumption that CaV3 channels,
which are functional in spermatogenic cells [3, 81] and also in
testicular sperm [88], are functional in mature spermatozoa.
However, the latter is now debated and the role of this hyper-
polarization is being re-examined.
The mouse sperm hyperpolarization involves activation of
K+ channels and inhibition of epithelial-like Na+ channels
(ENaCs) [59]. The cystic fibrosis transmembrane conductance
regulator (CFTR) has been suggested to activate during ca-
pacitation and somehow mediate ENaC-type channel inhibi-
tion contributing to the hyperpolarization [37]. Recent find-
ings show that amiloride, an ENaC inhibitor, genistein, a
CFTR agonist, and valinomycin, a K+ ionophore, are able to
mimic the capacitation-associated hyperpolarization. This
membrane potential change enhances the ability of a K+-
induced depolarization or of soluble ZP to increase [Ca2+]i
and induce the AR in non-capacitated sperm, although no
protein phosphorylation is promoted. These findings indicate
that sperm hyperpolarization by itself is the key to allow mice
sperm to acrosome react [30]. Future work will define if a
Ca2+ channel is involved in this process.
CaV channels and particularly CaV3.2 could contribute to
the rise in [Ca2+]i that accompanies capacitation. Notably,
bovine serum albumin (BSA) stimulates CaV3 currents from
mouse spermatogenic cells and shifts both their voltage-
dependent steady-state activation and inactivation [56]. BSA
also increases the window current that results from the
overlap of the steady-state activation and inactivation curves
of these CaV3 currents. As a result, a small persistent Ca2+
influx through CaV3 channels could contribute to the [Ca2+]i
rise that occurs during capacitation [35].
The AR
The head of spermatozoa from many species contains a
membrane-delimited organelle found overlying the sperm
nucleus. Certain physiological or pharmacological stimuli
trigger the fusion of this organelle and the closely apposed
plasma membrane at multiple sites, releasing the acrosomal
Pflugers Arch - Eur J Physiol (2014) 466:819831
contents to the extracellular media [18]. This single secretory
vesicle exocytosis process is called the AR and is essential for
fertilization in all sperm species possessing an acrosome. The
transformation of the sperm plasma membrane associated to
this reaction, which incorporates the inner acrosomal mem-
brane, is required for penetrating the outer envelope of the egg
and for fusion with its plasma membrane. External Ca2+ is
essential for the AR, and certain ion channel blockers, includ-
ing those selective for CaV channels, interfere with the reac-
tion [reviewed in 26, 42, 71].
Upon spawning, sea urchins release billions of their sperm
and eggs into the sea. Sperm interact with the outer layer of the
egg, the egg jelly, which triggers the AR. The egg jelly
contains a fucose sulfate polymer (FSP) that is the inducer
of the AR [126]. FSP binds to suREJ1, a 210-kDa membrane
glycoprotein, and triggers K+-dependent membrane potential
changes which involve increases in [Ca2+]i, [Na+]i and pHi
[reviewed in 26]. The sea urchin sperm AR is triggered by at
least two different Ca2+ channels [51, 53, 110]. A Ca2+-selec-
tive channel sensitive to verapamil and dihydropyridines is the
first to transiently open when FSP binds to suREJ1. Its phar-
macology is compatible with CaV channels. A second chan-
nel, insensitive to the latter blockers, opens 5 s later, is sensi-
tive to pHi, does not inactivate and results in the AR [52]. It is
likely that the second channel is of the SOC type [46]. Sperm
are devoid of endoplasmic reticulum, so it is likely that the
acrosome is the internal store which is known to contain InsP3
receptors [31, 142]. Furthermore, sea urchin sperm produce
InsP3 during the AR [reviewed in 27].
In Lytechinus pictus sperm, FSP induces a transient K+-
dependent hyperpolarization that is likely due to K+ channels
and blocked by TEA [45]. It was proposed that this hyperpo-
larization could be necessary to remove inactivation from CaV
channels [45], as it was later proposed for mammalian sperm
[40]. A CaV3 transcript is present in the S. purpuratus ge-
nome, but protein expression in sea urchin sperm has not been
demonstrated.
Ca2+ influx is the key to the mammalian sperm AR, and
several Ca2+-permeable channels have been proposed to par-
ticipate. As in sea urchin sperm and other marine species, the
mammalian sperm AR involves, after external Ca2+ uptake,
Ca2+ release from internal stores (the acrosome itself and
possibly residual nuclear vesicles found in the sperm neck
and midpiece) and the activation of store-operated Ca2+ chan-
nels [28, 46, 61, 95; reviewed in 18, 40].
The ZP, the extracellular coat surrounding the egg, and
progesterone, which is present in the oocyte vicinity, are two
physiologically relevant AR inducers that activate two or
more Ca2+-permeable channels most likely through distinct
pathways [reviewed in 27]. Findings derived from studies
using Ca2+-sensitive fluorescent dyes suggested the involve-
ment of CaV channels in the ZP-induced AR. The pharmaco-
logical characteristics of the initial Ca2+ transient triggered by
825
ZP are consistent with those displayed by the CaV3 currents in
mouse spermatogenic cells [reviewed in 27, 67, 71].
Notably, for a long time, ZP has been considered the
physiological inductor of the AR [132]. However, novel stud-
ies using transgenic mice expressing different GFPs in the
sperm acrosome and mitochondria have shown that sperma-
tozoa undergo the AR before contacting the ZP during in vitro
fertilization of cumulus-egg complexes [11, 69, 129]. These
findings pose new questions regarding the identity, location
and signaling pathway of the physiological inducer of the AR
and the ion channels involved in it. In spite of the fact that null
mice for either CaV3.1 or CaV3.2 are fertile, as stated before, it
will be necessary to generate the CaV3.1/CaV3.2 double
knockout to completely rule out the participation of CaV3
channels in the mouse sperm AR.
Although evidence has been presented for the presence of
CaV3 channel transcripts and protein in human sperm [66, 96,
125], and the ZP- and neoglycoprotein-induced AR pharma-
cology displays similarities with that of CaV3 channels [10,
12, 14, 70, 124], their functional participation in these cells
has not been established.
As indicated earlier, progesterone is secreted by the
cumulus in the female reproductive tract. In human
sperm, low-micromolar progesterone induces a rapid
[Ca2+]i increase followed by a plateau phase in sperm
population and can trigger the AR [and reviewed in 58,
102]. This hormone is now known to potently activate
CatSper [83, 119]. [Ca2+]i imaging in single human
sperm has revealed that progesterone induces an initial
transient increase beginning at the sperm flagella and
traveling towards the head [18, 79, 113]. In a fraction
of the sperm population, progesterone can induce
[Ca2+]i oscillations [75], which has been shown to be
sensitive to Ca V 3 and ryanodine receptor channel
blockers [1, 57].
Unfortunately, in pharmacological terms, CatSper is a pro-
miscuous channel with multiple ligands modulating its func-
tion [15]. It is sensitive to CaV3 channel blockers such as
mibefradil [15, 83] which complicates dissecting the contri-
bution of CaV3 channels in [Ca2+]i measurements.
CaV3 channels are subject to multiple forms of regulation
that have been reviewed in detail [98, 112]. Of interest regard-
ing the AR and the controversial role of CaV3 channels in
sperm physiology is the observation that CaM antagonists W7
and trifluoperazine inhibit mouse spermatogenic CaV3 cur-
rents in a concentration-dependent manner [86]. Both com-
pounds shift the voltage dependence of activation to more
positive values and slow activation and inactivation kinetics.
W5, a less potent homolog of W7 used as a control, does not
perturb these currents. The inhibitory effect of W7 is Ca2+-
dependent, suggesting a role for the Ca2+/CaM complex in
this regulation. These currents were not altered by inhibitors
of CaM-activated phosphatase, CaMKII and protein kinase A.
826
It is worth pointing out that the ZP-induced mouse sperm AR
and the transient [Ca2+]i increase associated to this reaction
are blocked by the CaM inhibitors [86]. In retrospective, these
findings are still consistent with the possible participation of
CaV3 channels in the mouse sperm AR and call our attention
to the need for a better understanding of the involvement of
CaM in the AR. Along this line, recent studies showed that
Ca2+ and CaM modulate CaV3.2/calcineurin interactions.
Calcineurins phosphatase activity is decreased when it binds
to CaV3.2 which diminishes the channels current density
[64]. As calcineurin is required in the human sperm AR
[21], CaM modulation would be consistent with the participa-
tion of CaV3.2 in mammalian sperm AR. From the informa-
tion stated above, we would have to conclude that more work
is needed to determine if CaV channels participate in the
signaling cascades leading to the AR in mammalian sperm.
Concluding remarks
The findings of Zeng et al. [144] with spermatozoa from the
double CatSper and Slo3 null mouse suggest that during
alkalinization, these two channels are the main and possibly
the only voltage- and pHi-activated cationic currents present
in the mouse corpus epididymal sperm principal piece. The-
se authors state that at a lower pHi, their conclusion may not
hold. Non-capacitated sperm have a pHi of around 6.8
which alkalinizes to ~7.27.4 after capacitation [reviewed
in 128]. Small currents buried in the background pipette
leak conductance at lower pHi values could nonetheless
have a profound influence on sperm physiology. As sper-
matozoa are small and have high input impedance, opening
or closing of a single channel might significantly contribute
to their resting potential [50, 144]. Because of these consid-
erations and the fact that in the Zeng et al. experiments,
sperm did not undergo other physiological changes associ-
ated to capacitation, which may alter ion channel behaviour,
one cannot rule out the presence and participation of a few
CaV channels or other cationic permeable channels in the
sperm plasma membrane particularly if they are located in
the head (see below).
In the voltage-clamp technique, current should be re-
corded ideally, from a membrane area of uniform potential,
i.e. from a population of channels experiencing the same
voltage. Whole-cell patch-clamp is ideal for small round
cells, where good space clamp can be achieved. However,
in long processes such as the sperm flagella, the membrane
potential distal to the recording electrode is not well
clamped, and therefore, current recorded by the voltage-
clamp electrode combines local trans-membrane current
with axial currents originating from locations experiencing
different potentials. Assuming a specific intracellular resis-
tivity value of 100 ohm cm (similar to that of a neurite) and a
Pflugers Arch - Eur J Physiol (2014) 466:819831
specific membrane resistivity of 20 kohm cm2, a flagellum of
0.5 m in diameter has a space constant () of ~500 m.
Therefore, a 150-m-long sperm flagellum would be reason-
ably space-clamped. However, because of the reduced cyto-
plasmic space inside the flagella and its packing with axone-
mal structural components, molecular mobility under such
crowded conditions is severely restricted compared with that
in the typical cell cytoplasm. Fluorescence recovery after
photo-bleaching (FRAP) experiments, using small fluorescent
tracers (calcein or carboxyfluorescein) in the sea urchin sperm
flagella, gave diffusion coefficients of ~60 m2/s, i.e., five
times less than in aqueous media (~300 m2/s) [122]. Assum-
ing a proportional increase of intracellular resistivity in the
flagellum (500 ohm cm), then the space constant falls to
~200 m, implying that near the tip of the mouse sperm
flagellum (100150 m), membrane voltage is attenuated by
4050 % from that applied at the cytoplasmic droplet. Mem-
brane currents recorded from such distant, poorly clamped
sources should be similarly attenuated. This situation gets
worst for the movement of small molecules between the
flagellum and the head: the fluorescence recovery rate is >60
times slower in the head than in the flagellum, suggesting the
presence of additional diffusion barriers around the neck
region of the spermatozoa (and even stronger space-clamp
problems). Altogether, these arguments point out that caution
must be exercised still regarding the exact set of ion channels
that determine their behaviour and influence fertilization.
Summarizing, there are still pending issues that need to be
addressed to establish if CaVs, and particularly CaV3 channels,
do participate in the mammalian sperm acrosome reaction. In
this regard, it will be important to carry out single-channel
recordings in the sperm head to determine if CaVs are func-
tionally present and if they participate in this important reac-
tion. Furthermore, it is very interesting and relevant to under-
stand why and how CaV3.2 channel expression gets turned off
as testicular spermatozoa complete their maturation if it is the
case and if it is possible that their expression gets turned on
again after capacitation just before the AR is triggered.
Sperm research is undergoing revolutionary changes.
Well-established paradigms such as the role of the zona
pellucida in inducing the sperm acrosome reaction are
being debated and re-examined. Novel strategies to
study ion channels are challenging to work with, but
this fascinating cells are revealing some of their unex-
pected properties and will help to define the complete
set of key ion transporters necessary for sperm matura-
tion, regulated swimming, capacitation and the AR.
Advances in temporal and spatial resolution of the
available imaging techniques are unveiling mechanistic
details of where, when and how intracellular Ca2+ and
pHi profoundly influence sperm physiology. This new
information is essential to advance our understanding of
reproduction, and species preservation and control.
Pflugers Arch - Eur J Physiol (2014) 466:819831
Acknowledgements This work was supported by the Direccin Gen-
eral de Asuntos del Personal Acadmico; by the Universidad Nacional
Autnoma de Mxico (DGAPA-UNAM) grants IN225406-3 and
IN222413 to to AD and AH-C, respectively; by the Consejo Nacional
de Ciencia y Tecnologa (CONACyT) grant 39908-Q to AD and RO3
TW 006121 to Pablo Visconti; and by the Secretara de Ciencia,
Tecnologa e Innovacin del Distrito Federal (SECITI) grant 039/2013
to AHC and AD.
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